UPLC-Q-TOF-MS Analysis of Chemical Constituents of the Kadsura coccinea Extract with Effect of Attenuating Lipid Accumulation in Vitro

Abstract

Objective/Background: This study aims to explore the Kadsura coccinea extract (KCE)'s effect on lipid accumulation in vitro and its chemical components characterizations, aiming at developing a new alternative plant medicinal resource to fight against non-alcoholic fatty liver disease (NAFLD). Methods: After toxicological evaluation of KCE on HepG₂ cells, Oil red O staining model and intracellular TGs quantification kit were used to examine the effects of KCE on lipid accumulation in vitro. The chemical components characterizations and potential active chemical constituents of the bioactive KCE were analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technology. Results: Among the chosen non-toxic concentrations of KCE (5, 10, 20 μg/mL), KCE can reduce the number and volume of lipid droplets in the oleic acid induced HepG₂ cells in a dose-dependent manner, and the results of TGs quantification were almost consistent with the results of the oil red O staining experiment. These data indicate the KCE has ameliorative effect on lipid accumulation in vitro. In addition, a total of 26 compounds from the KCE were tentatively identified, dibenzocyclooctadiene lignans (DCLs) including Kadsulignan L and Gomisin J could be the main supposed components. Conclusion: These findings support further investigation into Kadsura coccinea containing DCLs as a new alternative medicinal arsenal to battle against NAFLD.

Introduction

The most common causes of cirrhosis which contributes greatly to liver-related deaths accounting for 4% of all deaths worldwide are related to viral hepatitis, alcohol, and non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by pathological intrahepatic triglycerides (TGs) excess represents a burgeoning epidemic around the world. ¹ The mechanism underlying the pathogenesis is supposed to be related to the imbalance between lipid uptake/anabolism and output/catabolism in chronic states of over-nutrition. Due to its complex etiology reflecting multiple interactions between environmental and genetic factors, there is no official approved pharmacological therapy available for NAFLD currently except for weight reduction to meet the great demand. ² Traditional Chinese medicine (TCM) has undergone thousands of years of development and has also formed a unique theoretical system that acknowledged NALFD is related to deficiency and depression of Qi. The practice of TCM has been guided by the principle of “observing its pulse patterns, knowing where to go wrong, and treating according to the symptoms”. TCM and Western medicine have different perspectives on observing and solving problems, innovative concepts, and unique strengths. ³ In addition, many dietary strategies include the medicinal and edible homologous herb medicines such as some green tea have shown its preventive effects on the development of liver steatosis or its progression. ⁴ Therefore, exploring and developing the therapeutic candidates from these plant medicines including TCM is a big concern in the phytomedicine community.

Kadsuracoccinea(Lem.) A.C. (KC), known as Hei-lao-hu in TCM, a climbing shrub distributed in southwest China, belongs to the medicinally important genus Kadsura from the Schisandraceae family. Its fruits, leaves, stems and roots are applied as folk medicine in ethnic minorities such as Zhuang, Li, Miao and Tujia et al.^5,6 The fruits are edible and have unique shapes and high nutritional and medicinal values. Importantly, the root of the KC was legally recorded in the Chinese Pharmacopoeia 1977 as the Chinese name “Heilaohu” (Radix Kadsuraecoccineae)” to mainly target stagnant syndrome to activate blood circulation and dredge collaterals. It is commonly used to treat stomach and duodenal ulcer, chronic gastritis, abdominal pain and rheumatoid arthritis et al. ⁷ Studies in recent years have showed that some chemical components from KC have anti-inflammatory, anti-oxidation, and other pharmacological effects such as protein tyrosine phosphatase 1B (PTP1B) and acetylcholinesterase (AChE) inhibitory activities et al. ⁸ KC belongs to the genus Kadsura, which is phylogenetically close to Schisandra. The medicinal plant from genus Schisandra has been more widely used and studied for indication of liver disease, and several derived products including Wuzhi tablets (Schisandra sphenanthera extract) were marketed after regulatory approval by CFDA in China.^9–11 Thus, from a chemotaxonomic perspective, it was inferred that the roots of KC would show a phytochemical composition similar to the Schisandra chinensis and Schisandra sphenanthera, in which have yielded a number of lignans showed hepatoprotective activity. ¹² In pursuit of new potential medicinal resources and lead compound, some chemical components including some novel scaffolds from KC were successively separated and identified as dibenzocyclooctadiene lignans (DCLs), sesquiterpenoid, ascorbic acid derivatives, β-caryophyllene et al.^13–17 Nevertheless, the systemic investigation for the chemical compositions from roots of KC is deficient.

In recent years, with functional ingredients as the core to seek, construction and application of a technology system for discovery, enrichment, and quality control of active substances in TCM based on integration strategies is a hot topic. ¹⁸ Among these state-of-the-art technologies, quadrupole time-of-flight mass spectrometry technology (Q-TOF-MS) generating accurate measurements of mass, elemental composition and isotopic abundance to identify the possible molecular formula of each analyte with the mosaic library database, equipped with ultra-high-performance liquid chromatography (UPLC) systems separating solitary constituents rapidly with a high resolution, has become a powerful method for the rapid separation and identification of active ingredients in the complex traditional herb medicine as shown in our previous work. ¹⁹ In addition, in vitro model is one of the widely adopted methods for screening and evaluating candidate compounds in the early stage of investigational new drug development. Omega 9 oleic acid as the non-essential fatty acids-induced lipid accumulation in HepG₂ cells was to mimic the steatosis manifested as the intrahepatic fat excess of at least 5% of liver weight in the early stage of NAFLD progression. ²⁰ Combining the suitable in vitro model with UPLC/Q-TOF-MS can discover the novel active ligand towards specific drug target. ²¹ In the present study, we described the biological evaluation of the ethanolic extract from the roots of KC towards lipid storing inhibitory activities in hepatocytes. Meanwhile, UPLC-Q-TOF-MS technology as well as Masslynx 4.1 software and network database was adopted to analyze and confirm the chemical constituents of KC extract.

Materials and Methods

Materials Preparation

The root of KC was purchased from Guanyang County, Guilin City, Guangxi province and identified Kadsura coccinea (Lem.) A. C. Smith by associate professor Xuanxuan Cheng, School of Traditional Chinese Medicine, Guangdong Pharmaceutical University. The voucher specimen with code GDPU-2016GUI-KCL was deposited in the School of Pharmacy, Guangdong Pharmaceutical University. After sunshine for more than three months, the dried roots of KC (500 g) were crushed and extracted with 80% ethanol (3 L) twice at room temperature for 24 h at each time, then supernatant was filtered with filter paper, and then evaporated under reduced pressure at no more than 50 °C to remove the ethanol. The concentrated Kadsura coccinea extract (KCE) was stored in 4 °C refrigerator for future use. Before the experimental treatments, the stored solutions were diluted to different concentrations in the cell culture medium.

Reagents

The reagents for cell culture and treatments are as follows: Triglycerides (TGs) kits (Biosino Biotechnology & Science Inc., Beijing, China), Oil red O (Kanglang Biological Technology Co. Ltd, Shanghai, China), Dulbecco's Modified Eagle Medium (Invitrogen Co. Ltd, New York, USA), Fetal bovine serum (Hyclone Co. Ltd, Utah, USA), Trypsin (Amresco Co. Ltd, Ohio, USA), Sodium oleate and Penicillin-Streptomycin(Tokyo chemical industry Co. Ltd, Tokyo, Japan), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Thiazolyl blue tetrazolium bromide, MTT)(Sigma Co. Ltd, San Francisco, USA). In addition, acetonitrile (UPLC grade, Merck), Formic acid (UPLC grade), Ammonium formate (Merck) were purchased, phosphate-buffered saline was bought from Guangzhou Chemical Agent Factory (Guangzhou, China); Lockmass enkephalin (Sigma Company, USA, batch number: L9133-50MG) was obtained; standards of Gomisin J (Lot No. zzs17022206), Gomisin D (Lot No. zzs17022204), Gomisin E (Lot No. zzs17022205), Kadsulignan L (Lot No. zzs17022207) were purchased from Shanghai ZZBIO Co, LTD,. Other reagents were obtained from Guangzhou Chemical Agent Factory (Guangzhou, China).

Cell Culture

HepG₂ was provided by the Institute of Chinese Medical Science of Guangdong Pharmaceutical University, which is bought from the American Type Culture Collection (Mannassas, VA, USA). Cell culture is similar to the previous report of our work. ²¹ In short, after preparation and thawing frozen HepG₂cells, the operator carefully removed the supernatant and resuspend the cell pellet in fresh, pre-warmed DMEM with FBS, count the cells using a hemocytometer and adjust the cell density to the desired concentration 2 × 10⁵ cells/mL. Then cells were cultured in a 96-well plate with 2 × 10⁵ cells/mL (100 µl each well) in culturing medium including 13.5 g/L DMEM containing 10% FBS, double-antibiotic solution and high glucose. Place the culture plate in a cell culture incubator set to 37 °C with 5% CO₂ and check the cells daily and replace the culture medium every 2-3 days depending on the cell growth rate. Use the cultured HepG₂ cells for the following viability assay and oil-red staining experiments.

Cell Viability Assay

The effect of KCE with different concentration on the viability of HepG₂cells was evaluated using the MTT Detection Assay System. The MTT assay is a commonly used colorimetric assay to assess cell viability. It measures the metabolic activity of cells, which is an indirect indicator of cell viability. It involves the conversion of the yellow MTT dye into a purple formazan product by mitochondrial enzymes in metabolically active cells. The intensity of the purple color is proportional to the number of viable cells. For performing the MTT assay, HepG₂ cells were inoculated with 1.0 × 10⁵ cells per well into 96-well culture plates for 24 h incubation in an incubator containing 5% CO₂ at 37 °C. And then, the culture medium was replaced, cells were treated with various concentrations of KCE (1-1000 μg/mL), the control groups were prepared alongside. After 24 h incubation, carefully remove the culture medium from each well without disturbing the cell monolayer, and add 100 µl of the MTT solution to each well. Incubate the plate with the MTT solution for 4 h in the cell culture incubator. After the incubation, carefully remove the MTT solution from each well, put 100 µL of DMSO into each well to dissolve the formazan crystals. After measuring the absorbance at 485 nm using a microplate reader, the cell viability index was calculated to choose the appropriate concentration of KCE for the following experiments.

Oil Red O Staining and Determination of the Intracellular TG

Oil red O staining is a commonly used method to visualize and quantify intracellular TGs or lipid droplets in cultured cells or tissues. The procedure we adopted in this study is similar to the previous report of our colleagues. ²² Briefly, HepG₂ cells were plated in 6-well plates (2.5 × 10⁵ cells/mL) for 24 h incubation with 37 °C 5% CO₂. And then all wells cells were divided into 6 groups (with six parallel wells in each group) as the blank control group, model group, berberine (Ber) as the positive control group, and KCE groups (High, middle, low concentrations). After cell culture, fixation, washing and preparation, add the oil red O staining solution to the fixed cells, ensuring complete coverage, and incubate for 30 min at room temperature. The ingredients in each group are shown as follows: (1) KCE groups: high glucose DMEM culture medium containing 10% FBS and oleic acid with a final concentration of 0.2 mmol/L, KCE with high (20 μg/mL), middle (10 μg/mL) and low (5 μg/mL) concentrations, respectively. (2) Blank control group: high glucose DMEM culture medium containing 10% FBS.(3) Model group: high glucose DMEM culture medium containing 10% FBS and oleic acid with a final concentration of 0.2 mmol/L.(3) Ber as the positive control group: high glucose DMEM culture medium containing 10% FBS, oleic acid and Ber with a final concentration of 0.2 mmol/L and 10 μmol/L, respectively. Cell morphology and the distribution of lipid droplets were observed with the inverted microscope and images of the stained lipid droplets were captured.

In addition, measuring TGs content in HepG₂ cell lines is commonly done by commercial TG determination kits (Biosino Biotechnology & Science Inc., Beijing, China). The kits are typically based on enzymatic methods, where specific enzymes are used to catalyze reactions with TGs, producing a measurable signal that is directly proportional to the TGs concentration. The enzymatic method derived kit for TGs determination involves hydrolysis of TGs into glycerol, phosphorylation by glycerol kinase, oxidation induced production of hydrogen peroxide which reacts with a chromogenic substrate to produce a colored product. The color intensity is directly proportional to the concentration of TGs in the Cells. Similar to the reported procedures, ²³ the total protein was quantified by the BCA protein concentration determination kit method, and the intracellular concentration of TGs was measured by the TGs determination kits strictly. Then, the TG/total protein (mg.g⁻¹) in each well was calculated. All experiments were performed in triplicate.

UPLC-Q-TOF-MS Analysis for the Chemical Composition in KCE

Similar to our previous work,^19,21 UPLC-Q-TOF-MS was used to separate and analyze KCE constituents. Briefly, a 50 mm × 2.1 mm (Acquity 1.7 µm) UPLC BEH C-18 column (Waters, Milford, MA) was adopted for ingredients separation. The flow rate of the mobile phase was 0.3 mL/min at temperature 40°C with a gradient of MeCN-H₂O mixtures ranging from 2/98 to 55/45(v/v) containing 0.2% formic acid in a 12.5 min run. The injection volume was 10 μL each time. The full mass scanning range from m/z 100 to 1000 Da with TOFMS was operated in positive mode and negative mode with electrospray ionization. High purity nitrogen was used as the nebulizer and auxiliary gas; argon was used as the collision gas. The structure of each chemical was elucidated by tandem mass spectrometry fragmentation with collision energy ramp ranging from 10 to 30 eV. The other parameters were set as follows: capillary voltage: 3.2 kV, cone gas flow: 50 L/h, desolvation gas flow:500L/h, The source temperature and desolvation temperature were at 120 °C and 350 °C, respectively. Mass accuracy was maintained by using a lock spray with leucine enkephalin 500 pg/μL ([M + H]⁺m/z 556.2771, [M-H]⁻m/z 554.2615)) as reference. All the solvents for liquid chromatography and mass spectrometry in this study were of the highest grade commercially available.

1 mL of KCE was centrifuged for 5 min (12,000 r/min), the supernatant was taken for analysis. The repeatability was assessed by analyzing six independently prepared samples. The method precision was evaluated by analyzing the same sample for six consecutive times. The sample stability test was determined with one sample within one day. In this period, the solution was stored at room temperature and analyzed at 0, 1,2,4,12, 24 h, respectively.

Data Analysis

All experimental values are expressed in the figures as mean ± SD, and data were analyzed by two-tailed Student's t test. p < 0.05 was regarded as significantly different between different groups.

Results

Toxicological Evaluation of KCE to HepG₂ Cells

We preliminarily tested the effects of KCE in different concentrations (1-1000 μg/mL) on HepG₂ cell survival with the classic MTT method. Among the tested concentrations of KCE (1-1000 μg/mL), KCE exhibits some cytotoxicity with less than 95% viability only at high concentrations more than 80 μg/mL. To seek the non-toxic KCE concentration for the lipid droplets imaging and quantification, less than 50 μg/mL as the criteria was determined. Among the chosen concentrations of KCE (5, 10, 20 μg/mL) for lipid staining and quantification of the intracellular TGs, no significant cytotoxicity was noted.

Effect of KCE Treatment on in Vitro Oleic Acid Induced Model TGs

Oil red O is highly soluble in fats and can specifically stain TGs in tissues. As shown in Figure 1B model group, the cell lipid aggregation model to mimic the NAFLD manifestation induced by oleic acid has been successfully constructed. Compared to oleic acid induced model group, the 10 μmol/L Berberine treatment (Figure 1C) can slightly reduce lipid content as previous researcher reported. Interestingly, 5-20 μg/mL KCE can reduce the number and volume of lipid droplets in the oleic acid induced HepG₂cells in a dose-dependent manner. The effective potency triggered by the positive control berberine is between low and medium doses of KCE, but it is significantly lower than that of the high-dose 20 μg/mL KCE group. In another parallel experiment, the TG content of each group after treatment was determined by the quantitative reagent kit of TGs. As demonstrated in Figure 2, the results of TGs quantification were almost consistent with the results of the above oil red O staining experiment. In addition, the decrease of TGs content in the HepG₂ cells is also a dose-dependent manner in the low, middle and high dose KCE groups.

Figure 1.

Oil red O staining maps of HepG₂ cells (10 × 40):A: control group cells, B: induced group cells, C: 10 μmol/L berberine-treated cells, D: 5 μg/mL KCE-treated cells, E: 10 μg/mL KCE-treated cells, F: 20 μg/mL KCE-treated cells.

Figure 2.

Effect of KCE on the content of TG in oleic acid-induced HepG₂ cells. Note: KCE groups with low (5 μg/mL), middle (10 μg/mL) and high (20 μg/mL) concentrations are indicated as KCE-L,KCE-M, KCE-H for short respectively. ^###P < 0.001, compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the model group.

Constituents Identification of KCE by UPLC-Q-TOF-MS

UPLC-Q-TOF-MS was used to analyze the chemical constituents of KCE. The method validation demonstrated that the relative standard deviation (R.SD) of the main peaks retention time in the sample was no more than 0.3%.The total ion current chromatograms in both positive and negative ion modes were shown in Figure 3. By UPLC-Q-TOF-MS, the mass spectrum data of each compound in KCE were obtained. The chemical constituents were analyzed and identified by searching in database and matching, and comparing the references and the standards.

Figure 3.

The profile for base peak ion （BPI）chromatogramof KCE in positive(A) andnegative(B) ion mode with electrospray ionizationby UPLC/Q-TOF-MS.

The identification results of the compounds are detailed in Table 1 with the corresponding references.^24–40 Among them, there were 16 lignans (peaks 3, 4, 6, 7, 10, 11, 14, 15, 19, 20-26), 2 triterpenoids, 1 glycosides, and 7 others compounds. In this study, most of the identified lignans in KCE are dibenzocyclooctadiene lignans except for peak 30.The structural skeleton of these lignans was shown in Figure 4. Some special lignans of biphenyl cyclooctadiene, such as 6, 9-oxo-bridged biphenyl cyclooctadiene (peak 25) were found.

Figure 4.

The structural skeleton of dibenzocyclooctadiene lignans.

Table 1.

Constituents Identification of KCE by UPLC/Q-TOF-MS.

No.	TR(min)	Detected ions	Mass (m/z)		REppm	Formula	MSMSFragmentationsm/z (Abundance %)	Identification
			Det.	Cal.
1	2.42	[M-H]−	359.1122	359.1131	−2.5	C15H20O10	359(10), 197(70), 182(70), 153(42), 138(100), 123(40)	24
2	2.28	[M + H]+	387.1433	387.1444	−2.8	C21H22O7	387(10), 369(10), 348(26), 318(100), 208(20)	Praeruptorin A 25
3	2.84	[M + H2O]+	450.1882	450.1890	−1.8	C23H28O8	450(100), 318(50), 301(10), 295(20),	Schisandrol A 26
		[M-H]−	431.1712	431.1706	1.4		431(40), 385(30), 367(5), 299(10), 175(100)
4	3.32	[M + H2O]+	436.2086	436.2097	−2.5	C23H30O7	436(10), 257(100), 183(75), 165(50), 137(50)	Gomisin H 27
		[M + COOH]−	453.1167	453.1186	−4.2		453(70), 423(100), 397(45), 393(15), 363(15)
5	3.68	[M-H]−	433.1869	433.1862	1.6	C23H30O8	433(10), 387(80), 357(40), 255(40), 179(50), 161(100)	–
6	4.31	[M + Na + H]+	618.2089	618.2077	1.9	C32H34O11	618(60), 565(10), 421(100), 403(40), 341(10), 209(60)	Schizanrin F 28
		[M-H]−	593.2034	593.2023	1.9		593(10), 549(20), 563(20), 533(100)
7	4.42	[M-H]−	441.1565	441.1549	3.6	C24H26O8	441(80), 397(20), 330(80), 263(15), 205(20), 119(100)	Schiarisanrin B 29
8	4.67	[M + H2O]+	482.2528	482.2516	2.5	C25H36O8	482(80), 465(30), 387(20), 333(35), 295(30), 259(10), 171(30), 153(100)	–
9	4.82	[M + H2O]+	482.2530	482.2516	2.9	C25H36O8	482(100), 465(50), 406(48), 371(20), 333(30)	Isomer of Peak 18
10	5.19	[M + H2O]+	524.2072	524.2046	2.9	C29H32O9	524(60), 477(100), 385(30), 331(80),	Kadsuralignan K 30
		[M + COOH]−	551.1945	551.1917	5.1		551(50), 417(20), 377(15), 301(25)
11	5.38	[M + H]+	557.2414	557.2387	4.8	C30H37O10	520(80), 503(10), 341(22), 305(20), 195(100)	Interiotherin C 31
12	6.95	[M + COOH]−	403.2492	403.2484	−2.0	C23H34O3	403(30), 357(20), 327(100), 311(15)	Schiglausin H 32
13	7.12	[M-H]−	575.2846	575.2856	−1.7	C31H44O10	575(5), 529(15), 397(60), 161(100)	Kadcoccilactone I 33
14	7.28	[M + H]+	387.1797	387.1808	−2.8	C22H26O6	387(40), 355(100), 299(40), 263(15)	Kadsuralignan H 30
15	7.66	[M + H]+	403.1745	403.1757	−3.0	C22H26O7	403(20), 386, 348(100), 295(30)	Binankadsurin A 34
16	7.66	[M-H]−	221.1914	221.1905	4.1	C15H26O	221(50), 206(40), 174(20), 146(15)	–
17	8.33	[M-H]−	329.2330	329.2328	0.6	C18H34O5	329(100), 293(10), 229(40), 211(60), 171(80)	Tianshic acid 35
18	9.58	[M-H]−	343.2289	343.2273	4.7	C22H32O3	343(60), 329(10), 293(100), 236(20)	Seco-coccinic acid H 36
19	9.87	[M + H]+	417.1541	417.1549	−1.9	C22H24O8	417(10), 385(10), 347(100), 317(45), 287(35)	Schisantherin P 37
20	9.88	[M + COOH]−	373.1665	373.1651	3.8	C20H24O4	373(80), 227(10), 166(10), 139(100)	Anwulignan 38
21	10.13	[M + H]+	389.1975	389.1964	2.8	C22H28O6	389(100), 357(50), 331(10), 319(20), 287(10), 274(30)	Gomisin J 27
		[M-H]−	387.1815	387.1808	1.8		387(18), 372(48), 357(100), 331(30), 325(30)
22	10.33	[M + H]+	531.2219	531.2230	−2.1	C28H34O10	531(65), 485(70), 401(80), 331(10)	Gomisin D 27
		[M + COOH]−	575.2121	575.2129	−1.4		575(10), 529(60), 399(100)
23	10.90	[M + H]+	415.1745	415.1757	−2.9	C23H26O7	415(10), 357(15), 342(5), 329(100), 326(21), 300(16)	Kadsulignan L39,40
24	11.90	[M + Na]+	523.1937	523.1944	-1.3	C27H32O9	523(28), 502(100), 470(10), 415(20), 385(80)	Oxo-kadsuralignan J 30
25	12.04	[M + Na]+	567.2181	567.2206	-4.4	C29H36O10	567(25), 528(20), 483(100), 440(30),	Kadsuralignan G 41
26	12.09	[M + H]+	515.2272	515.2281	-1.7	C28H34O9	515(100), 469(10), 403(70), 385(20),	Gomisin E 27

Note：“Det.” means detected ion mass; “Cal.” means calculated ion mass; “RE” means relative error between detected ion mass and calculated ion mass. RE = [Mass Det.-Mass Cal.]/Mass Cal.×10⁶; “–” means not identified yet.

In this study, it was found that the fragmentation ion produced by loss of C7-C8 and its substituent in the eight-membered ring of dibenzocyclooctadiene lignans (Figure 4) was characteristic ion in ESI source mass spectrometry. In addition, the ring opening and ring shrinking cracking of the eight-membered ring in the parent structure of dibenzocyclooctadiene lignans took place.

Take peak 23 as an example to describe the process of identification and analysis. Peak 23(t_R= 10.90 min) with a molecular ion of [M + H]⁺at m/z 415.1745 (Figure 5) and a calculated molecular formula of C₂₃H₂₇O₇under ESI positive ion mode. Its fragment ions at m/z 415, 397, 357, 342, 329, 326 and 300 produced in TOF-MS/MS under ESI positive ion mode (Figure 6). The quasi-molecular ion peak was [M + H]⁺ at m/z 415, and further produced fragment ion [M + H-H₂O]⁺ at m/z 397.The fragment ions [M + H-C₄H₁₀]⁺ at m/z 357 were produced because of the cracking of C7-C8 bond and its substituent (−2CH₃) of the eight-membered ring structural skeleton, which probably split into seven-membered ring and five-membered ring due to its unstable structure generated the fragmentation ions at m/z 387 and 329 respectively. Peak 23 was tentatively assigned as a biphenyl cyclooctadiene.

Figure 5.

The base peak ion (BPI) chromatogram of standard Kadsulignan L (a) and KCE(b), MS spectrum of Kadsulignan L in positive ion mode by UPLC/Q-TOF-MS.

Figure 6.

The MS/MS spectrum and possible Mass fragmentation pathway of Peak 23 represented as Kadsulignan L.

As the result, peak 23 was identified and confirmed as Kadsulignan L^39,40 based on comparison of the retention time, and mass spectral data with those of the reference standard. Its possible fragmentation pathway was shown in Figure 6. Other lignans were identified by the similar way.

The standards of Gomisin J, Gosimin D, Gomisin E as samples were used for verifying the results of the putative identification. Take the Gomisin J for an example, specifically, according to MS spectra information, it was obviously observed that ions of [M + H]⁺ and [M-H]⁻ at m/z 389.1975 and 387.1815 of peak 21 in KCE sample (Figure 7), and corresponding to the ions in standard were 389.1958 and 387.1819 under positive and negative respectively; as the results, the ions had the same chemical formulas of C₂₂H₂₉O₆ (m/z 389.1964) and C₂₂H₂₇O₆(387.1808) in both the standard and KCE sample, by comparing the MS, MS/MS spectra, we could infer that they were essentially the same.

Figure 7.

The comparison of MS, MS/MS spectra of Gomisin J identified in KCE to the standards both in positive and negative ion modes.

Discussions

Some researchers have evaluated the cytotoxicity of lignans Heilaohulignan C, kadsuralignan I, longipedunin B, kadusurain A from KC ethyl acetate fraction on HepG₂ Cells,^5,13 all the IC₅₀ values of pure compound are more than 10 μg/mL. In our EtOH extract, HepG₂ cells showed less than 95% viability at concentrations more than 80 μg/mL. Therefore, we choose the concentrations of KCE 5, 10, 20 μg/mL for the later oil red O staining and determination of the intracellular TG. The accurate lignan content calculation in the KC roots and its extract depends on the standard reference available needed to be developed.

Oleic-acid is frequently used to induce the accumulation of lipid droplets in HepG_2. ²² In the current study, oil red O imaging demonstrated that KCE in gradient concentrations (5, 10, 20 μg/mL) processing significantly mitigated lipid agglomeration and fusion in HepG₂cells induced by oleic acid. These data are consistent with the intracellular TG content determination, which also showed a dose-dependent manner. These results suggest that KCE could improve abnormal lipid metabolism in the liver characterized in NAFLD initiation and progression. However, the detailed mechanism involved in the improvement needs to be investigated in further.

The putative active components of KCE related to this effect were investigated by UPLC-Q-TOF-MS analysis. As the results, 26 compounds from KCE were identified, dibenzocyclooctadiene lignans were the main supposed components contained in KCE according to the literature.^{13–16,40,42,43} The follow-up separation and purification of these supposed compounds are warranted to confirm its structure by other technologies such as nuclear magnetic resonance.

It is noteworthy that lignans were frequently reported that have the potentiality to treat NAFLD. Schisandrin B, are presentative DCL isolated from the fruit of Schisandra chinensis, was found to possess antihyperlipidemic, antioxidant, anti-endoplasmic reticulum stress, and anti-inflammatory activities in cultured hepatocytes in vitro and in rodent livers in vivo. ⁴⁴ And sesamin, another lignan from the dietary seeds, can ameliorates hepatic steatosis and inflammation in rats on a high-fat diet via nuclear receptor LXRα and PPARα which is hot pharmacological target towards NAFLD. ⁴⁵ Taking together, the KCE's effect on intrahepatic lipid accumulation could be attributed to these abundant DCL such as Gomisin D, E, H, J, and Kadsulignan L, K et al according to their similar structure potentially as the pharmacophore. However, the definite contributive chemical constituents for the KCE bioactivity are still ambiguous though this study can offer an extract with preliminary ameliorative effect on lipid accumulation. As a part of our continual investigation, ⁴⁶ the available compounds after identification will be tested in the above in vitro NAFLD model to disclose the pharmacodynamic substance from KCE, to facilitate to develop the scientific quality standard for the KCE as the medicinal usage.

Conclusions

In this study, the effects of KCE on lipid accumulation were assessed, and chemical profiles of KCE were analyzed by UPLC/Q-TOF-MS. The KCE has ameliorative effect on lipid accumulation in the model in vitro. DCLs including Kadsulignan L and Gomisin J could be the supposed principal constituents. Given the existing data, the KCE might be prepared as arsenal to battle against NAFLD and further studies need to be warranted.