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

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.

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. Flavanoids (#1–78) isolated from Jing liqueur.

NO	Fr.	tR, HPLC Condition	MF	Compound Name	Reference (See SM)
1	V	C.1, ACN, 33%	C20H18O6	Noranhydroicaritin	Komatsu et al. 1970
2	V	Sepherdex LH20	C25H26O6	Broussoflavonol F	Fang et al. 1995
3	IV	71 min, C.1, ACN, 18% 110 min	C26H28O11	Epimedoside C	Li et al. 1990
4	IV	68 min, C.2, ACN, 30–35%, 80 min	C27H30O11	Icariside I	Mizuno et al. 1987
5	V	56 min, C.1, ACN, 24%	C33H40O15	Ieariline	Liang et al. 1988
6	V	C.1, ACN, 33%	C27H30O11	Caohuoside C	Li et al. 1995
7*	V	C.1, ACN, 30%	C26H28O11	4′-O-demethyl caohuoside C (New)	New
8	V	C.1, ACN, 35%	C26H28O11	Phelodendrozide	Wang et al. 2010
9	V	C.1, ACN, 33%	C26H28O10	baohuoside II	Dong et al. 1994
10	IV	53.5 min, C.2, ACN, 17% 100 min	C32H38O16	Hexandraside E	Leu et al. 2006
11	V	C.2, ACN, 40–50%, 48 min	C26H28O10
12	V	49 min, C.2, ACN, 34%	C32H38O14	Baohuoside IV	Li and Liu 1990
13	V	C.1, ACN, 35%	C32H38O15	Icarisoside B	Fukai et al. 1988
14	V	C.1, ACN, 35%	C32H38O15		Zhao et al. 2010
15	V	C.1, MeOH, 64%	C34H40O15		Tu et al. 2011
16	V	C.1, ACN, 33%	C31H36O14	Ikarisoside F	Fukai et al. 1988
17	IV	75 min, C.2, ACN, 30–35%, 80 min	C33H40O14		Ueda et al. 1992
18	V	35.5 min, C.2 ACN, 36%	C32H38O14		Zhao et al. 2010
19	V	39 min, C.2, ACN, 34%	C33H40O15	Baohuoside VII	Li et al. 1988
20	V	Sepherdex LH20	C25H26O5	5,7, 4′-trihydroxy-8, 3′-diprenylflavone	Guo et al. 2006
21	V	C.1, MeOH, 65%	C27H30O10	Icariside II	Zhang et al. 2006
22	V	C.1, MeOH, 65%	C32H38O14	Sagittatoside B	Mizuno et al. 1988
23	V	C.1, ACN, 24%	C39H50O19	Epimedin C	Mizuno et al. 1988
24	V	C.1, ACN, 24%	C33H40O15	Sagittatoside A	Mizuno et al. 1988
25	V	C.1, MeOH, 65%	C33H40O14	2″-O-rhamnosyl icariside II	Zhao et al. 2016
26	V	C.1, MeOH, 64%	C33H38O14	3″′-carbonyl-2″-β-l-quinovosyl icariside II	Zhang et al. 2006
27	V	C.1, ACN, 33%	C16H12O4		Asahina et al. 1935
28	IV	85.5 min, C.1, ACN, 18% 110 min	C39H50O20		Das and Tripathi 2002
29	V	Sepherdex LH20	C15H10O5	Versulin	Geissman et al. 1946
30	V	Sepherdex LH20	C15H10O7	Xanthaurine	Bao et al. 2004
31	V	C.1, ACN, 35%	C27H30O11	Koreanoside E	Li et al. 2015
32	V	C.1, MeOH, 64%	C34H42O15		Hu et al. 2010
33	V	C.1, MeOH, 64%	C34H42O14
34	V	C.1, ACN, 24%	C39H50O20	Epimedin A	Han, Lee 2017
35	V	C.1, ACN, 24%	C39H50O20	Maohuoside B	Li et al. 2006
36	V	C.1, ACN, 24%	C38H48O19	Epimedin B	Guo and Xiao 2003
37	V	C.1, ACN, 24%	C39H50O19	Hexandraside D	Mizuno et al. 1991
38	V	C.1, ACN, 28%	C39H48O19		Zhao et al. 2008
39	V	C.1, ACN, 28%	C39H50O19		Ueda et al. 1992
40	V	29 min, C.1, ACN, 22–33%, 60 min	C39H50O20	Hexandraside F	Wang et al. 2007
41	V	33 min, C.1, ACN, 22–33%, 60 min	C38H48O19		Zhao et al. 2010
42	IV	85.5 min, C.2, ACN, 18% 110 min	C16H12O6	Diosmetin	Takeda et al. 2007
3	V	77.5 min, C.2, ACN, 16–20%, 100 min	C38H48O20	Rouhuoside	Li et al. 1990
44	V	47 min, C.2, ACN, 22–33%, 60 min	C38H48O20	Diphylloside A/Ikarisoside C	Jia et al. 1998
45	IV	39 min, C.2, MeOH, 50–60% 80 min	C39H48O21		Jin et al. 2013
46	V	C.1, ACN, 28%	C39H48O20		Jin et al. 2013
47	III	41.5 min, C.1, MeOH, 6–8.5%, 65 min	C34H44O20		Li et al. 2012
48	V	38.5 min, C.2, ACN, 22–24%, 75 min	C15H10O4	Isoaurostatin/4′,7-Dihydroxyisoflavone	Xu et al. 1979
49	V	Sepherdex LH20	C16H12O4	Formononetin	Reiners 1966
50	V	47 min, C.2, ACN, 22–24%, 75 min	C16H12O5	Biochanin A, Olmelin	Nilsson 1961
51	V	47 min, C.2, ACN, 22–24%, 75 min	C16H12O5	Calycosin, Cyclosin	Markham et al. 1968
52	V	C.2, ACN, 20–22%, 75 min 55.5 min	C16H12O5	7,4′-Dihydroxy-3′-methoxyisoflavone	Hirakura et al. 1997
53	V	C.2, ACN, 20–22%, 75 min 55.5 min	C23H24O10	8-O-Methylretusin-7-O-β-d-glucopyranoside	Rukachaisirikul 2002
54	V	69 min, C.1, MeOH, 35%, 80 min	C23H24O10		Clarke et al. 2004
55	IV	17 min, C.2, ACN, 15–18%, 80 min	C21H20O9	Daidzoside	Xiao et al. 2016
56	IV	17min, C.2, ACN, 15–18%, 80 min	C22H22O10	3′-Methoxydaidzin	Hirakura et al. 1997
57	IV	38 min, C.2, ACN, 20%, 90 min	C21H20O10	Genistoside	Yuan et al. 2008
58	V	66.5 min, C.2, ACN, 22–24%, 75 min	C22H22O10	Calycosin 7-glucoside	Markham et al. 1968
59	IV	59 min, C.1, ACN, 18% 110 min	C22H22O9	Ononoside	Lebreton et al. 1967
60	IV	14.5 min, C.2, MeOH, 20%	C27H30O14	Daidzein 7,4′-diglucoside	Li et al. 2014
61	IV	33.5 min, C.2, MeOH, 28%, 100 min	C26H28O14	Neobacin	Breytenbach 1986
62	IV	19 min, C.2, ACN, 15–18%, 80 min	C22H22O10	Caragiside B	Nisar et al. 2011
63	V	C.1, MeOH, 35%, 75 min	C22H22O9	Isoononin	Liu et al. 2005
64	IV	24 min, C.2, MeOH, 20%	C26H28O14	Ambocin	Breytenbach 1986
65	IV	C.2, ACN, 15–18%, 80 min	C21H20O9	Neopuerarin	Zhang et al. 2009
66	IV	42 min, C.2, ACN, 20%, 90 min	C21H20O9		Ma et al. 2017
67	IV	18 min, C.2, MeOH, 20%	C21H20O10	8-C-Glucosyl-7,3′,4′-trihydroxy isoflavone	Wong et al. 2017
68	IV	63 min, C.2, MeOH, 20%, 56 min, 20–30% 40 min	C26H28O13		Chen et al. 2009
69	IV	C.2, ACN, 15–18%, 80 min	C21H20O9	Neopuerarin A	Zhang et al. 2009
70	III	39 min, C.2, MeOH, 6–8.5%, 65 min	C28H32O15		Wang et al. 2006
71	III	26 min, C.2, MeOH, 28%, 50 min	C21H20O10		Pistelli et al. 1998
72	IV	54 min, C.2, ACN, 20%, 90 min	C22H22O10		Ohshima et al. 1988
73	IV	58.5 min, C.2, ACN, 20%, 90 min	C26H28O13	Puerarin apioside	Ingham et al. 1986
74	IV	67.5 min, C.2, ACN, 20%, 90 min	C26H28O13		Kinjo et al. 1987
75	III	45 min, C.2, MeOH, 6–8.5%, 65 min	C26H28O14		Peng et al. 2011
76	IV	73 min, C.2, MeOH, 28% 100 min	C23H22O10	Acetyldaidzin	Ohta et al. 1979
77	IV	24 min, C.2, ACN, 15–18%, 80 min	C23H22O11		Zhou et al. 2013
78	IV	74 min, C.1, ACN, 18% 110 min	C23H28O11	Astraganoside	Liu 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.

NO	Fr.	tR, HPLC Condition	MF	Compound	Reference (See SM)
79	V	58 min, C.2, ACN, 57%, 30 min, 57–62% 20 min, 62–70% 29 min	C30H48O3	Oleanolic acid	Tan et al. 2002
80	V	26 min, C.2, ACN, 57%, 30 min, 57–62% 20 min, 62–70% 29 min	C30H48O4	Sumaresinolic acid	Chan et al. 1992
81	V	C.1, MeOH, 72%	C36H58O8	3-O-β-Glc-oleanolic acid	Dubois et al. 1990
82	IV	34 min, C.2, MeOH, 60–70%, 60 min	C48H76O19	Calendulaglycoside B	Vidal-Ollivier et al. 1989
83	V	C.1, MeOH, 72%	C36H56O9	Calenduloside E	Zhang et al. 2013
84	V	C.2, ACN, 30–35%, 60 min, 35–40% 20 min	C48H74O18	Papyrioside LG
85	V	27 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 min	C42H66O14	Chikusetsusaponin Iva	Yang et al. 1995
86	V	C.2, ACN, 30–35%, 60 min, 35–40% 20 min	C42H64O14		Kuroda et al. 2006
87	IV	36.5 min, C.2, MeOH, 65–68%, 40 min	C54H86O23	Scheffleraside II	Mshvildadze et al. 2001
88	V	C.2, ACN, 30–35%, 60 min, 35–40% 20 min	C49H78O19	Chikusetsusaponin V methyl ester	Kondo et al. 1971
89	V	C.2, ACN, 30–35%, 60 min, 35–40% 20 min	C48H76O19	Ginsenoside Ro	Matsuda et al. 1990
90	V	C.2, ACN, 30–35%, 60 min, 35–40% 20 min	C54H86O24	Calendulaglycoside A	Vidal-Ollivier et al. 1989
91	V	C.2, ACN, 30–35%, 60 min, 35–40% 21 min	C53H84O23	Elatoside C	Yoshikawa et al. 1993
92	V	24 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 min	C47H74O18	Pseudoginsenoside RT1	Morita et al.
93	V	55 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 min	C43H68O14	Silphioside A	Jiang et al. 1992
94	V	46 min, C.1, ACN, 35–42%, 80 min, 42–100% 5 min	C48H76O18	Umbellatoside B	Sosa et al. 2011
95	V	20 min, C.2, ACN, 36%, 40 min	C42H66O14	Wedelin	Matos et al. 1983
96	V	27.8 min, C.2, ACN, 57%, 30 min, 57–62% min, 62–70% 29 min	C30H48O4	6β-Hydroxyursolic acid	Sakakibara et al. 1983
97	V	C.2, ACN, 35%	C30H48O6	3β,6β,19α,24-tetrahydro xyurs-12-en-28-oic acid	Fang et al. 1996
98	IV	71 min, C.2, ACN, 18% 110 min	C36H58O11		Abe et al. 1987
99	V	38 min, C.2, ACN, 20–22%, 75 min	C36H58O11		Abe et al. 1987
100	V	20min, C.2, ACN, 36%, 40 min	C42H66O14	Wedelin	Matos et al. 1983
101	IV	8 min, C.2, MeOH, 10–14%, 40 min	C37H55N3O16	Lycibarbarspermidine L	Zhou et al. 2016
102	IV	C.2, MeOH, 10–14%, 40 min	C37H55N3O16	Lycibarbarspermidine M	Zhou et al. 2016
103	IV	C.2, MeOH, 10–14%, 40 min	C37H53N3O16	Lycibarbarspermidine E	Zhou et al. 2016
104	IV	C.2, MeOH, 10–14%, 40 min	C37H53N3O16		Jin et al. 2015
105	IV	C.2, MeOH, 10–14%, 40 min	C31H43N3O11	Lycibarbarspermidine D	Zhou et al. 2016
106	IV	C.2, MeOH, 10–14%, 40 min	C31H43N3O11	Lycibarbarspermidine A	Zhou et al. 2016
107	IV	26.5min, C.2, ACN, 20%, 90 min	C13H14N2O2	Tetrahydroharman-3-carboxylic acid	Tsuchiya et al. 1999
108	III	23 min, C.2, MeOH, 15%, 95 min	C13H14N2O3		Herraiz et al. 2004
109	III	39 min, C.2, MeOH, 17%, 80 min	C13H14N2O2		Herraiz et al. 1993
110	III	36.5 min, C.2, MeOH, 15%, 95 min	C14H17NO3	3α-Benzoyloxynortropan-6β-ol	Al-Said et al. 1986
111	IV	12 min, C.2, ACN, 20%, 90 min	C17H21NO5	Confoline	Aripova et al. 1996
112	II	25 min, C.2, MeOH, 5%, 40 min	C6H7NO2		Hiermann et al. 2002
113	III	47.5 min, C.2, MeOH, 17–20%, 100 min	C10H13NO4		Chin et al. 2003
114	III	7.5 min, C.2, MeOH, 5–15%, 60 min	C6H5NO2	Nicotinic acid	Krehl et al. 1946
115	IV	18 min, C.2, MeOH, 20%	C8H15NO2		Singer et al. 1935
116	IV	8 min, C.2, MeOH, 10–14%, 40 min	C14H22NO4+	Codonopyrrolidium B	He et al. 2014
117	III	17 min, C.2, MeOH, 10%, 80 min	C8H17N3O3		Gegauer et al. 2003
118	III	19 min, C.2, MeOH, 10%, 80 min	C6H13NO2		Perrin et al. 2000
119	IV	20 min, C.2, ACN, 15–18%, 80 min	C10H8O4	Scopoletin	Best 1948
120	IV	23 min, C.2, ACN, 15–18%, 80 min	C20H24O8	Vellein	Maruyama et al. 2009
121	IV	33.5min, C.2, MeOH, 28%, 100 min	C8H10O2	Phenethyl alcohol	Wang et al. 1982
122	IV	35.5 min, C.2, ACN, 20%, 90 min	C8H8O4	Pisolithin B	Benecke et al. 1984
123	V	43.5 min, C.2, ACN, 22–33%, 60 min	C9H8O2	trans-β-Carboxystyrene	Billmann et al. 1909
124	IV	21 min, C.2 ACN, 15–18%, 80 min	C10H10O4	Ferulic acid	Henderson and Farmer 1955
125	V	60 min, C.2 ACN, 22–24%, 75 min	C11H12O4		Oonuma et al. 1993
126	V	C.1, ACN, 28%	C11H12O3		Newman et al. 1952
127	V	C.1, ACN, 28%	C12H14O4	Ethyl ferulate	Nakayama et al. 1996
128	III	71 min, C.1, ACN, 12–25%, 43 min, 25–30% 12 min	C9H8O4	Caffeic acid	Baerheim 1951
129	IV	55 min, C.1, MeOH, 45–48%, 80 min	C11H12O4	Ethyl caffeoate	Mao et al. 2011
130	III	37min, C.2, MeOH, 28%, 50 min	C9H8O3	trans-p-Hydroxycinnamic acid	King et al. 1952
131	III	40 min, C.2, MeOH, 28%, 50 min	C10H10O4	Isoferulic acid	Qiao and Chen 1991
132	III	48.5 min, C.2, MeOH, 15%, 95 min	C15H18O8	p-Coumaric acid β-glucoside	Runeckles, Woolrich 1963
133	III	48.5 min, C.2, MeOH, 15%, 95 min	C16H20O9	Glucosidoferulic acid	Ibrahim et al. 1970
134	IV	22 min, C.2, MeOH, 20%	C10H10O4		Muratake et al. 2013
135	III	30 min, C.2, MeOH, 6–8.5%, 65 min	C14H20O7		Fujimatu et al. 2003
136	IV	73.5 min, C.2, MeOH, 20%, 56 min, 58.5 min, 20–30%, 40 min	C35H46O20	Echinacoside	Frezza et al. 2017
137	III	29.5 min, C.2, MeOH, 30–40%, 90 min	C37H48O21	Tubuloside A	Kobayashi et al. 1987
138	IV	80 min, C.2, MeOH, 28% 100 min	C29H36O15	Verbascoside	Pham et al. 1988
139	V	60.5 min, C.2, ACN, 16–20%, 100 min	C20H26O12		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.

and

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.

NO	Fr.	tR, HPLC Condition	MF	Compound	Reference (See SM)
140	V	C.1, ACN, 60%	C16H14O4		Letcher and Nhamo 1973
141	V	C.2, ACN, 40–50%, 48 min	C17H16O4	Batatasin I	Gonnet et al. 1973
142	V	C.1, ACN, 30%	C15H10O4		Morton et al. 1941
143	V	C.1, ACN, 30%	C16H12O5		Wang et al. 2011
144	V	C.1, ACN, 33%	C16H12O5		Wu et al. 2003
145	V	C.1, ACN, 30%	C15H10O5		Lee et al. 1994
146	V	C.1, ACN, 30%	C16H12O4	Digitolutein	Koumaglo et al. 1992
147	V	34 min, C.2, ACN, 40%	C15H10O3		Bistrzycki and Zen-Ruffinen 1920
148	V	34 min, C.2, ACN, 40%	C15H10O5		Yang et al. 1992
149	IV	19 min, C.2, MeOH, 20%, 56 min, 20–30% 40 min	C14H8O4		Varbanov et al. 1986
150	V	29 min, C.2, ACN, 22–27%, 60 min	C13H8O4	1,3-Dihydroxyxanthone	Liang et al. 1982
151	III	39 min, C.2, MeOH 6–8.5%, 65 min	C12H16O8		Baba et al. 1995
152	IV	15 min, C.2, MeOH, 20%	C16H18O9	Biflorin	Zhang et al. 1997
153	III	46 min, C.2, MeOH, 15%, 95 min	C16H18O9	Isobiflorin	Tanaka et al. 1993
154	III	37.5 min, C.2, MeOH, 6–8.5%, 65 min	C16H18O9	Undulatoside A	Itoh et al. 2003
155	III	37.5 min, C.2, MeOH, 6–8.5%, 65 min	C16H18O9		Wang et al. 2011
156	III	55 min, C.2, MeOH, 17%, 80 min	C26H34O12		Yan et al. 2008
157	V	89 min, C.2, ACN, 16–20%, 100 min	C22H26O8	Syringaresinol	Abu Zarga 1986
158	V	54 min, C.2, ACN, 22–33%, 60 min	C18H16O5		Shi et al. 2007
159	IV	25.5 min, C.2, ACN, 15–20%, 50 min	C18H14O8	Prolithospermic acid	Dai et al. 2010
160	III	29 min, C.2, MeOH, 28%, 50 min	C18H14O8	7-Epiblechnic acid	Wang et al. 2010
161	III	29 min, C.2, MeOH, 28%, 50 min	C18H14O8	Blechnic acid	Wada et al. 1992
162	V	69 min, C.2, MeOH, 35%, 80 min	C23H26O10		Guo et al. 2014
163	V	36 min, C.2, ACN, 22–24%, 75 min	C7H6O3	p-Salicylic acid	Sager and Schooley 1945
164	V	63.5 min, C.2, ACN, 22–24%, 75 min	C9H10O3		Heim et al. 1957
165	V	41 min, C.2, ACN, 20–22%, 75 min	C8H8O3	p-Methoxy benzoic acid	Reitberg and Schentag 1983
166	III	67 min, C.2, MeOH, 12–25%, 43 min, 25–30% 12 min	C8H8O4		Parham et al. 1954
167	III	49.5 min, C.2, ACN, 13–15%, 90 min	C8H8O4	Vanillic acid	Sammons and Williams 1946
168	IV	20min, C.2, MeOH, 20%	C7H6O3	Rancinamycin IV	Li et al. 2004
169	IV	44 min, C.2, MeOH, 20%, 56 min, 20–30% 40 min	C7H6O2		Rivers 1947
170	II	39.5 min, C.2, MeOH, 7–9%, 70 min	C14H18O9		Yang et al. 2013
171	V	57 min, C.2, ACN, 20–22%, 75 min	C9H12O3	1,3,5-Trimethoxybenzene	Allain et al. 1980
172	IV	24min, C.2, MeOH, 20%	C8H10O2	p-Hydroxyphenetole	Rosenwald 1951
173	II	22 min, C.2, MeOH, 2%, 60 min	C13H18O8	Tachioside	Sano et al. 1991
174	IV	73 min, C.2, MeOH, 28% 100 min	C9H10O4
175	III	77 min, C.2, MeOH, 12–25%, 43 min, 25–30% 12 min	C7H6O3	Salicylic acid	Ichniowski and Hueper 1946
176	III	78 min, C.2, MeOH, 25–30%, 60 min, 30–35% 40 min	C16H26O8	Bodinierin	Xie et al. 2006
177	III	80 min, C.2, MeOH, 25–30%, 60 min, 30–35% 40 min	C16H26O8	Kankanoside O	Liu et al. 2016
178	III	75 min, C.2, MeOH, 20–25%, 80 min	C16H28O7		Fan et al. 2011
179	V	21 min, C.2, ACN, 16–20%, 100 min	C27H44O8	(25R)-20,26-Dihydroxyecdysone	Suksamrarn 1998
180	III	43min, C.2, MeOH, 30–40%, 90 min	C27H44O7	26-Hydroxyecdysone	Li et al. 2006
181	IV	C.2, ACN, 15–18%, 80 min	C27H44O7	3-epi-20-Hydroxyecdysone	Thompson et al. 1974
182	III	47.5 min, C.2, MeOH, 6–8.5%, 65 min	C16H22O10		Li et al. 1999
183	II	51 min, C.2, MeOH, 2%, 60 min	C16H22O11	Desacetylasperulosidic acid	Inouye et al. 1974
184	II	47.5 min, C.2, MeOH, 7–9%, 70 min	C16H24O10	Mussaenosidic acid	Kohda et al. 1989
185	III	9.5 min, C.2, MeOH, 5–15%, 60 min	C11H13N3O5	9-Deazainosine	Liu et al. 2005
186	V	38 min, C.2, MeOH, 35%, 80 min	C12H16O2	Sedanonic acid lactone	Mitsuhashi and Nomura 1966
187	V	46 min, C.2, ACN, 20–22%, 75 min	C12H16O4	Senkyunolide I	Huang et al. 2013
188	IV	65.5 min, C.2, ACN, 17% 100 min	C12H16O4	Senkyunolide H	Huang et al. 2013
189	V	C.2, ACN, 60%	C17H24O2	Falcarindiol	Lechner 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.

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. Relative Nrf2 activity induced by compounds (40 μg/mL) isolated from Jing liqueur.

Cpd	Fr.	Relative Nrf2 Activity (Fold Induction)	Cpd	Fr.	Relative Nrf2 Activity (Fold Induction)	Cpd	Fr.	Relative Nrf2 Activity (Fold Induction)
Control	-	1.0	125	V	9.5	145	V	3.0
50	V	4.0	126	V	16.0	146	V	3.0
51	V	11.5	127	V	10.5	168	IV	3.0
53	V	4.0	128	V	3.0	171	V	3.5
55	IV	3.0	129	IV	2.2	186	V	3.5
58	V	6.5	143	V	6.0	SF (5 μM)	-	15.0
78	IV	4.0	144	V	3.5

). 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 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.

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.