Diterpenoid Alkaloids Isolated from Delphinium brunonianum and Their Inhibitory Effects on Hepatocytes Lipid Accumulation

This research aimed to excavate compounds with activity reducing hepatocytes lipid accumulation from Delphinium brunonianum. Four novel diterpenoid alkaloids, brunodelphinine B–E, were isolated from D. brunonianum together with eleven known diterpenoid alkaloids through a phytochemical investigation. Their structures were elucidated by comprehensive spectroscopy methods including HR-ESI-MS, NMR, IR, UV, CD, and single-crystal X-ray diffraction analysis. The inhibitory effects of a total of 15 diterpenoid alkaloids on hepatocytes lipid accumulation were evaluated using 0.5 mM FFA (oleate/palmitate 2:1 ratio) to induce buffalo rat liver (BRL) cells by measuring the levels of triglyceride (TG), total cholesterol (TC), alanine transaminase (ALT), aspartate transaminase (AST), and the staining of oil red O. The results show that five diterpenoid alkaloids—brunodelphinine E (4), delbruline (5), lycoctonine (7), delbrunine (8), and sharwuphinine A (12)—exhibited significant inhibitory effects on lipid accumulation in a dose-dependent manner and without cytotoxicity. Among them, sharwuphinine A (12) displayed the strongest inhibition of hepatocytes lipid accumulation in vitro. Our research increased the understanding on the chemical composition of D. brunonianum and provided experimental and theoretical evidence for the active ingredients screened from this herbal medicine in the treatment of the diseases related to lipid accumulation, such as non-alcoholic fatty liver disease and hyperlipidemia.


Introduction
The metabolic diseases caused by lipid accumulation, such as non-alcoholic fatty liver disease (NAFLD) and obesity, affect an increasing number of people. The global prevalence of NAFLD continues to increase and is now estimated as being up to 25% [1,2]. Despite extensive research on NAFLD, there are still no FDA-approved effective drugs as of now. Although many lipid-lowering drugs (statins), insulin sensitizers (metformin), and antioxidants (vitamin E) have been shown to improve NAFLD, there are still several adverse effects [3]. Therefore, it is of great significance to explore drugs with safety and effective lipid accumulation reducing activity from plants for treatment of the metabolic diseases.
Delphinium brunonianum Royle, belonging to genus Delphinium (Ranunculaceae family), is a perennial herbaceous plant and mainly distributed at an altitude of 4000-6000 m from Tibet Autonomous Region of China to Nepal and Afghanistan [4]. The dried aerial parts of D. brunonianum, as a traditional Chinese aboriginal medicine named "Qiagaobei" in Tibetan, have been widely used for a long time in the treatment of jaundice, influenza, skin itching, and snake bites due to its properties of cooling blood, clearing heat, and detoxification. Traditional Chinese medicines with the above properties are more likely to have anti-inflammatory, antiviral, and antibacterial activity.
Phytochemical research has presented that alkaloids, flavonoids, and sterols were the predominant composition of D. brunonianum. Among them, diterpenoid alkaloids are the characteristic constituents having complex structural features, which mainly include lycoctonine-type C19 and atisine-type C20 diterpenoid alkaloids. The two types of diterpenoid alkaloids are different in terms of skeletal structure. The former, as the main type of diterpenoid alkaloid in D. brunonianum, is different from the latter in the number of carbon atoms. Additionally, the latter have an exocyclic double bond on the 16-carbon. Pharmacological studies have proven that diterpenoid alkaloids have many bioactivities with antihypertensive, anti-bacterial, anti-epileptic, diuretic, and anti-inflammatory effects, etc. [5][6][7]. Research showed that several diterpenoid alkaloids may exert anti-inflammatory effects through NF-κB/MAPK and Nrf2/HO-1 [8].
The extract of D. brunonianum possesses efficacy in the regulation of metabolic disorders in high fructose-induced rats [9]. Moreover, our previous research exhibited the D. brunonianum extract, which is enriched with diterpenoid alkaloids, could alleviate NAFLD by reducing the accumulation of lipids in the liver. So far, fourteen lycoctoninetype diterpenoid alkaloids, three atisine-type diterpenoid, and eleven amide alkaloids have been isolated from D. brunonianum [6,[10][11][12]. Therefore, information on the chemical composition of diterpenoid alkaloids in D. brunonianum is limited.
To further discover structurally and biologically intriguing active diterpenoid alkaloids in D. brunonianum, a phytochemical study of D. brunonianum was performed as part of our continuous work, which resulted in the isolation of fifteen diterpenoid alkaloids (as shown in Figure 1), including four novel diterpenoid alkaloids (named brunodelphinine B-E) and eleven known ones. Furthermore, these isolated compounds were evaluated for their inhibitory effects on lipid accumulation in free fatty acid (FFA)-induced BRL cells, aiming to screen a series of entities with potential for development as drugs to treat NAFLD.
The relative configuration of compound 1 was deduced by NOESY spectrum. The Compound 3 was isolated as a yellow amorphous powder and showed a positive reaction with Dragendorff's reagent. Its molecular formula was determined as C23H35NO8 by HR-ESI-MS at m/z 454.2439 [M + H] + (calculated for C23H36NO8, 454.2446), corresponding to 7 degrees of unsaturation. The IR spectrum showed absorptions for hydroxyl (3447 cm −1 ) groups. The 1 H and 13 C NMR spectroscopic data (Table 1) of compound 3 were essentially identical with those of compound 2, suggesting that 3 also belonged to lycoctonine-type C19-diterpenoid alkaloid. The only difference between 3 and 2 was the presence of a methylenedioxy group (δH 5.18 (2H, d, J = 4.9 Hz), δC 95.0) in the former, instead of the carbonyl group (δC 209.3) in the latter. This was also supported by 2D-NMR data; the location of one carbonyl group could be assigned at C-7 due to the HMBC correlation of H-6 with C-7 (δC 209.3) and H-15 with C-7 (δC 209.3). By comparison with compound 2, it was found that compound 3 is also a C19-diterpenoid alkaloids containing deazoethyl group. Finally, 3 was identified as 7,17-seco, 7-keto, 8-hydroxy-brunodelphinine C (Figure 2), named brunodelphinine D.
Careful comparison of the 13 C NMR spectral data with the known compound sharwuphinine A (12) revealed that compound 2 might be a deazoethyl compound that contained a nitrone structure and a nitrogen-oxygen double bond with C-19 (δ H 6.78 (1H, d, J = 1.6 Hz), δ C 138.0). Our results confirmed that compound 2 is a lycoctonine-type C19-diterpenoid alkaloid. The  Compound 3 was isolated as a yellow amorphous powder and showed a positive reaction with Dragendorff's reagent. Its molecular formula was determined as C 23  were essentially identical with those of compound 2, suggesting that 3 also belonged to lycoctonine-type C19-diterpenoid alkaloid. The only difference between 3 and 2 was the presence of a methylenedioxy group (δ H 5.18 (2H, d, J = 4.9 Hz), δ C 95.0) in the former, instead of the carbonyl group (δ C 209.3) in the latter. This was also supported by 2D-NMR data; the location of one carbonyl group could be assigned at C-7 due to the HMBC correlation of H-6 with C-7 (δ C 209.3) and H-15 with C-7 (δ C 209.3). By comparison with compound 2, it was found that compound 3 is also a C19-diterpenoid alkaloids containing deazoethyl group. Finally, 3 was identified as 7,17-seco, 7-keto, 8-hydroxy-brunodelphinine C ( Figure 2), named brunodelphinine D.

Cell Viability of Fifteen Isolated Compound and Positive Drug in BRL Cells
The cell viability (%) of the isolated diterpenoid alkaloids in the concentration range of 1-500 µM and the time-effectiveness of them for 24 h and 48 h were evaluated using CCK-8 assay ( Figure 4). The results show that the cell viability of isolated compounds 1-15 and positive (ATC) decreased after 48 h treatment and was generally lower than that of cells treated for 24 h at the same concentration. The IC 50 values of 15 compounds and positive drug were calculated by SPSS 23.0 software. The results show that the IC 50 values of compounds (1, 2, 8, and 11) were in the range of 300-500 µM, while others were more than 500 µM (Table 2). Therefore, through the entire experiments, the maximum concentration of tested diterpenoid alkaloids was limited to 10 µM, a concentration about one-tenth to one-fiftieth of IC 50 of them. In addition, optimum dosage of modeling agent (FFA) was also selected as the concentration of 0.5 mM through CCK-8 assay ( Figure S77).   We selected FFA-induced BRL cells (oleate and palmitate ratio was 2:1) to establish a cellular NAFLD model, which is a widely used method by researchers for studying diseases related to hepatocytes lipid accumulation [18]. The TG secretion was measured to preliminarily evaluate the inhibitory effect on lipid accumulation of 15 isolated alkaloid compounds. The results show that TG content in the model group was 3 times that of the normal control group, indicating that BRL cell stimulated by 0.5 mM FFA for 24 h could successfully establish a hepatocytes model of lipid accumulation. Compounds 1-15 were tested at a concentration of 10 μM in FFA-induced BRL cells to determine their inhibitory effects on hepatocytes TG secretion ( Figure 5). Atorvastatin calcium (ATC) is a statin cholesterol-lowering drug and widely used in the clinical treatment of hyperlipidemia and NAFLD, etc. [19,20]. Therefore, we used atorvastatin calcium (ATC, 10 μM) as a positive control drug. Compared with the model group, compounds 1, 2, 4-8, 10-12, 14, and 15  We selected FFA-induced BRL cells (oleate and palmitate ratio was 2:1) to establish a cellular NAFLD model, which is a widely used method by researchers for studying diseases related to hepatocytes lipid accumulation [18]. The TG secretion was measured to preliminarily evaluate the inhibitory effect on lipid accumulation of 15 isolated alkaloid compounds. The results show that TG content in the model group was 3 times that of the normal control group, indicating that BRL cell stimulated by 0.5 mM FFA for 24 h could successfully establish a hepatocytes model of lipid accumulation. Compounds 1-15 were tested at a concentration of 10 µM in FFA-induced BRL cells to determine their inhibitory effects on hepatocytes TG secretion ( Figure 5). Atorvastatin calcium (ATC) is a statin cholesterol-lowering drug and widely used in the clinical treatment of hyperlipidemia and NAFLD, etc. [19,20]. Therefore, we used atorvastatin calcium (ATC, 10 µM) as a positive control drug. Compared with the model group, compounds 1, 2, 4-8, 10-12, 14, and 15 had the effect of inhibiting the lipid accumulation (TG secretion) in BRL cells stimulated by FFA (p < 0.05), and compound 12 showed the strongest inhibition effect on the level of TG (p < 0.01).
The activity of reducing lipid accumulation (TG secretion) of compounds 4, 5, 7, 8, and 12 was better than that of the positive control drug (10 µM). Therefore, we selected these five compounds for the further experiments. In addition to TG level, TC level and the liver injury indicators (ALT and AST) as typical indicators of NAFLD metabolic phenotype are also necessary for the five isolated compounds to be measured. According to the results, compounds 4, 5, 7, 8, and 12 ameliorated the indicators of TG, TC, ALT, and AST levels to varying degrees in FFA-induced BRL cells (Figure 6), showing that these five compounds have the potential to be developed as drugs for treatment of NAFLD.

Oil Red O Staining of Lipid Droplets and Quantitative Analysis in FFA-Induced BRL Cells
Combined with the oil red O staining and quantitative analysis assay, the effect of reducing lipid accumulation of compounds 4, 5, 7, 8, and 12 was performed in FFA-induced BRL cells. Oil red O staining showed that the above five compounds can significantly reduce lipid accumulation in FFA-induced BRL cells compared to the model group ( Figure 7A,B). Quantitative analysis of lipid accumulation was also analyzed by ImageJ significant at TG, TC, ALT, and ALT levels ( ∆ p < 0.05), while the medium (5 µM) and high (10 µM) doses of compound 4 were statistically significant at TG, TC, and AST levels ( ∆ p < 0.05). The low (1 µM) and high (10 µM) doses of compound 5 and 12 were statistically significant at TG, ALT, and ALT levels ( ∆∆ p < 0.01). The low (1 µM) and high (10 µM) doses of compound 7 were statistically significant at TG and AST levels ( ∆ p < 0.05). In pairwise comparison between different dose groups, compound 5 had a significant difference in ALT level ( ∆ p < 0.05), as did compound 8 in AST level ( ∆ p < 0.05).

Oil Red O Staining of Lipid Droplets and Quantitative Analysis in FFA-Induced BRL Cells
Combined with the oil red O staining and quantitative analysis assay, the effect of reducing lipid accumulation of compounds 4, 5, 7, 8, and 12 was performed in FFA-induced BRL cells. Oil red O staining showed that the above five compounds can significantly reduce lipid accumulation in FFA-induced BRL cells compared to the model group ( Figure 7A,B). Quantitative analysis of lipid accumulation was also analyzed by ImageJ 1.   According to the network pharmacology method, a total of 81 targets were screened, and these targets were considered as potential targets of five compounds for the treatment of NAFLD. At the same time, Cytoscape 3.8.0 software was used to construct a com-

Prediction of Underlying Mechanism for Five Isolated Compounds in the Treatment of NAFLD
According to the network pharmacology method, a total of 81 targets were screened, and these targets were considered as potential targets of five compounds for the treatment of NAFLD. At the same time, Cytoscape 3.8.0 software was used to construct a compoundstargets network diagram ( Figure 8A). The protein interaction (PPI) network diagram ( Figure 8B) shows that PPARG, MTOR, ACE, PPARA, NOS3, etc., play very important roles in protein interaction, indicating that these targets may be potential therapeutic targets. It is worth noting that PPARG has the strongest protein interaction among the screened targets, suggesting that the inhibition of lipid synthesis may be the underlying mechanism by which five compounds exert their effect on reducing lipid accumulation.
Molecules 2022, 27, x FOR PEER REVIEW 12 of 18 gamma (PPARγ, also known PPARG) and sterol regulatory element-binding protein 1C (SREBP1C), which in turn lead to cellular lipid accumulation. Therefore, PPARγ and SREBP1C as the lipogenic regulators play an important role in the occurrence and development of NAFLD [21][22][23]. Compared with the other 14 compounds, compound 12 has the strongest inhibitory effect on TG level (p < 0.01). Therefore, we selected compound 12 as a representative for subsequent potential mechanism studies. Based on network pharmacology predicted results and studies in the literature, the relative mRNA levels of PPARγ and SREBP1C in FFA-induced BRL cells were analyzed by real-time PCR system. Our results show that compound 12 decreased relative mRNA levels of PPARγ and SREBP1C in a dose-dependent manner ( Figure 8C). It indicated that compound 12, as a representative of diterpenoid alkaloids isolated from D. brunonianum, may inhibit fat accumulation by regulating the expression of lipid synthesis transcription factors, such as PPARγ and SREBP1C.

Effects of Compound 12 on Fatty Acid Synthesis in FFA Mixture-Induced BRL Cells
Studies have shown that many enzymes involved in lipid synthesis are upregulated by lipogenic transcription factors, such as peroxisome proliferator-activated receptor gamma (PPARγ, also known PPARG) and sterol regulatory element-binding protein 1C (SREBP1C), which in turn lead to cellular lipid accumulation. Therefore, PPARγ and SREBP1C as the lipogenic regulators play an important role in the occurrence and development of NAFLD [21][22][23]. Compared with the other 14 compounds, compound 12 has the strongest inhibitory effect on TG level (p < 0.01). Therefore, we selected compound 12 as a representative for subsequent potential mechanism studies. Based on network pharmacology predicted results and studies in the literature, the relative mRNA levels of PPARγ and SREBP1C in FFA-induced BRL cells were analyzed by real-time PCR system. Our results show that compound 12 decreased relative mRNA levels of PPARγ and SREBP1C in a dose-dependent manner ( Figure 8C). It indicated that compound 12, as a representative of diterpenoid alkaloids isolated from D. brunonianum, may inhibit fat accumulation by regulating the expression of lipid synthesis transcription factors, such as PPARγ and SREBP1C.

Plant Material
The aerial parts of D. brunonianum Royle were collected from Linzhi (Tibet Autonomous Region, China) and were identified by Professor Ga Wu (Tibetan Traditional Medical College, Lasa, China). The voucher specimen was deposited at the Department of College of Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China.

Extraction and Isolation
Dried and powdered aerial parts of D. Brunonianum Royle (20 kg) were repeatedly extracted with methanol (MeOH) at room temperature for five times, and each extraction time was for 24 h. The extracted solution was evaporated in vacuum to afford the crude methanol extraction. The concentrated extraction was suspended in water and partitioned in different solvents to obtain petroleum extract (650 g), chloroform extract (240 g), EtOAc extract (42 g), and n-butanol extract (400 g), respectively.
The crude chloroform fraction was subjected to CC with silica gel (100-200 mesh) and eluted with cyclohexane (CYH)/ethyl acetate (EtOAc) gradient system (5:0 to 1:0) and EtOAc/MeOH gradient system (4:1 to 1:1); the gradient system gave fractions 1-9. Fr.5 was separated to Sephadex LH-20 CC (CHCl 3 /MeOH, 1:1) to afford subfractions (Fr.5-1 and Fr.5-2). Fr.5-2 was subjected to normal phase silica gel column with CYH/EtOAc gradient system (3:1 to 1:3), ODS middle pressure column eluted with MeOH/H 2 O (30-80%) to obtain compounds 5 (10 g), 6 (22 mg), 7 (1034 mg), 8 (2800 mg), 9 (254 mg). Fr. 6 was subjected to Sephadex LH-20 CC (CHCl 3 /MeOH, 1:1) to afford subfractions (Fr.6-1 and Fr.6-2). Fr.6-1 was purified by ODS middle pressure column eluted with MeOH/H 2 O (60%) to obtain 10 (93 mg), and Fr.6-1 was subjected to normal phase silica gel column CYH/EtOAc (1:5) to 3.6. Effects of Fifteen Isolated Compounds on the Inhibition of Lipid Accumulation in FFA-Induced BRL Cells 3.6.1. TG, TC, ALT, and AST Quantification of Fifteen Isolated Compounds in FFA-Induced BRL Cells BRL cells were grown in 24-well plates until 70-80% confluence. Subsequently, 0.5 mM FFA (oleate and palmitate in a final ratio of 2:1) was applied for 24 h to establish an in vitro model of hepatocytes lipid accumulation in the model group, treated with 0.5 mM FFA combination with 1, 5, or 10 µM isolated compounds for 24 h in alkaloids groups. A portion of 1% bovine serum albumin (BSA) was added in BRL cells of the control group. Then, the medium of each group was collected for determination of ALT and AST levels by commercial kits. After removing the medium, 200 µL of 0.25% trypsin was added to digest the cells in 37 • C for 2 min. Then, 500 µL of DMEM that contained 10% fetal bovine serum (FBS) was added to stop digesting. Cells suspension was centrifuged at 800 r/min for 3 min. After adding 200 µL of ethanol, cells were collected and crushed by ultrasonic cell disruptor at 4 • C. Part of the cell fragmentation solution was used for the determination of cell protein content, the other part was used to determine TG and TC levels by commercial kits according to the manufacturer's instruction [25].

Oil Red O Staining Assay
BRL cells were plated in 96-well plates until reaching 70-80% confluence and treated with 0.5 mM FFA mixture and the tested compounds for 24 h. Controls were incubated for the same period in complete medium.
After 24 h of incubation, the cells were fixed with 5% formalin solution for 30 min and then incubated with oil red O working solution for 15 min at room temperature. After that, the cells were washed once with 60% isopropanol and three times with water. The nuclei were stained with hematoxylin solution for 2 min at room temperature. Then, the operation of oil red O staining was referred to the previous research [26]. Finally, the red oil droplets were observed using an optical microscope. The ChemDraw software (14.0) was used to draw the structural formulas of the five compounds (4, 5, 7, 8, and 12), and their respective SMILE formulas were saved. The pharmmapper database (http://www.lilab-ecust.cn/pharmmapper/ (accessed on 14 February 2022)) and swisstarget database (http://swisstargetprediction.ch/ (accessed on 14 February 2022)) were used to predict the potential targets of five compounds. All targets were aggregated, and duplicate targets were removed to obtain potential targets for five compounds. In OMIM (https://www.omim.org/ (accessed on 14 February 2022)), TTD (http://db.idrblab.net/ttd/ (accessed on 15 February 2022)), GeneCards (https://www. genecards.org/ (accessed on 15 February 2022)), DisGeNET (https://www.disgenet.org/ dbinfo (accessed on 15 February 2022)) and DrugBank (https://go.drugbank.com/drugs (accessed on 15 February 2022)) databases, with "Non-alcoholic liver disease" as the search term, NAFLD-related targets were searched. The predicted targets of the five compounds were intersected with the related targets of NAFLD, and the intersection genes were considered as possible targets for the treatment of NAFLD by the five compounds.

Target Protein Interaction (PPI) Core Network Construction
The potential therapeutic targets were imported to the String (https://string-db.org/ (accessed on 16 February 2022)) database, and the multiple proteins tool was used to define the species as "Homo sapiens" to obtain protein interaction relationships. The results were imported into Cytoscape 3.8.0 software in CSV format for visual analysis, and a protein interaction network diagram was constructed.

Effects of Compound 12 on Fatty Acid Synthesis in FFA Mixture-Induced BRL Cells
Total RNA was extracted from BRL cells with the use of the Trizol reagent. The expression levels of PPARγ and SREBP1C mRNA in FFA-induced BRL cells were analyzed by Applied Biosystems StepOne Real-Time PCR (RT-PCR) System. In addition, the PCR amplification was performed for 40 repetitive thermal cycles with SYBR green (95 • C for 15 s, 60 • C for 15 s, and 72 • C for 32 s). Data were normalized by the amount of β-actin mRNA. Primers are listed in Table 3.

Statistical Analysis
All data are expressed as mean ± SEM of six independent experiments. SPSS 23.0 software was used for statistical analysis by one-way ANOVA method. A p value less than 0.05 was considered statistically significant.

Conclusions
Natural products extracted from medical plants are a rich source of biologically active substances, which play an important and irreplaceable role in the drug discovery field [27][28][29]. In our present research, fifteen compounds were isolated from the CHCl 3 extraction of D. brunonianum, including four undescribed compounds (1-4) and eleven known compounds (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Among the known compounds, uraphine (10) was isolated from genus Delphinium for the first time, and delpheline (6), anthranoyllycoctonine (11), sharwuphinine A (12), shawurensine (14), and delavaine B (15) were first discovered in D. brunonianum. The inhibitory effects of isolated compounds (1-15) on hepatocytes lipid accumulation were also evaluated in FFA-induced BRL cells. Our results indicate that five selected compounds (4, 5, 7, 8, and 12) showed strong inhibitory effects on hepatocytes lipid accumulation in a dose-dependent manner (1, 5, and 10 µM). Compound 12, as a representative of diterpenoid alkaloids isolated from D. brunonianum, may inhibit fat accumulation by regulating the expression of lipid synthesis transcription factors PPARγ and SREBP1C. In summary, the compounds isolated in this study could serve as a potential for treatment of diseases caused by lipid accumulation, such as NAFLD and hyperlipidemia diseases. In addition, this study enriches the chemical constituents and activity of D. brunonianum and provides a reference for further research on the potential mechanism of diterpenoid alkaloids in improving hepatocytes lipid accumulation.

Data Availability Statement:
The data presented in this study are available in this article.