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Case Report

Compound Analysis of Jing Liqueur and nrf2 Activation by Jing Liqueur—One of the Most Popular Beverages in China

1
Department of Pharmaceutical Sciences, Daniel K Inouye College of Pharmacy, University of Hawaii at Hilo, 200 W. Kawili Street, Hilo, HI 96720, USA
2
Institute of TCM and Natural Products, School of Pharmaceutical Sciences, Wuhan University, 185 Donghu Road, Wuhan 430071, China
3
Jing Brand Research Institute, Jing Brand Co., Ltd., Daye 435100, China
*
Authors to whom correspondence should be addressed.
Submission received: 19 November 2019 / Revised: 13 December 2019 / Accepted: 24 December 2019 / Published: 31 December 2019
(This article belongs to the Special Issue Chemical Contaminants and Residues in Beverages)

Abstract

:
The aim of this study is to identify the minor compounds in Jing liqueur, determine the concentration of metals, amino acids, and polysaccharides, and evaluate their Nrf2 activity and cytotoxicity. Jing liqueur that contains Chinese medicine is one of the best-selling liqueurs in China, which is also marketed in the United States. Totally, we have isolated 189 minor compounds including one new molecule (7) from a concentrated Jing liqueur, with the concentrations of most isolated compounds at micromolar levels. The structures of all these compounds were determined by using MS and NMR (1D and 2D) or by comparison of their chemical and physical data with reported values in the literatures. Besides, the concentrations of iron (0.52 mg/L), zinc (0.21 mg/L), calcium (11.0 mg/L), L-proline (2.33 mg/L), L-arginine (1.73 mg/L), total amino acids (9.84 mg/L), and total polysaccharides (337.4 mg/L) were determined. Jing liqueur, the five fractions and most of the compounds isolated from Jing liqueur were screened for their activities in the Nrf2-ARE and MTT assays. At 5.2 mg/mL the crude enhanced the Nrf2 activity. At 80 μg/mL, fraction IV weakly but fraction V strongly activated Nrf2. Among the compounds screened in the Nrf2 assay, eighteen activated Nrf2 at 40 μg/mL and compounds 51 and 126 from fraction V were the most active. The crude, all the five fractions, and Nrf2 activators were not cytotoxic toward HepG2 cells. In conclusion, Jing liqueur contains different classes of compounds including flavonoids, terpenoids, alkaloids, coumarins, cinnamic acid or coumaric acid, and phenyl ethanol (or acetic acid) derivatives, benzoquinone, naphthoquinone, anthraquinones or phenanphrene derivatives, xanthones, chromone, and γ-pyrone derivatives, lignans, other aromatic compounds, and others. Jing liqueur and the eighteen compounds, which were isolated from Jing liqueur, could activate Nrf2 without any cytotoxicity.

Graphical Abstract

1. Introduction

Jing liqueur [1,2,3] is a popular health beverage in China, which contains biologically active components from several tonic traditional Chinese herbal medicines. About 30 years ago, Jing Brand Co., Ltd. (Daye City, Hubei Province, China) began to sell its products overseas. Nowadays, Jing liqueur is sold in more than 20 countries and districts, including Hong Kong, Macau, Japan, South Korea, Australia, and the United States etc. The liqueur is manufactured using modern bioengineering technology to prepare extracts from Chinese herbal medicines such as Astragalus membranaceus, Cistanche deserticola, Dioscorea opposita, Lycium barbarum, Epimedium brevicornum, Cinnamomum cassia, Syzygium aromaticum, Angelica sinensis, and Imperata cylindrica, several of which are also used as foods or dietary supplements. These Chinese herbal medicines are carefully selected and prepared according to their applications.
A. membranaceus is a very common traditional Chinese medicine (TCM) widely used as an immunostimulant, cardiotonic, hepatoprotective, antidiabetic, antitumor, and antiviral drug [4]. C. deserticola is a famous Chinese Materia Medica (CMM) used for the treatment of kidney deficiency, infertility, and chronic constipation [5]. D. opposita is a famous tonic Chinese medicine with beneficial effects on spleen, lung, and kidney in addition to the antidiarrheal activity. The tuber is also a favorite food in China used in a stir-fry or in soups [6]. L. barbarum a traditional food and medicine in East Asia has become increasingly popular in Europe and North America in recent years. L. barbarum is used in folk medicine to increase longevity and is reported to have beneficial effects on blurry vision and diminished visual acuity, infertility, abdominal pain, dry cough, fatigue, and headache [6]. L. barbarum is also a very popular ingredient in Chinese cuisine, which is consumed in soups, as porridge with rice, and added to numerous meat and vegetable dishes [7]. E. brevicornum is one of the most commonly used traditional Chinese medicines, and has the reported benefits of reinforcing the “kidney yang,” strengthening the tendons and bones, and relieving rheumatic conditions. It is also used to treat impotence, seminal emission, weakness of the limbs, rheumatoid arthralgia, and hypertension [8]. C. cassia is used in traditional Chinese medicine for various ailments including abdominal pain, vomiting, diarrhea, dysmenorrhea, blood stasis, bruises, and traumatic bleeding. It is also used as an appetite stimulant and a flavoring agent [9]. S. aromaticum is a Chinese medicine that is used as an aromatic stomachic agent, to relieve abdominal bloating, increase gastric secretions, aid in digestion, and reduce nausea and vomiting. It is also one of the most ancient and valuable spices of the Orient [10]. A. sinensis is one of the most important drugs in traditional Chinese medicine, and is commonly used for treating gynecopathias, including anemia, dysmenorrhea, amenorrhea, premenstrual, and menopausal syndromes. It is also used in the management of cancer, cardiovascular diseases, and Alzheimer’s disease. Chicken soup made with Radix Angelica is a popular dish in China [11]. I. cylindrica is also a traditional Chinese medicine used in treating hot blood, blood vomiting, blood stasis, hematuria, fever, polydipsia, damp heat jaundice, edema, reduced urine output, and painful urination [12].
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that controls the expression of antioxidant and phase II detoxifying enzymes. Nrf2 is widely recognized for its cytoprotective role, and has defensive properties against neurodegenerative, airway, and cardiac diseases [13]. Nrf2 is also targeted for the prevention of cancer and other chronic diseases such as diabetes, where oxidative stress and inflammation contribute to pathogenesis [14]. Also, Nrf2 activation through cell lineage-specific Keap1 disruption is important for the improvement of autoimmune diseases [15]. In these settings, transient activation of Nrf2 by compounds such as sulforaphane or curcumin can stimulate the expression of Nrf2 target genes to combat oxidative and electrophilic stress, reorganize cortical actin, reduce stress fibers formation, and improve the integrity of cell-cell junctions [16]. On one hand, Nrf2 activators could be used for the prevention of chemical carcinogenesis, whereas Nrf2 inhibitors could be used for cancer treatment [17].
Although the phytochemical composition and pharmacological properties of these nine Chinese herbal medicines have been independently evaluated, Jing liqueur containing extracts from these nine herbal medicines has not been previously investigated much. It has been reported that Jing liqueur showed anti-inflammatory [1], immune enhancement [3], anti-fatigue [2,3] properties, and enhancing Shen-Yang (kidney Yang) or invigorating the vital activities of kidney [3]. We argue that the anti-inflammation and immune enhancement of Jing liqueur may be due to or at least partially due to the Nrf2 activation by these traditional Chinese herbal medicines used in Jing liqueur. Hence, we also decided to evaluate Jing liqueur for its effect on Nrf2 besides the analysis of minor compounds. In this study, we isolated one hundred eighty nine (189) minor compounds from Jing liqueur including a new flavonoid (7), and determined their structures based on the MS data and NMR spectra. In addition, we determined the concentrations of iron, zinc, calcium, L-proline, L-arginine, total amino acids, and total polysaccharides. We also evaluated the effects of the crude Jing liqueur, the five fractions, and majority of the isolated compounds on the Nrf2 activity in a cell-based assay, and investigated the cytotoxicity of the crude Jing liqueur, the five fractions and the identified Nrf2 activators in a MTT assay. At 40 μg/mL, eighteen compounds demonstrated Nrf2 activation without any cytotoxicity, and compound 51 was slightly less active while compound 126 was more active than the positive control SF (5 μM), indicating that compounds 51 and 126 might be responsible for or partially account for the Nrf2 activation.

2. Materials and Methods

2.1. Plant Materials

Plant materials were collected in 2017 by researchers at Jing Brand Research Institute. Voucher specimens (JP20170219, A. membranaceus, Min County, Dingxi City, Gansu Province, China; JP20170259, C. deserticola, Hetian City, Xinjiang Uygur Autonomous Region, China; JP20170014, D. opposita, Jiaozuo City, Henan Province, China; JP20170214, L. barbarum, Zhongning County, Ningxia Hui Autonomous Region, China; JP20170244, E. brevicornum, Min County, Dingxi City, Gansu Province, China; JP20170143, C. cassia, Fangchenggang City, Guangxi Zhuang Autonomous Region, China; JP20170265, S. aromaticum, Indonesia; JP20170133, A. sinensis, Min County, Dingxi City, Gansu Province, China; and JP20170000, I. cylindrica, Louzhou City, Sichuan Province, China) are deposited at the herbarium of Jing Brand Research Institute, Daye City, Hubei Province, People’s Republic of China.

2.2. Preparation of Jing Liqueur

Jing liqueur was prepared at Jing Brand Co., Ltd., using the company’s proprietary technology. Following is a basic description of the process: (a). The raw Chinese herbal medicine (Astragalus membranaceus, Cistanche deserticola, Dioscorea opposita, Lycium barbarum, Epimedium brevicornum, Cinnamomum cassia, Syzygium aromaticum, Angelica sinensis, and Imperata cylindrica) was washed, dried, and sliced into pieces according to the protocols as described in the Chinese Pharmacopoeia. (b). Pieces of each herbal medicine were added to the “Xiaoqu white liqueur” with an alcohol content of 35% according to the company’s process recipe. After percolation, filtration, and evaporation, various concentrated mother liquids were obtained. (c). The concentrated mother liquids were added to the “Xiaoqu white liqueur” with an alcohol content of 35% for precise blending according to a standardized process recipe. (d). Certain amount of white sugar was added to adjust the taste. (e). The finished product was kept in a storage tank and stored for one year. After quality control, Jing liqueur was filled into small bottles and packaged for shipping to commission merchants.

2.3. Concentration of Jing Liqueur

Before white sugar was added, one hundred sixty liters (160 L) of Jing Brand “Xiaoqu white liqueur” (a semi-finished product after step c in the Section 2.2) was concentrated under vacuum to yield a syrup-like liquid, which was about 232 g if completely dried and was used for the separation and purification of minor compounds.

2.4. HP20 Open Column, Preparative and Semi-Preparative HPLC

To generate five fractions for the Nrf2 and cytotoxicity assay, 20 mL Jing liqueur was dried to yield a sample (1.66 g) in a pilot study. The sample was dissolved in 10 mL water, and loaded onto an open column (HP20 6.6 g, 1.5 × 6.0 cm). HP20 is based on a unique rigid polystyrene/divinylbenzene matrix, in which a controlled pore size distribution and large surface area offer excellent resolution and the capacity for a wide range of molecules. The separation mechanism of a HP20 column is very similar to that of the C18 reverse chromatography—the most polar compounds are eluted out of the column with water first, while the most non-polar compounds will be eluted out of the column with methanol. Hence, a gradient solvent system from 100% water to 100% methanol (0, 20, 50, 80, and 100% MeOH/H2O) was used for the HP20 open column separation, and the eluents were dried using SpeedVac to yield five fractions (Fr. I: 1.5 g; Fr. II: 134 mg; Fr. III: 93.0 mg; Fr. IV: 8.0 mg; Fr. V: 1.3 mg). Separation of large amount of 160 L Jing Brand “Xiaoqu white liqueur” sample was scaled up accordingly. Fraction I was mainly composed of saccharides, which was not chemically investigated in this study. Fractions II, III, IV, and V each were first separated with a Thermo Scientific Ultimate 3000 preparative high performance liquid chromatography (HPLC) system (Column: Phenomenex Luna C18, 100 Å, 100 × 21.2 mm, 5 μm; Flow-rate: 10 mL/min) and then an Agilent 1100 semi-preparative HPLC system (Column: Phenomenex Luna C18 or C8, 100 Å, 250 × 10 mm, 5 μm; Flow-rate: 3 mL/min) to get pure compounds (Scheme 1).

2.5. LC/MS Condition for the Analysis

System: Agilent 1260 HPLC coupled to 6120 quadrupole LC/MS or Agilent 1260 HPLC coupled to an Agilent 6530 Accurate-Mass Q-TOF LC/MS in positive or negative modes. Column: Phenomenex C18, 100 Å, 100 × 4.6 mm, 5 μm; Flow-rate: 0.2 mL/min; Solvent A: water 0.1% formic acid, Solvent B: acetonitrile 0.1% formic acid, loading at 10% B, increasing the solvent gradient to 100% B in 20 min, and then re-equilibrating the HPLC column over 7 min in 10% B. The molecular weights of all the isolated compounds were obtained through LC/MS analysis.

2.6. NMR Experiments

NMR spectra including 1D (one dimension) and 2D (two dimensions) experiments were recorded in acetone-d6 or MeOH-d4 or CDCl3 or DMSO-d6 on a Bruker 400 MHz NMR, which plays a major role in the structural determination of the isolated compounds.

2.7. Analysis of Metals, Amino Acids, and Total Polysaccharides

Iron (GB 5009, 90-2016), zinc (GB 5009, 14-2017), calcium (GB 5009, 92-2016), and amino acids (GB 5009, 124-2016) were analyzed according to the methods as described in the National Food Safety Standards, People’s Republic of China. The concentration of total polysaccharides was measured according to the methods as published in the literatures [18].

2.8. Cell Culture and Condition

Nrf2 Antioxidant Pathway ARE Reporter—Hep G2 cell line was purchased from BPS Bioscience (San Diego, CA, USA). Cells were propagated at 37 °C in a humidified incubator with 5% CO2, in Eagle’s minimum essential medium (MEM, Corning, New York, NY, USA) with non-essential amino acids and supplemented with 10% fetal bovine serum (FBS, Invitrogen, Waltham, MA, USA), penicillin and streptomycin (Thermo Fisher, Waltham, MA, USA). Cells were trypsinized and split every 6 to 7 days.

2.9. Chemicals Exposure and Luciferase Assay to Measure the Nrf2 Activation

Sterile DMSO (dimethyl sulfoxide) stock solutions of crude, HP20 fractions and DL-sulforaphane (Sigma # S4441) were prepared in DMSO. The HepG2-Nrf2 stable cell line was seeded into 96-well plates at 4 × 104 per well in a final volume of 100 μL MEM. 24 h after seeding, media was replaced with fresh MEM and the cells were treated with the crude extract or fractions or pure compounds. Plates were incubated for 24 h, then 100 μL ONE-Step Luciferase reagent (BPS Bioscience) was added to each well and the assay was performed according to manufacturer’s instructions. Luminescence was detected using a luminometer (LUMIstar Galaxy BMG, Offenburg, Germany) and data are expressed as relative luminescence units (RLU) emitted from total assays. DL-sulforaphane was used as a positive control at a concentration of 5 μM. All experiments were performed in triplicate.

2.10. Cell Viability Assay

Cell viability was assessed by methylthiazoltetrazolium (MTT) assay (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer’s instruction. Briefly, cells (4 × 104) were seeded into a 96-well plate in 100 μL MEM and allowed to adhere overnight. Culture medium was replaced, and cells were treated with crude or fractionated samples (Fr. I-V) or pure compounds for 24 h treatments. The medium of each well was replaced by 200 μL fresh medium plus 50 μL of the MTT solution (5 mg/mL in PBS). The plates were incubated at 37 °C for 4 h. The absorbance being proportional to cell was subsequently measured at 570 nm in each well using a Bio-Rad 680 plate reader (Hercules, CA, USA). DL-sulforaphane was also used as a control at a concentration of 5 μM. All experiments were performed in triplicate.

2.11. Statistical Analysis

Values are expressed as the mean ± standard error of the mean p values < 0.05 were considered statistically significant. All analyses were performed with the Student t-test using GraphPad Prism 5.1 (GraphPad, La Jolla, CA, USA).

3. Results

3.1. Isolation and Structure Elucidation of Minor Compounds from Jing Liqueur

In order to analyze the minor compounds in Jing liqueur, we concentrated 160 L of Jing liqueur Brand “Xiaoqu white liqueur,” and separated the extract with HP20 (See Section 2.4 and Scheme 1) into five fractions (Fraction I: 100% H2O; Fraction II: 20% MeOH/H2O; Fraction III: 50% MeOH/H2O; Fraction IV: 80% MeOH/H2O; Fraction V: 100% MeOH). Each of the fractions (II–V) was further separated with C18 preparative HPLC (Fraction II: 5–25% MeOH/H2O in 42 min; Fraction III: 15–42% MeOH/H2O in 42 min; Fraction IV: 23–80% MeOH/H2O in 50 min; Fraction V: 50–10% MeOH/H2O in 56 min), and subfractions (one subfraction per min) were collected. Then the subfractions were purified with semi-preparative HPLC to get the pure compounds (See Table 1, Table 2 and Table 3 for retention times of the 189 pure compounds and semi-preparative HPLC conditions including columns, flow-rates, and solvent systems). In total, one hundred eighty nine (189) compounds, including flavonoids, terpenoids, alkaloids, coumarins, cinnamic acid or coumaric acid, and phenyl ethanol (or acetic acid) derivatives, benzoquinone, naphthoquinone, anthraquinones or phenanphrene derivatives, xanthones, chromone, and γ-pyrone derivatives, lignans, other aromatic compounds, and others were isolated and identified. The chemical structures were determined by using LC-MS and NMR as shown in Figure 1 and Figure 2. The HPLC conditions, molecular formulas, sources of the corresponding plants, and references of the characterized compounds are summarized in Table 1, Table 2 and Table 3. All the MS, NMR spectra, and references for the 189 compounds isolated from Jing liqueur are listed in the Supplementary Material. These 189 compounds were simply categorized into nine classes.
(1). Flavonoids: Seventy-eight flavonoids (#1–78, Table 1, Figure 1) have been isolated. They are either aglycones or glycosides of flavone, flavonol, flavanone, isoflavone, and isoflavanone derivatives. The new compound (7) was obtained as a light yellowish powder. Its molecular formula was determined as C26H28O11 based on NMR and high resolution ESI mass spectrometry (HRESIMS) data (m/z 517.1710 [M + H]+, calcd for C26H29O11, 517.1710). Its 1H NMR spectrum in CD3OD exhibited signals similar to those of caohuoside C (6) [19]. The only difference between 6 and 7 was the presence of the 1H NMR signal for a methoxy group at 4′-position in 6, but absence in 7. Hence, the new compound (7) was determined as 4′-O-demethyl caohuoside C (Table 1 and Table S1, Figure 1 and Figure S1). (2). Terpenoids: Twenty-two triterpenoids (#79–100) have been isolated from Jing liqueur (Table 2, Figure 1). These compounds are either simple oleanane or ursane derivatives, or glycosides of oleanolic acid. The sugars were connected to either 3-position, or 28-position, or both of the aglycones. Eighteen of these twenty-two triterpenoids are saponins, among which seventeen are oleanane glycosides and one is ursane glycoside [20]. (3). Alkaloids: Eighteen alkaloids including derivatives of spermidine, indole, tropane, pyrrole, piperidine, and alanyllysine (#101–118) have been isolated from Jing liqueur (Table 2, Figure 2). (4). Coumarins, cinnamic acid or coumaric acid, and phenyl ethanol (or acetic acid) derivatives: Twenty-one coumarins, cinnamic acid or coumaric acid, and phenyl ethanol (or acetic acid) derivatives (#119–139) were isolated from Jing liqueur (Table 2, Figure 2), including ten cinnamic acid analogs. (5). Benzoquinone, naphthoquinone, anthraquinones, or phenanphrene derivatives: Ten benzoquinone, naphthoquinone, anthraquinones, or phenanphrene derivatives (#140–149) were isolated from Jing liqueur (Table 3, Figure 2), eight of which were anthraquinones. (6). Xanthones, chromone, and γ-pyrone derivatives: Six xanthones, chromone, and γ-pyrone derivatives (#150–155) were isolated (Table 3, Figure 2). (7). Lignans: Seven lignans (#156–162) were isolated (Table 3, Figure 2). (8). Other aromatic compounds: Thirteen small aromatic compounds (#163–175) were isolated (Table 3, Figure 2). They were either benzoic acid or phenolic derivatives. (9). Other compounds: Fourteen other compounds (#176–189) were isolated, including three monoterpenoid glycosides, three cholesterol analogs, three iridoid glycosides, one nucleoside analog, three γ-lactone, and one alkyne derivatives (Table 3, Figure 2). In summary, one hundred eighty nine (189) compounds have been isolated, including ninety two (92) from fraction V, fifty seven (57) from fraction IV, thirty four (34) from fraction III, and six (6) from fraction II.

3.2. Analysis of Metals, Amino Acids, and Total Polysaccharides

Besides the isolation and structure determination of the above 189 compounds from Chinese herbal medicines, we determined the concentrations of two amino acids (L-proline, 2.33 mg/L; and L-arginine, 1.73 mg/L), total amino acids (9.84 mg/L), and three metals (iron, 0.52 mg/L; zinc, 0.21 mg/L; and calcium, 11.0 mg/L). The total amount of polysaccharides, the main component in fraction I was also determined (337.4 mg/L).

3.3. Nrf2 Activation

Results showed that a 5.2 mg/mL crude extract of Jing liqueur increased Nrf2 activity by approximately 7–8-fold (Figure 3). We also tested the five fractions from Jing liqueur. Nrf2 was strongly activated by fraction V at 80 μg/mL, weakly activated by fraction V and IV at 40 and 80 μg/mL, respectively (Figure 4), indicating that fraction V contains most of the Nrf2 activators in Jing liqueur. Fraction V at 20 μg/mL, fraction IV at 40 μg/mL, and both fractions III and II at 80 μg/mL marginally activated Nrf2, while fraction I was inactive. Next, we screened almost all the 189 isolated molecules except twenty one (21) because of their insufficiency. Eighteen (18) compounds showed Nrf2 activation when compared with the negative control (Figure 5). Among these eighteen Nrf2 activators (Figure 1, Figure 2 and Figure 5), four (55, 78, 129, and 168) from fraction IV, and the other fourteen (50, 51, 53, 58, 125, 126, 127, 128, 143, 144, 145, 146, 171, and 186) from fraction V (Table 4). Compounds 55, 78, 129, and 168 from fraction IV and 50, 53, 58, 128, 143, 144, 145, 146, 171, and 186 from fraction V were weakly active. When comparing the activity of the active compounds from fraction V, compounds 51, 125, 126, and 127 showed much stronger activity than the other Nrf2 activators, which robustly enhanced the Nrf2 expression at 40 μg/mL (Table 4).

3.4. Cytotoxicity Evaluation

In order to evaluate the cytotoxicity of Jing liqueur, we used the MTT assay to measure the activity of the crude and the five fractions. We tested the five fractions (I–V) at 20, 40, and 80 μg/mL, and none exhibited cytotoxicity as shown by the MTT results (Figure 6). The Nrf2 activators identified in this study were also evaluated for the activity against HepG2 in our MTT assay, and none of them showed any cytotoxicity at 40 μg/mL.

4. Discussion

One hundred eighty nine compounds have been isolated from Jing liqueur. Most of them are aromatic compounds including 78 flavonoids, 21 coumarins, cinnamic acid, or coumaric acid, and phenyl ethanol (or acetic acid) derivatives, 10 benzoquinone, naphthoquinone, anthraquinones, or phenanphrene derivatives, 6 xanthones, chromone, and γ-pyrone derivatives, 7 lignans, and 13 small aromatic compounds. The three major types of compounds are flavonoids, terpenoids, and alkaloids, and they have a broad range of biological activities including anti-oxidant, anti-inflammatory, antibacterial, and anticancer properties etc. These aromatic compounds, especially flavonoids, anthraquinones, cinnamic acid derivatives, lignans, and some other small molecule aromatic compounds, are probably the main anti-oxidant components in Jing liqueur.
L-proline and L-arginine are two of the six conditionally essential amino acids [21,22]. These amino acids and elements are important for heart muscle, immune function, blood production, blood pressure regulation, and prevention of osteoporosis etc. Iron is an essential element for blood production [23]. Zinc is extremely important for the body’s defense (immune) system to work properly, and plays a role in cell division, cell growth, wound healing, and the breakdown of carbohydrates [24]. Calcium helps to form and maintain healthy teeth and bones, which is important for the prevention of osteoporosis [25,26]. Olysaccharides are the most abundant type of compounds in Jing liqueur. We have a good reason to argue that olysaccharides together with flavonoids and terpenoids may account for some other biological activities besides the anti-oxidant property of Jing liqueur.
Fractions II–V were active in the Nrf2 assay at 80 μg/mL, fraction V was much more active than fractions II–IV, and fraction IV was more active than fractions II and III. Since most of the 189 compounds were isolated from fractions IV and V, majority of which are flavonoids, cinnamic acid derivatives, lignans, and other aromatic compounds, we argued that it is likely that Nrf2 activators in Jing liqueur are aromatic molecules. We evaluated the 168 compounds that were enough for the screening, and eighteen compounds activated Nrf2. Among these eighteen Nrf2 activators four were isolated from fraction IV, and the other fourteen were separated from fraction V. Clearly, most of the active compounds and the four most active Nrf2 activators (51 and 125127) identified in this study were isolated from fraction V, which was consistent with our screening result of the five fractions with fraction V being the most active fraction. Almost all these eighteen Nrf2 activators are aromatic molecules except 186, including six flavonoids (50, 51, 53, 55, 58, and 78), five cinnamic acid derivatives (125129), four anthraquinones (143146), 3,4-dihydroxybenzaldehyde (168), 1,3,5-trimethoxybenzene (171), and (E)-3-butylidene-4,5,6,7-tetrahydroisobenzofuran-1(3H)-one (186). Jing liqueur, the five fractions at 80 μg/mL and the eighteen Nrf2 activators at 40 μg/mL were evaluated for their antiproliferative activity against HepG2, and none showed any cytotoxicity.
This study is significant because it was the first time to extensively investigate Jing liqueur chemically that has not been previously interrogated although the phytochemical components and biological activities of these nine Chinese plants as a single herbal medicine have been investigated. The Nrf2 activators especially compounds 51, 125, 126, and 127 identified in this study could be used as biomarkers for quality control. Jing liqueur was reported to exhibit anti-inflammatory [1], immune enhancement [3], anti-fatigue [2,3] properties, and invigorating the vital activities of kidney [3]. Our study showed that the Nrf2 activation by Jing liqueur may account for the observed anti-inflammatory activity and immune enhancement of Jing liqueur. These experiments also demonstrated that to drink certain volume of Jing liqueur equivalent to the highest concentration tested in these experiments should be safe regarding the cytotoxicity of the metabolites of the herbal medicine in Jing liqueur. Hence, adequate consumption of Jing liqueur may offer health benefits mainly or partially because of the transient activation of Nrf2 considerably by the above-mentioned eighteen Nrf2 activators present in Jing liqueur. Of course, excessive drinking is not encouraged.

5. Conclusions

We isolated 189 compounds from fractions II–V of Jing liqueur, one of which (7) was a minor new flavonoid. Out of these 189 compounds, 78 are flavonoids, revealing the Jing liqueur is rich in phenolic compounds. The concentrations of most compounds were at micromolar levels (corresponding to μg/L levels). Fraction I was mainly composed of polysaccharides. The concentration of total polysaccharides was very high (337.4 mg/L), which may be worthy of further study for the components and functions. Both iron and zinc were less than 1 mg/L while the concentration of calcium was much higher (11 mg/L). L-proline and L-arginine were at mg/L levels. We also demonstrated that the crude extract of Jing liqueur, fractions II–V activated the Nrf2 transcription factor pathway, and fraction V was much more active than fractions II–IV, indicating that fraction V contains more Nrf2 activators than fractions II–IV. Screening of compounds demonstrated that most (14) of the eighteen active compounds including the two most potent Nrf2 activators (51 and 126) were isolated from fraction V. Nrf2 is an important defense mechanism for mitigating oxidative and electrophilic stress. Despite the “dark side” [27], Nrf2 activation is believed to have many beneficial effects on human health including inhibition of systemic inflammation, cancer prevention, relief of diabetes-induced cardiac oxidative stress, and neuroprotection. The activation of Nrf2 is highly consistent with the traditional use of the herbal medicines present in Jing liqueur, which itself is known for its tonic effects including general health and well-being promotion. Many of the reported beneficial properties of Jing liqueur including anti-inflammatory [1], immune enhancement [2], and anti-fatigue [2,3] properties could at least partially be justified by the presence of different types of compounds including Nrf2 activators. The crude extract and the five fractions were not cytotoxic against HepG2 cells at 80 μg/mL, and the compounds that activated Nrf2 were also not active in our MTT cytotoxicity assay at 40 μg/mL, the highest concentration of compounds tested in the Nrf2 assay. Further investigation of Jing liqueur on the Nrf2 pathway is warranted.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/2306-5710/6/1/1/s1. Figure S1: Structure of 7. Table S1: 1H and 13C NMR data of 7 (400 MHz, CD3OD). Figure S2: HR-ESI-MS spectrum of 7. Figure S3: 1H-NMR spectrum of 7 (400 MHz, CD3OD). Figure S4: COSY (Correlation Spectroscopy) spectrum of 7 (400 MHz, CD3OD). Figure S5: HSQC (Heteronuclear Single Quantum Coherence) spectrum of 7 (400 MHz, CD3OD). Figure S6: HMBC (Heteronuclear Multiple Bond Correlation) spectrum of 7 (400 MHz, CD3OD). MS, NMR, and references of the other known compounds.

Author Contributions

Y.-S.C., J.X., M.C., Y.L. and S.C. designed research; Y.-S.C., J.X., M.C., Y.Y., A.M., D.W. and X.W. performed research; Y.-S.C. and S.C. analyzed data; and Y.-S.C. and S.C. wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful to Jing Brand Research Institute, Jing Brand Co., Ltd. for financial support.

Acknowledgments

We thank Aaron Jacobs for his advice and help in the Nrf2 assay and manuscript preparation.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

Traditional Chinese medicine (TCM); Chinese Materia Medica (CMM); nuclear factor erythroid 2-related factor 2 (Nrf2); antioxidant responsive element (ARE); high performance liquid chromatography (HPLC); nuclear magnetic resonance (NMR); liquid chromatography–mass spectrometry (LC-MS); methylthiazoltetrazolium (MTT).

References

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Scheme 1. Flow chart of experimental design and numbers of pure compounds isolated from fractions II–V (See Table 1, Table 2 and Table 3 for retention times of the 189 pure compounds and high performance liquid chromatography (HPLC) conditions including columns, flow-rates, and solvent systems).
Scheme 1. Flow chart of experimental design and numbers of pure compounds isolated from fractions II–V (See Table 1, Table 2 and Table 3 for retention times of the 189 pure compounds and high performance liquid chromatography (HPLC) conditions including columns, flow-rates, and solvent systems).
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Figure 1. Structures of compounds 1100 isolated from Jing liqueur.
Figure 1. Structures of compounds 1100 isolated from Jing liqueur.
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Figure 2. Structures of compounds 101189 isolated from Jing liqueur.
Figure 2. Structures of compounds 101189 isolated from Jing liqueur.
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Figure 3. The effect of the crude on Nrf2 in ARE reporter-Hep G2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with 5.2 mg/mL concentration of crude Jing liqueur for additional 24 h. The negative control cells were treated with 0.2% DMSO (dimethyl sulfoxide), and positive control cells were treated with 5 μM DL-sulforaphane (SF). Luciferase activity was determined. * p < 0.05.
Figure 3. The effect of the crude on Nrf2 in ARE reporter-Hep G2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with 5.2 mg/mL concentration of crude Jing liqueur for additional 24 h. The negative control cells were treated with 0.2% DMSO (dimethyl sulfoxide), and positive control cells were treated with 5 μM DL-sulforaphane (SF). Luciferase activity was determined. * p < 0.05.
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Figure 4. The effect of five fractions on ARE-luciferase reporter activity in ARE reporter-HepG2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with 20, 40, 80 μg/mL concentrations of each fraction for additional 24 h. The negative control cells were treated with 0.2% DMSO, and positive control cells were treated with 5 μM DL-sulforaphane (SF). Luciferase activity was determined. Mean ARE-luciferase reporter activity represents the average of three-independent experiments ± S.E.M. * p < 0.05; ** p < 0.01, *** p < 0.001. (a: 20 μg/mL; b: 40 μg/mL; c: 80 μg/mL).
Figure 4. The effect of five fractions on ARE-luciferase reporter activity in ARE reporter-HepG2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with 20, 40, 80 μg/mL concentrations of each fraction for additional 24 h. The negative control cells were treated with 0.2% DMSO, and positive control cells were treated with 5 μM DL-sulforaphane (SF). Luciferase activity was determined. Mean ARE-luciferase reporter activity represents the average of three-independent experiments ± S.E.M. * p < 0.05; ** p < 0.01, *** p < 0.001. (a: 20 μg/mL; b: 40 μg/mL; c: 80 μg/mL).
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Figure 5. The effect of pure compounds on ARE-luciferase reporter activity in ARE repoter-HepG2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with compounds (40 μg/mL each) for additional 24 h. The negative control cells were treated with 0.2% DMSO, and positive control cells were treated with 5 μM DL-sulforaphane (SF). Luciferase activity were determined. Mean ARE-luciferase reporter activity represents the average of three-independent experiments ± S.E.M. ** p < 0.01, *** p < 0.001.
Figure 5. The effect of pure compounds on ARE-luciferase reporter activity in ARE repoter-HepG2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with compounds (40 μg/mL each) for additional 24 h. The negative control cells were treated with 0.2% DMSO, and positive control cells were treated with 5 μM DL-sulforaphane (SF). Luciferase activity were determined. Mean ARE-luciferase reporter activity represents the average of three-independent experiments ± S.E.M. ** p < 0.01, *** p < 0.001.
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Figure 6. Evaluation of cytotoxicity activity of five fractions in ARE reporter-HepG2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with 20, 40, 80 μg/mL concentrations of each fraction for additional 24 h. The negative control cells were treated with 0.2% DMSO cells. Cell viability was estimated with the methylthiazoltetrazolium (MTT) assay. Mean cytotoxicity activity represents the average of three-independent experiments ± S.E.M. (a: 20 μg/mL; b: 40 μg/mL; c: 80 μg/mL).
Figure 6. Evaluation of cytotoxicity activity of five fractions in ARE reporter-HepG2 cells. Cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated for 24 h. The cells were further treated with 20, 40, 80 μg/mL concentrations of each fraction for additional 24 h. The negative control cells were treated with 0.2% DMSO cells. Cell viability was estimated with the methylthiazoltetrazolium (MTT) assay. Mean cytotoxicity activity represents the average of three-independent experiments ± S.E.M. (a: 20 μg/mL; b: 40 μg/mL; c: 80 μg/mL).
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Table 1. Flavanoids (#1–78) isolated from Jing liqueur.
Table 1. Flavanoids (#1–78) isolated from Jing liqueur.
NOFr.tR, HPLC ConditionMFCompound NameReference (See SM)
1VC.1, ACN, 33%C20H18O6NoranhydroicaritinKomatsu et al. 1970
2VSepherdex LH20C25H26O6Broussoflavonol FFang et al. 1995
3IV71 min, C.1, ACN, 18% 110 minC26H28O11Epimedoside CLi et al. 1990
4IV68 min, C.2, ACN, 30–35%, 80 minC27H30O11Icariside IMizuno et al. 1987
5V56 min, C.1, ACN, 24%C33H40O15IearilineLiang et al. 1988
6VC.1, ACN, 33%C27H30O11Caohuoside CLi et al. 1995
7*VC.1, ACN, 30%C26H28O114′-O-demethyl caohuoside C (New)New
8VC.1, ACN, 35%C26H28O11PhelodendrozideWang et al. 2010
9VC.1, ACN, 33%C26H28O10baohuoside IIDong et al. 1994
10IV53.5 min, C.2, ACN, 17% 100 minC32H38O16Hexandraside ELeu et al. 2006
11VC.2, ACN, 40–50%, 48 minC26H28O10
12V49 min, C.2, ACN, 34%C32H38O14Baohuoside IVLi and Liu 1990
13VC.1, ACN, 35%C32H38O15Icarisoside BFukai et al. 1988
14VC.1, ACN, 35%C32H38O15 Zhao et al. 2010
15VC.1, MeOH, 64%C34H40O15 Tu et al. 2011
16VC.1, ACN, 33%C31H36O14Ikarisoside FFukai et al. 1988
17IV75 min, C.2, ACN, 30–35%, 80 minC33H40O14 Ueda et al. 1992
18V35.5 min, C.2 ACN, 36%C32H38O14 Zhao et al. 2010
19V39 min, C.2, ACN, 34%C33H40O15Baohuoside VIILi et al. 1988
20VSepherdex LH20C25H26O55,7, 4′-trihydroxy-8, 3′-diprenylflavoneGuo et al. 2006
21VC.1, MeOH, 65%C27H30O10Icariside IIZhang et al. 2006
22VC.1, MeOH, 65%C32H38O14Sagittatoside BMizuno et al. 1988
23VC.1, ACN, 24%C39H50O19Epimedin CMizuno et al. 1988
24VC.1, ACN, 24%C33H40O15Sagittatoside AMizuno et al. 1988
25VC.1, MeOH, 65%C33H40O142″-O-rhamnosyl icariside IIZhao et al. 2016
26VC.1, MeOH, 64%C33H38O143″′-carbonyl-2″-β-l-quinovosyl icariside IIZhang et al. 2006
27VC.1, ACN, 33%C16H12O4 Asahina et al. 1935
28IV85.5 min, C.1, ACN, 18% 110 minC39H50O20 Das and Tripathi 2002
29VSepherdex LH20C15H10O5VersulinGeissman et al. 1946
30VSepherdex LH20C15H10O7XanthaurineBao et al. 2004
31VC.1, ACN, 35%C27H30O11Koreanoside ELi et al. 2015
32VC.1, MeOH, 64%C34H42O15 Hu et al. 2010
33VC.1, MeOH, 64%C34H42O14
34VC.1, ACN, 24%C39H50O20Epimedin AHan, Lee 2017
35VC.1, ACN, 24%C39H50O20Maohuoside BLi et al. 2006
36VC.1, ACN, 24%C38H48O19Epimedin BGuo and Xiao 2003
37VC.1, ACN, 24%C39H50O19Hexandraside DMizuno et al. 1991
38VC.1, ACN, 28%C39H48O19 Zhao et al. 2008
39VC.1, ACN, 28%C39H50O19 Ueda et al. 1992
40V29 min, C.1, ACN, 22–33%, 60 minC39H50O20Hexandraside FWang et al. 2007
41V33 min, C.1, ACN, 22–33%, 60 minC38H48O19 Zhao et al. 2010
42IV85.5 min, C.2, ACN, 18% 110 minC16H12O6DiosmetinTakeda et al. 2007
3V77.5 min, C.2, ACN, 16–20%, 100 minC38H48O20RouhuosideLi et al. 1990
44V47 min, C.2, ACN, 22–33%, 60 minC38H48O20Diphylloside A/Ikarisoside CJia et al. 1998
45IV39 min, C.2, MeOH, 50–60% 80 minC39H48O21 Jin et al. 2013
46VC.1, ACN, 28%C39H48O20 Jin et al. 2013
47III41.5 min, C.1, MeOH, 6–8.5%, 65 minC34H44O20 Li et al. 2012
48V38.5 min, C.2, ACN, 22–24%, 75 minC15H10O4Isoaurostatin/4′,7-DihydroxyisoflavoneXu et al. 1979
49VSepherdex LH20C16H12O4FormononetinReiners 1966
50V47 min, C.2, ACN, 22–24%, 75 minC16H12O5Biochanin A, OlmelinNilsson 1961
51V47 min, C.2, ACN, 22–24%, 75 minC16H12O5Calycosin, CyclosinMarkham et al. 1968
52VC.2, ACN, 20–22%, 75 min 55.5 minC16H12O57,4′-Dihydroxy-3′-methoxyisoflavoneHirakura et al. 1997
53VC.2, ACN, 20–22%, 75 min 55.5 minC23H24O108-O-Methylretusin-7-O-β-d-glucopyranosideRukachaisirikul 2002
54V69 min, C.1, MeOH, 35%, 80 minC23H24O10 Clarke et al. 2004
55IV17 min, C.2, ACN, 15–18%, 80 minC21H20O9DaidzosideXiao et al. 2016
56IV17min, C.2, ACN, 15–18%, 80 minC22H22O103′-MethoxydaidzinHirakura et al. 1997
57IV38 min, C.2, ACN, 20%, 90 minC21H20O10GenistosideYuan et al. 2008
58V66.5 min, C.2, ACN, 22–24%, 75 minC22H22O10Calycosin 7-glucosideMarkham et al. 1968
59IV59 min, C.1, ACN, 18% 110 minC22H22O9OnonosideLebreton et al. 1967
60IV14.5 min, C.2, MeOH, 20%C27H30O14Daidzein 7,4′-diglucosideLi et al. 2014
61IV33.5 min, C.2, MeOH, 28%, 100 minC26H28O14NeobacinBreytenbach 1986
62IV19 min, C.2, ACN, 15–18%, 80 minC22H22O10Caragiside BNisar et al. 2011
63VC.1, MeOH, 35%, 75 minC22H22O9IsoononinLiu et al. 2005
64IV24 min, C.2, MeOH, 20%C26H28O14AmbocinBreytenbach 1986
65IVC.2, ACN, 15–18%, 80 minC21H20O9NeopuerarinZhang et al. 2009
66IV42 min, C.2, ACN, 20%, 90 minC21H20O9 Ma et al. 2017
67IV18 min, C.2, MeOH, 20%C21H20O108-C-Glucosyl-7,3′,4′-trihydroxy isoflavoneWong et al. 2017
68IV63 min, C.2, MeOH, 20%, 56 min, 20–30% 40 minC26H28O13 Chen et al. 2009
69IVC.2, ACN, 15–18%, 80 minC21H20O9Neopuerarin AZhang et al. 2009
70III39 min, C.2, MeOH, 6–8.5%, 65 minC28H32O15 Wang et al. 2006
71III26 min, C.2, MeOH, 28%, 50 minC21H20O10 Pistelli et al. 1998
72IV54 min, C.2, ACN, 20%, 90 minC22H22O10 Ohshima et al. 1988
73IV58.5 min, C.2, ACN, 20%, 90 minC26H28O13Puerarin apiosideIngham et al. 1986
74IV67.5 min, C.2, ACN, 20%, 90 minC26H28O13 Kinjo et al. 1987
75III45 min, C.2, MeOH, 6–8.5%, 65 minC26H28O14 Peng et al. 2011
76IV73 min, C.2, MeOH, 28% 100 minC23H22O10AcetyldaidzinOhta et al. 1979
77IV24 min, C.2, ACN, 15–18%, 80 minC23H22O11 Zhou et al. 2013
78IV74 min, C.1, ACN, 18% 110 minC23H28O11AstraganosideLiu et al. 2007
C.1: C18, 5 μm, 250 × 10 mm, flow (3 mL/min); C.2: C8, 5 μ, 250 × 10 mm, flow (3 mL/min), see Section 2.4; *: new compound, MS and NMR data (Table S1 and Figures S2–S7).
Table 2. Terpenoids (#79–100), alkaloids (#101–118), coumarins, cinnamic acid or coumaric acid, and phenyl ethanol (or acetic acid) derivatives (#119–139) isolated from Jing liqueur.
Table 2. Terpenoids (#79–100), alkaloids (#101–118), coumarins, cinnamic acid or coumaric acid, and phenyl ethanol (or acetic acid) derivatives (#119–139) isolated from Jing liqueur.
NOFr.tR, HPLC ConditionMFCompoundReference (See SM)
79V58 min, C.2, ACN, 57%, 30 min, 57–62% 20 min, 62–70% 29 minC30H48O3Oleanolic acidTan et al. 2002
80V26 min, C.2, ACN, 57%, 30 min, 57–62% 20 min, 62–70% 29 minC30H48O4Sumaresinolic acidChan et al. 1992
81VC.1, MeOH, 72%C36H58O83-O-β-Glc-oleanolic acidDubois et al. 1990
82IV34 min, C.2, MeOH, 60–70%, 60 minC48H76O19Calendulaglycoside BVidal-Ollivier et al. 1989
83VC.1, MeOH, 72%C36H56O9Calenduloside EZhang et al. 2013
84VC.2, ACN, 30–35%, 60 min, 35–40% 20 minC48H74O18Papyrioside LG
85V27 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 minC42H66O14Chikusetsusaponin IvaYang et al. 1995
86VC.2, ACN, 30–35%, 60 min, 35–40% 20 minC42H64O14 Kuroda et al. 2006
87IV36.5 min, C.2, MeOH, 65–68%, 40 minC54H86O23Scheffleraside IIMshvildadze et al. 2001
88VC.2, ACN, 30–35%, 60 min, 35–40% 20 minC49H78O19Chikusetsusaponin V methyl esterKondo et al. 1971
89VC.2, ACN, 30–35%, 60 min, 35–40% 20 minC48H76O19Ginsenoside RoMatsuda et al. 1990
90VC.2, ACN, 30–35%, 60 min, 35–40% 20 minC54H86O24Calendulaglycoside AVidal-Ollivier et al. 1989
91VC.2, ACN, 30–35%, 60 min, 35–40% 21 minC53H84O23Elatoside CYoshikawa et al. 1993
92V24 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 minC47H74O18Pseudoginsenoside RT1Morita et al.
93V55 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 minC43H68O14Silphioside AJiang et al. 1992
94V46 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 minC48H76O18Umbellatoside BSosa et al. 2011
95V20 min, C.2, ACN, 36%, 40 minC42H66O14WedelinMatos et al. 1983
96V27.8 min, C.2, ACN, 57%, 30 min, 57–62% min, 62–70% 29 minC30H48O46β-Hydroxyursolic acidSakakibara et al. 1983
97VC.2, ACN, 35%C30H48O63β,6β,19α,24-tetrahydro xyurs-12-en-28-oic acidFang et al. 1996
98IV71 min, C.2, ACN, 18% 110 minC36H58O11 Abe et al. 1987
99V38 min, C.2, ACN, 20–22%, 75 minC36H58O11 Abe et al. 1987
100V20min, C.2, ACN, 36%, 40 minC42H66O14WedelinMatos et al. 1983
101IV8 min, C.2, MeOH, 10–14%, 40 minC37H55N3O16Lycibarbarspermidine LZhou et al. 2016
102IVC.2, MeOH, 10–14%, 40 minC37H55N3O16Lycibarbarspermidine MZhou et al. 2016
103IVC.2, MeOH, 10–14%, 40 minC37H53N3O16Lycibarbarspermidine EZhou et al. 2016
104IVC.2, MeOH, 10–14%, 40 minC37H53N3O16 Jin et al. 2015
105IVC.2, MeOH, 10–14%, 40 minC31H43N3O11Lycibarbarspermidine DZhou et al. 2016
106IVC.2, MeOH, 10–14%, 40 minC31H43N3O11Lycibarbarspermidine AZhou et al. 2016
107IV26.5min, C.2, ACN, 20%, 90 minC13H14N2O2Tetrahydroharman-3-carboxylic acidTsuchiya et al. 1999
108III23 min, C.2, MeOH, 15%, 95 minC13H14N2O3 Herraiz et al. 2004
109III39 min, C.2, MeOH, 17%, 80 minC13H14N2O2 Herraiz et al. 1993
110III36.5 min, C.2, MeOH, 15%, 95 minC14H17NO33α-Benzoyloxynortropan-6β-olAl-Said et al. 1986
111IV12 min, C.2, ACN, 20%, 90 minC17H21NO5ConfolineAripova et al. 1996
112II25 min, C.2, MeOH, 5%, 40 minC6H7NO2 Hiermann et al. 2002
113III47.5 min, C.2, MeOH, 17–20%, 100 minC10H13NO4 Chin et al. 2003
114III7.5 min, C.2, MeOH, 5–15%, 60 minC6H5NO2Nicotinic acidKrehl et al. 1946
115IV18 min, C.2, MeOH, 20%C8H15NO2 Singer et al. 1935
116IV8 min, C.2, MeOH, 10–14%, 40 minC14H22NO4+Codonopyrrolidium BHe et al. 2014
117III17 min, C.2, MeOH, 10%, 80 minC8H17N3O3 Gegauer et al. 2003
118III19 min, C.2, MeOH, 10%, 80 minC6H13NO2 Perrin et al. 2000
119IV20 min, C.2, ACN, 15–18%, 80 minC10H8O4ScopoletinBest 1948
120IV23 min, C.2, ACN, 15–18%, 80 minC20H24O8VelleinMaruyama et al. 2009
121IV33.5min, C.2, MeOH, 28%, 100 minC8H10O2Phenethyl alcoholWang et al. 1982
122IV35.5 min, C.2, ACN, 20%, 90 minC8H8O4Pisolithin BBenecke et al. 1984
123V43.5 min, C.2, ACN, 22–33%, 60 minC9H8O2trans-β-CarboxystyreneBillmann et al. 1909
124IV21 min, C.2 ACN, 15–18%, 80 minC10H10O4Ferulic acidHenderson and Farmer 1955
125V60 min, C.2 ACN, 22–24%, 75 minC11H12O4 Oonuma et al. 1993
126VC.1, ACN, 28%C11H12O3 Newman et al. 1952
127VC.1, ACN, 28%C12H14O4Ethyl ferulateNakayama et al. 1996
128III71 min, C.1, ACN, 12–25%, 43 min, 25–30% 12 minC9H8O4Caffeic acidBaerheim 1951
129IV55 min, C.1, MeOH, 45–48%, 80 minC11H12O4Ethyl caffeoateMao et al. 2011
130III37min, C.2, MeOH, 28%, 50 minC9H8O3trans-p-Hydroxycinnamic acidKing et al. 1952
131III40 min, C.2, MeOH, 28%, 50 minC10H10O4Isoferulic acidQiao and Chen 1991
132III48.5 min, C.2, MeOH, 15%, 95 minC15H18O8p-Coumaric acid β-glucosideRuneckles, Woolrich 1963
133III48.5 min, C.2, MeOH, 15%, 95 minC16H20O9Glucosidoferulic acidIbrahim et al. 1970
134IV22 min, C.2, MeOH, 20%C10H10O4 Muratake et al. 2013
135III30 min, C.2, MeOH, 6–8.5%, 65 minC14H20O7 Fujimatu et al. 2003
136IV73.5 min, C.2, MeOH, 20%, 56 min, 58.5 min, 20–30%, 40 minC35H46O20EchinacosideFrezza et al. 2017
137III29.5 min, C.2, MeOH, 30–40%, 90 minC37H48O21Tubuloside AKobayashi et al. 1987
138IV80 min, C.2, MeOH, 28% 100 minC29H36O15VerbascosidePham et al. 1988
139V60.5 min, C.2, ACN, 16–20%, 100 minC20H26O12 Wende and Fry 1997
C.1: C18, 5 μm, 250 × 10 mm, flow (3 mL/min); C.2: C8, 5 μ, 250 × 10 mm, flow (3 mL/min), see Section 2.4.
Table 3. Benzoquinone, naphthoquinone, anthraquinones, or phenanphrene derivatives (#140–149), xanthones, chromone, and γ-pyrone derivatives (#150–155), lignans (#156–162), other aromatic compounds (#163–175), and other types of compounds (#176–189) isolated from Jing liqueur.
Table 3. Benzoquinone, naphthoquinone, anthraquinones, or phenanphrene derivatives (#140–149), xanthones, chromone, and γ-pyrone derivatives (#150–155), lignans (#156–162), other aromatic compounds (#163–175), and other types of compounds (#176–189) isolated from Jing liqueur.
NOFr.tR, HPLC ConditionMFCompoundReference (See SM)
140VC.1, ACN, 60%C16H14O4 Letcher and Nhamo 1973
141VC.2, ACN, 40–50%, 48 minC17H16O4Batatasin IGonnet et al. 1973
142VC.1, ACN, 30%C15H10O4 Morton et al. 1941
143VC.1, ACN, 30%C16H12O5 Wang et al. 2011
144VC.1, ACN, 33%C16H12O5 Wu et al. 2003
145VC.1, ACN, 30%C15H10O5 Lee et al. 1994
146VC.1, ACN, 30%C16H12O4DigitoluteinKoumaglo et al. 1992
147V34 min, C.2, ACN, 40%C15H10O3 Bistrzycki and Zen-Ruffinen 1920
148V34 min, C.2, ACN, 40%C15H10O5 Yang et al. 1992
149IV19 min, C.2, MeOH, 20%, 56 min, 20–30% 40 minC14H8O4 Varbanov et al. 1986
150V29 min, C.2, ACN, 22–27%, 60 minC13H8O41,3-DihydroxyxanthoneLiang et al. 1982
151III39 min, C.2, MeOH 6–8.5%, 65 minC12H16O8 Baba et al. 1995
152IV15 min, C.2, MeOH, 20%C16H18O9Biflorin Zhang et al. 1997
153III46 min, C.2, MeOH, 15%, 95 minC16H18O9IsobiflorinTanaka et al. 1993
154III37.5 min, C.2, MeOH, 6–8.5%, 65 minC16H18O9Undulatoside AItoh et al. 2003
155III37.5 min, C.2, MeOH, 6–8.5%, 65 minC16H18O9 Wang et al. 2011
156III55 min, C.2, MeOH, 17%, 80 minC26H34O12 Yan et al. 2008
157V89 min, C.2, ACN, 16–20%, 100 minC22H26O8SyringaresinolAbu Zarga 1986
158V54 min, C.2, ACN, 22–33%, 60 minC18H16O5 Shi et al. 2007
159IV25.5 min, C.2, ACN, 15–20%, 50 minC18H14O8Prolithospermic acidDai et al. 2010
160III29 min, C.2, MeOH, 28%, 50 minC18H14O87-Epiblechnic acidWang et al. 2010
161III29 min, C.2, MeOH, 28%, 50 minC18H14O8Blechnic acidWada et al. 1992
162V69 min, C.2, MeOH, 35%, 80 minC23H26O10 Guo et al. 2014
163V36 min, C.2, ACN, 22–24%, 75 minC7H6O3p-Salicylic acidSager and Schooley 1945
164V63.5 min, C.2, ACN, 22–24%, 75 minC9H10O3 Heim et al. 1957
165V41 min, C.2, ACN, 20–22%, 75 minC8H8O3p-Methoxy benzoic acidReitberg and Schentag 1983
166III67 min, C.2, MeOH, 12–25%, 43 min, 25–30% 12 minC8H8O4 Parham et al. 1954
167III49.5 min, C.2, ACN, 13–15%, 90 minC8H8O4Vanillic acidSammons and Williams 1946
168IV20min, C.2, MeOH, 20%C7H6O3Rancinamycin IVLi et al. 2004
169IV44 min, C.2, MeOH, 20%, 56 min, 20–30% 40 minC7H6O2 Rivers 1947
170II39.5 min, C.2, MeOH, 7–9%, 70 minC14H18O9 Yang et al. 2013
171V57 min, C.2, ACN, 20–22%, 75 minC9H12O31,3,5-TrimethoxybenzeneAllain et al. 1980
172IV24min, C.2, MeOH, 20%C8H10O2p-HydroxyphenetoleRosenwald 1951
173II22 min, C.2, MeOH, 2%, 60 minC13H18O8TachiosideSano et al. 1991
174IV73 min, C.2, MeOH, 28% 100 minC9H10O4
175III77 min, C.2, MeOH, 12–25%, 43 min, 25–30% 12 minC7H6O3Salicylic acidIchniowski and Hueper 1946
176III78 min, C.2, MeOH, 25–30%, 60 min, 30–35% 40 minC16H26O8BodinierinXie et al. 2006
177III80 min, C.2, MeOH, 25–30%, 60 min, 30–35% 40 minC16H26O8Kankanoside OLiu et al. 2016
178III75 min, C.2, MeOH, 20–25%, 80 minC16H28O7 Fan et al. 2011
179V21 min, C.2, ACN, 16–20%, 100 minC27H44O8(25R)-20,26-DihydroxyecdysoneSuksamrarn 1998
180III43min, C.2, MeOH, 30–40%, 90 minC27H44O726-HydroxyecdysoneLi et al. 2006
181IVC.2, ACN, 15–18%, 80 minC27H44O73-epi-20-HydroxyecdysoneThompson et al. 1974
182III47.5 min, C.2, MeOH, 6–8.5%, 65 minC16H22O10 Li et al. 1999
183II51 min, C.2, MeOH, 2%, 60 minC16H22O11Desacetylasperulosidic acidInouye et al. 1974
184II47.5 min, C.2, MeOH, 7–9%, 70 minC16H24O10Mussaenosidic acidKohda et al. 1989
185III9.5 min, C.2, MeOH, 5–15%, 60 minC11H13N3O59-DeazainosineLiu et al. 2005
186V38 min, C.2, MeOH, 35%, 80 minC12H16O2Sedanonic acid lactoneMitsuhashi and Nomura 1966
187V46 min, C.2, ACN, 20–22%, 75 minC12H16O4Senkyunolide IHuang et al. 2013
188IV65.5 min, C.2, ACN, 17% 100 minC12H16O4Senkyunolide HHuang et al. 2013
189VC.2, ACN, 60%C17H24O2FalcarindiolLechner et al. 2004
C.1: C18, 5 μm, 250 × 10 mm, flow (3 mL/min); C.2: C8, 5 μ, 250 × 10 mm, flow (3 mL/min), see Section 2.4.
Table 4. Relative Nrf2 activity induced by compounds (40 μg/mL) isolated from Jing liqueur.
Table 4. Relative Nrf2 activity induced by compounds (40 μg/mL) isolated from Jing liqueur.
CpdFr.Relative Nrf2 Activity (Fold Induction)CpdFr.Relative Nrf2 Activity (Fold Induction)CpdFr.Relative Nrf2 Activity (Fold Induction)
Control-1.0125V9.5145V3.0
50V4.0126V16.0146V3.0
51V11.5127V10.5168IV3.0
53V4.0128V3.0171V3.5
55IV3.0129IV2.2186V3.5
58V6.5143V6.0SF (5 μM)-15.0
78IV4.0144V3.5

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Cai, Y.-S.; Xu, J.; Chen, M.; Wang, D.; Yang, Y.; Manavalan, A.; Wu, X.; Liu, Y.; Cao, S. Compound Analysis of Jing Liqueur and nrf2 Activation by Jing Liqueur—One of the Most Popular Beverages in China. Beverages 2020, 6, 1. https://0-doi-org.brum.beds.ac.uk/10.3390/beverages6010001

AMA Style

Cai Y-S, Xu J, Chen M, Wang D, Yang Y, Manavalan A, Wu X, Liu Y, Cao S. Compound Analysis of Jing Liqueur and nrf2 Activation by Jing Liqueur—One of the Most Popular Beverages in China. Beverages. 2020; 6(1):1. https://0-doi-org.brum.beds.ac.uk/10.3390/beverages6010001

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Cai, You-Sheng, Jian Xu, Mosi Chen, Daoqing Wang, Yuejun Yang, Arulmani Manavalan, Xiaohua Wu, Yuancai Liu, and Shugeng Cao. 2020. "Compound Analysis of Jing Liqueur and nrf2 Activation by Jing Liqueur—One of the Most Popular Beverages in China" Beverages 6, no. 1: 1. https://0-doi-org.brum.beds.ac.uk/10.3390/beverages6010001

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