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

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.

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.

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.

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.

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). Scheme 1. Flow chart of experimental design and numbers of pure compounds isolated from fractions II-V (See Tables 1-3 for retention times of the 189 pure compounds and high performance liquid chromatography (HPLC) conditions including columns, flow-rates, and solvent systems).

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.

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.

Analysis of Metals, Amino Acids, and Total Polysaccharides
Iron (GB 5009, 90-2016), zinc (GB 5009, , 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 [ Tables 1-3  Tables 1-3  Tables 1-3  Tables 1-3 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.

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 × 10 4 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.

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 × 10 4 ) 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.

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).

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).

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.

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,

Cell viability (% of control)
Treatments 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 125-127) 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 (125-129), four anthraquinones (143-146), 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.

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.