Original Papers
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Authors
Ping Jiao 1, Julie Tseng-Crank 1, Brandon Corneliusen 1, Mesfin Yimam 1, Mandee Hodges 1, Mei Hong 1, Catherine Maurseth 1, Misun Oh 2, Hyunjin Kim 2, Min Chu 1, 2, Qi Jia 1, 2
Affiliations
1 2
Key words " Atractylodes lancea l " Asteraceae l " pancreatic lipase l " lipase inhibitor l " atractylodin l " polyacetylenes l " DIO mice l
Unigen USA, Seattle, WA, USA Unigen Korea, Cheonam Si, Chungnam, South Korea
Abstract !
The ethanol extract of Atractylodes lancea rhizome displayed significant lipase inhibition with an IC50 value of 9.06 µg/mL in a human pancreatic lipase assay from high-throughput screening. Bioassay-guided isolation led to the identification of one new polyacetylene, syn-(5E,11E)-3-acetoxy4-O-(3-methylbutanoyl)-1,5,11-tridecatriene7,9-diyne-3,4-diol (7), along with six known compounds (1–6). The structure of compound 7 was determined based on the analysis of NMR and MS data. Among these seven lipase inhibitors, the major compound atractylodin (1) showed the highest lipase inhibitory activity (IC50 = 39.12 µM). The antiobesity effect of the ethanol extract of Atractylodes lancea rhizome was evaluated in a high-fat diet-induced obesity mice model at daily dosages of 250 mg/kg and 500 mg/
Introduction ! received revised accepted
Dec. 17, 2013 March 6, 2014 March 10, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368354 Published online March 31, 2014 Planta Med 2014; 80: 577–582 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Ping Jiao Unigen USA 3005 1st Avenue Seattle, WA 98121 USA Phone: + 1 36 04 86 82 00 Fax: + 1 20 64 41 55 17 [email protected]
Obesity is becoming one of the most serious global health problems, with prevalence at an increasing rate both in adults and children. Obesity can increase the risk factors of many diseases such as diabetes mellitus type 2, high blood pressure, high blood cholesterol, high triglyceride levels, and some forms of cancer [1–3]. Despite a significant amount of research done in this field, obesity still remains a tough medical challenge. Therefore, efforts for the development of antiobesity drugs and therapeutic interventions have been continued intensively. Clearly defined core lipid metabolic pathways involving well-characterized enzymes provide potential opportunities and rich sources of targets for therapeutic interventions and obesity treatments [4, 5]. Pancreatic lipase has been validated as one of the important targets in the lipid metabolic process [6].
kg body weight for 4 weeks, and treatment with this extract demonstrated a moderate efficacy at the 500 mg/kg dose level.
Abbreviations !
AL‑EE: DGGMR: DIO: HFD: hPL: OE: SAR:
ethanol extract of Atractylodes lancea rhizome 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin)-ester diet-induced obesity high-fat diet human pancreatic lipase organic extract structure-activity relationship
Supporting information available online at http://www.thieme-connect.de/products
Pancreatic lipase is considered the primary enzyme involved in the hydrolysis of fat molecules in the digestive system and the release of monoglycerides and free fatty acids. By targeting pancreatic lipase, the approach is to specifically inhibit triglyceride digestion, thus reducing the dietary fat absorption and calorie intake from fat. Orlistat, a competitive pancreatic lipase inhibitor, is marketed as a long-term obesity treatment drug. It is a derivative of a potent natural pancreatic lipase inhibitor, lipstatin, isolated from the bacterium Streptomcyces toxytricini. Orlistat decreases the dietary fat absorption through the gastrointestinal system by inhibiting lipase activities, predominantly pancreatic lipase, to prevent weight gain and to induce weight loss [7, 8]. The most reported side effect of Orlistat is oily spotting/loose stool (sometimes referred to as treatment effects). The success of Orlistat has driven the development of lipase inhibitors from natural sources [9–12]. Pancreatic lipase inhibitors reported from
Jiao P et al. Lipase Inhibition and …
Planta Med 2014; 80: 577–582
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Lipase Inhibition and Antiobesity Effect of Atractylodes lancea
Original Papers
Fig. 1 Chemical structures of compounds 1–6 from the rhizome of Atractylodes lancea.
plants are mainly classified into three class groups, saponins, polyphenols, and terpenoids [6]. There is always a need and requirements for novel natural lipase inhibitors with better efficacy and less side effects. In order to discover natural lipase inhibitors, a high-throughput hPL inhibition assay was developed by utilizing recombinant hPL. A total of 8563 plant extracts from our in-house phytologic library collection were screened against hPL. The OE of the Chinese traditional herbal medicine Atractylodes lancea (Thunb.) DC. (Asteraceae) showed a relatively high lipase inhibition in this screening. Six known lipase inhibitors and one new compound were isolated and identified from this extract via bioassayguided purification. AL‑EE was prepared and evaluated in a high-fat DIO animal model for the prevention of weight gain effects. To the best of our knowledge, this is the first report of pancreatic lipase inhibitory activity from the A. lancea rhizome extract.
Results and Discussion !
A. lancea OE showed 84.77 % inhibition on hPL at the 120 µg/mL level in the primary screening. Bioassay-guided isolation led to the discovery of seven polyacetylenic compounds with pancreatic lipase inhibitory activity. Among all seven compounds, atractylodin (1) was the most active lipase inhibitor and compound 7 was identified as a novel polyacetylenic compound. A. lancea rhizome is one of the commonly used Chinese traditional herbs, also known as cangzhu in Chinese. Cang refers to the dark color of the outer skin layer of the rhizome. Zhu was recorded in Shennong Bencao Jing, one of the earliest Chinese dispensatory, with the description as being boiled with water, nourishing, free from hunger, and extending oneʼs life span [13]. It was included in many ancient formulas and is still being used in a lot of decoctions today. The essential oil is claimed to be the major active ingredient for this herbal material. The main function is for the treatment of indigestion and stomach disorders which occurs by strengthening the spleen, supplementing Qi, and removing dampness [13, 14]. A. lancea rhizome is rich in volatile oil with the presence of atractylodin as the main characteristic compound. During our primary screening, lipase activity was found in A. lancea OE extract with 84.77 % inhibition at a concentration of 120 µg/mL. To clarify the active components in the organic ex-
Jiao P et al. Lipase Inhibition and … Planta Med 2014; 80: 577–582
tract, bioassay-guided isolation was carried out, leading to the isolation of a mixture of atractylodin and β-eudesmol in a ratio of 2 : 1 based on NMR and LC‑MS analyses, with a high lipase inhibition (IC50 = 11.35 µg/mL). Further purification confirmed that atractylodin (1), the major compound in the crude extract, was the active compound responsible for the lipase inhibition. To further study the chemical profile of A. lancea, an additional five minor known polyacetylenic compounds and one related new compound were isolated and identified from AL‑EE. The structures of the five known compounds, including 1-(2-furanyl)-7-nonene-3,5-diyne-1,2-diacetate (2), (3Z,5E,11E)-3,5,11tridecatriene-7,9-diyne-1-O-acetate (3), (3Z,5E,11E)-3,5,11-tridecatriene-7,9-diyne-1,2-diacetate (4), 4,6,12-tetradecatriene8,10-diyne-1,3-diacetate (5), and (5E,11E)-1,5,11-tridecatriene7,9-diyne-3,4-diacetate (6), were established based on the analysis and comparison of NMR and MS data with literature reports " Fig. 1). [15–19] (l New compound 7 was recognized as a new member of the " Fig. 2). polyacetylene class by analysis of UV and NMR data (l Compound 7 showed maximal UV absorptions at wavelengths of 311, 291, 276, 260, and 231 nm, indicating the presence of the same ene-diyne-ene chromophore as observed in compound 6. The molecular weight 328 was determined by GC‑MS. The molecular formula C20H24O4 (9 degrees of unsaturation) was assigned for 7 based on the analysis of NMR and HR‑ESI‑MS data. The 1H NMR spectrum showed a terminal methyl group at 1.83 ppm coupling with two trans-configured olefinic protons at 6.34 and 5.58 ppm, which is consistent with all the other six acetylenes. Two terminal vinylic protons at 5.34 and 5.31 ppm were coupled with another olefinic proton at 5.72 ppm with coupling constants of 17.36 Hz and 10.76 Hz, respectively. The geometry of the C5–C6 double bond at 5.82 and 6.13 ppm was assigned as E on the basis of the coupling constant of 15.89 Hz. 1H-1H COSY spectrum together with chemical shift data revealed a spin sys" Fig. 3. Only one singlet methyl peak tem C1–C6 as shown in l was observed at 2.09 ppm, indicating the presence of one acetoxy group. The HMBC correlation of H-3 to the carboxyl carbon at 169.61 ppm enabled the connection of the acetate to C-3. One isovalerate unit was identified based on the analysis of 13C and 1 H, and COSY NMR spectra. Similarly, HMBC correlations of H-4 to the carboxyl carbon C-1′ allowed the assignment of this isovalerate unit attached to C4. Isovalerate polyacetylenes were also reported from A. chinensis and A. macrocephala [20, 21].
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Fig. 2 Chemical structure of isolated compound 7.
Fig. 3
Key HMBC and COSY correlations of compound 7.
Table 1 Pancreatic lipase inhibitory activity of A. lancea extracts and compounds.
Orlistat AL‑EE OE1-F12 Atractylodin (1) Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7
IC50 (µg/mL)
IC50 (µM)
0.035 9.06 11.35 7.12 14.2 9.1 22.3 16.7 23.9 15.6
0.07 – – 39.12 47.33 39.91 77.97 55.67 83.57 47.56
In addition, atractyldoin (1) and its analogues could serve as a chemical platform for synthetic chemists to conduct SAR studies in order to search for desirable antiobesity drug candidates.
Materials and Methods !
Plant material The dried rhizome of A. lancea was collected from Xiangyang, Hubei, China. The plant material was identified by professor ShouYuen Zhao from Si-Chuan Chinese Traditional Medicine Research Institutes. A voucher of specimen A. lancea (P00541) was deposited at the plant library of Unigen, Seattle, WA, USA. The recollected A. lancea rhizome was obtained from an herbal store in Seattle, characterized, and confirmed in comparison with the original voucher specimen.
Apparatus and chemicals used for the phytochemical study The UV and MS data were collected from an LC‑MS/PDA system, MS M-8000 (Hitachi) and PDA L-4500A (Hitachi), and GC‑MS instrument 6890 N (Agilent Technologies). HR‑ESI‑MS was recorded on an LTQ-Orbitrap high-resolution mass spectrometer (Thermo Scientific). The proton and carbon NMR spectra were recorded on a VNMRS-500 spectrometer (Agilent Technologies). The extracts were produced by Dionex accelerated solvent extractor ASE 300 (Thermo Scientific). Isolation was carried on on a Hitachi high-throughput purification system HTP D7000 (Hita-
Jiao P et al. Lipase Inhibition and …
Planta Med 2014; 80: 577–582
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Although the absolute configurations of C-3 and C-4 of compound 7 could not be identified, a syn-configuration was assigned based on a relatively larger 3J coupling constant of 6.3 Hz. According to a literature report, the coupling constants of the vicinal protons in this type of compound were observed in the range of 5–8 Hz in the syn (or threo) form, while smaller coupling constants around 4 Hz were observed for the anti (or erythro) configuration [17]. Thus, the structure of 7 was determined as syn-(5E,11E)-3-acetoxy-4-O-(3-methylbutanoyl)-1,5,11-trideca" Fig. 2). triene-7,9-diyne-3,4-diol (l The pancreatic lipase inhibitory effects were evaluated by mea" Table 1). suring the IC50 values for individual compounds (l Among those seven compounds, atractylodin (1) substantially in" Fig. 4). hibited pancreatic lipase with an IC50 value of 39.12 µM (l The content of atractylodin of the ethanol extract was quantified " Fig. 5. as 11.16 % by weight using HPLC analysis as shown in l Due to its high content in the ethanol extract, atractylodin definitely contributed to the lipase inhibitory activity of the material. Compounds 2-6 showed similar or relatively weaker lipase inhibition compared with atractylodin. The inhibitory potency of the new compound 7 was determined with an IC50 value at 47.56 µM. Enyne compounds from marine sponges and algae have also been reported as lipase inhibitors [22, 23]. Although it is difficult to make a conclusion on the SAR based on the current activity of the polyacetylenic compounds isolated from A. lancea, this enediyne-ene skeleton could be a good template for lipase inhibitor drug design. The antiobesity effect of AL‑EE with an IC50 value at 9.06 µg/mL " Fig. 4) was evaluated in a high-fat diet-induced obesity mice (l model at daily dosages of 250 mg/kg and 500 mg/kg body weight. A progressive increase in body weight gain was observed when the mice were provided with a 60% kcal HFD for 4 weeks. The known pancreatic lipase inhibitor Orlistat-treated group showed statistically significant reductions in body weight gain as of day 7 during the treatment in compared to the vehicle-treated HFD " Fig. 6). Similarly, a trend in reduction in body weight group (l gain was also observed for mice treated with AL‑EE, with statistical significance on days 21 and 24 for the 500 mg/kg dosing group. When compared with the vehicle-treated HFD mice, these tendencies in reducing body weight gain for mice treated with AL‑EE at a dose of 500 mg/kg were found to have p values of 0.06, 0.08, and 0.09 on days 10, 14, and 17 after oral treatment, respectively. Both AL‑EE treatment groups at 500 mg/kg and 250 mg/kg showed the tendency to prevent the HFD-induced increases in the body weight of mice. We have successfully developed a reliable and robust highthroughput hPL inhibition assay. Our results reveal for the first time that AL‑EE showed hPL inhibitory activity, with atractylodin (1) as the major active compound. AL‑EE also prevented body weight gain in a high-fat diet mice model in comparison to the HFD controlled group. This extract could be considered a potential active ingredient for obesity and associated lipid dysfunction.
Original Papers
chi) and HPLC L-6200A (Hitachi). Orlistat (purity ≥ 98 %) was purchased from Sigma-Aldrich.
Plant extraction and isolation
Fig. 4 Human pancreatic lipase inhibition of the ethanol extract of A. lancea and compound 1 (atractylodin). Orlistat was used as a positive control.
Fig. 5 HPLC chromatogram of Atractylodes lancea ethanol extract at a wavelength of 320 nm. (Color figure available online only.)
Fig. 6 Effects of Atractylodes lancea rhizome ethanol extract on body weight in mice fed a high-fat diet for 28 days. Each point represents the mean ± SEM, and values significantly different from the controls are indicated by * p < 0.05.
Jiao P et al. Lipase Inhibition and … Planta Med 2014; 80: 577–582
A total of 59.4 grams of dried rhizome of A. lancea powder were loaded into two 100 mL stainless steel tubes and extracted twice with an organic solvent mixture (methylene chloride/methanol in a ratio of 1 : 1) under heat (80 °C) and pressure (1500 psi). The extract solution was automatically filtered and collected. The combined OE solution was evaporated with a rotary evaporator to give crude OE (14.1 g, 23.7 %). The A. lancea OE (13 g) was subjected to two silica gel columns (Biotage SNAP cartridge KP‑Sil 100 g) in parallel and eluted with a gradient solvent system of 25 % EtOAc in hexanes for 5 min, then increased to 100% EtOAc in 25 min, followed by introducing MeOH from 50% to 100 % MeOH in 20 min, and ended in washing with 100% MeOH for 16 min at a flow rate of 5 mL/min using a Hitachi high-throughput purification L7260 system. Eighty-eight fractions were collected for each column and combined to 10 fractions. Fraction 1 (600 mg) was further processed by a 10-µm Luna C18 column (30 × 250 mm, Phenomex) on an HPLC system L6200A eluted with 80% MeOH/H2O for 10 min, and increased to 100 % MeOH in 20 min at a flow rate of 10 mL/min with a UV wavelength of 250 nm to afford 12 fractions/compounds. Fraction 12 (tR = 35.12 min) was identified as a mixture of atractylodin (1) and β-eudesmol in a ratio of 2 : 1 (OE1-F12, 76.49 mg) based on NMR and LC‑MS analyses. Further bioassay-guided isolation and purification were carried out on the ethanol extract in order to get pure atractylodin and other analogues due to its complexity. The recollected A. lancea rhizome material (1.15 kg) was extracted with ethanol by reflux to give an ethanol extract (AL‑EE, 205.2 g, 17.8 %). The ethanol extract (44.1 g) was partitioned between hexanes and H2O. The hexane fraction (6.1 g) was isolated by a silica gel column (Biotage SNAP cartridge KP‑Sil 100 g) with a gradient elution by hexanes/ EtOAc/MeOH on the HPLC system L6200A at a flow rate of 5 mL/ min to yield 12 fractions. Atractylodin (1) (1.2 g) was obtained from fraction 6 (~ 500–600 mL). Fractions 7, 8, and 10 (~ 600– 700, 700–800, 1000–1100 mL) were further isolated by RP-HPLC to give compounds 2–6. Compound 7 (8.8 mg) was isolated by a 10-µm Luna C18 RP-HPLC column (30 × 250 mm, Phenomex) eluted with 70 % MeOH/H2O for 5 min, and increased to 100 % MeOH in 30 min, and 100 % MeOH for another 15 min at a flow rate of 10 mL/min, with a UV wavelength of 235 nm and tR at 31.67 min by injection of 114 mg of fraction 8. Syn-(5E,11E)-3-acetoxy-4-O-(3-methylbutanoyl)-1,5,11-tridecatriene-7,9-diyne-3,4-diol (7): colorless solid; UV (MeOH) λmax 311, 291, 276, 260, 231 nm; GC‑MS m/z 328 [M]+; HR‑ESI‑MS m/ z 351.15 836 [M + Na]+ (calcd. for C20H24O4Na: 351.15 668); 1H NMR (500 MHz, CDCl3) δ ppm 0.96 (6H, d, J = 6.60 Hz, H-4′, H-5′), 1.83 (3H, dd, J = 6.85, 1.50 Hz, H-13), 2.09 (3H, s, OCOCH3), 2.10 (1H, m, H-3′), 2.22 (2H, d, J = 6.85 Hz, H-2′), 5.31 (1H, d, J = 10.76 Hz, H-1a), 5.34 (1H, d, J = 17.36 Hz, H-1b), 5.38 (1H, t, J = 6.34 Hz, H-3), 5.47 (1H, t, J = 6.80 Hz, H-4), 5.58 (1H, d, J = 15.41 Hz, H-11), 5.72 (1H, ddd, J = 17.12, 10.64, 6.24 Hz, H-2), 5.82 (1H, d, J = 16.14 Hz, H-6), 6.13 (1H, dd, J = 15.89, 6.60 Hz, H5), 6.34 (1H, dq, J = 15.91, 6.85 Hz, H-12); 13C NMR (126 MHz, CDCl3) δ ppm 18.95 (CH3, C-13), 20.92 (CH3, OCOCH3), 22.35 (2CH3, C-4′, C-5′), 25.69 (CH, C-3′), 43.29 (CH2, C-2′), 71.91 (C, C9), 72.90 (CH, C-4), 74.03 (CH, C-3), 76.18 (C, C-8), 77.74 (C, C-7), 81.42 (C, C-10), 109.70 (CH, C-11), 113.40 (CH, C-6), 119.87 (CH2,
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C-1), 131.58 (CH, C-2), 139.09 (CH, C-5), 144.18 (CH, C-12), 169.61 (C, OCOCH3), 171.62 (C, C-1′)
Quantification of atractylodin (1) in the ethanol extract of Atractylodes lancea rhizome HPLC analysis of AL‑EE was performed to quantify atractylodin (1) content using a Hitachi HPLC D-7000 system with a 10-µm Luna RP‑C18 column (4.6 × 250 mm, Phenomenex) and diode array detector. The column was eluted at a condition of 70 % MeOH in H2O to 100% MeOH in 15 min, and 100 % MeOH for 8 min. This ethanol extract was shown to contain 11.16 % atractylodin " Fig. 5). (l
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treated animals received 0.5 % methylcellulose only. Regular rodent diet-fed (T2018, Harlan Teklad Diets) nonobese mice were included as a control in this study. The animals were maintained in a temperature controlled room (22.2 °C) on a 12-hour lightdark cycle. Body weight measurements were taken twice a week. All animal experiments were conducted according to institutional guidelines congruent with the guide for the care and use of laboratory animals. All procedures performed were in compliance with the Animal Welfare Act and US Department of Agriculture regulations (IACUC approval date: 01/06/2011; protocol No.: UAS-071205).
The coding region of hPL cDNA (Invitrogen) was cloned into the pcDNA3.1 plasmid vector for mammalian cell transfection, driven by the CMV promoter to create recombinant hPL. A native signal peptide sequence at the N-terminus ensured a proper secretion of hPL from the transfected 293 cells into the culture medium. The hPL protein produced excellent enzyme activity using a commercially available triglyceride lipase assay kit (Randox Laboratories), with DGGMR as the colorimetric substrate. A porcine co-lipase, which is necessary for pancreatic lipase activity, was supplied as part of the substrate and buffer formulation. A lipase inhibitory test was established utilizing recombinant hPL and a commercially available substrate (DGGMR, Nuclin Diagnostics). Orlistat (Sigma-Aldrich, purity ≥ 98 %) was used as a positive control in the assay at 2.4 µM. The reaction mixture containing the test sample was no greater than 3 % DMSO (20 µL) and the enzyme solution (100 µL, containing assay buffer at pH 8.4) was incubated for 10 minutes. The colorimetric substrate (50 µL) was added and the reactions were incubated at 37 °C for 30 min. Each sample was tested in triplicate and the 580 nm optical density (OD580) was read for each sample. IC50 values were determined using the same method with serial dilutions. All IC50 values were determined using SigmaPlot (Version 11.0) by a four parameter logistic nonlinear regression model with the sample absorbencies over the log concentrations of the test samples. The lipase inhibition activity was evaluated for compounds 1 (HPLC purity ≥ 98%), 2 (HPLC purity ≥ 95 %), 3 (HPLC purity ≥ 90%), 4 (HPLC purity ≥ 90%), 5 (HPLC purity ≥ 90 %), 6 (HPLC purity ≥ 80 %), and new compound 7 (HPLC purity ≥ 98 %) by calculating their IC50 values.
Animals and induction of obesity A HFD-induced obesity animal model was developed and used for the evaluation of potential antiobesity therapeutic agents. Male C57BL/6J mice were purchased from Jackson Laboratories at the age of 8 weeks with a body weight of 25.6–34.2 g. One week after an acclimation period, the animals were provided with high-fat (60% kcal) rodent pellets (D12492, Research Diets, Inc.) and water ad libitum for 8 weeks. Once the induction of obesity was confirmed (with a 40.9 % increase in body weight gain), the mice were randomized to treatment groups of Orlistat (Alli, GSK, 30 mg/kg), AL‑EE (250 mg/kg or 500 mg/kg), or vehicle with 9 animals in each group. The test compounds were suspended in 10 ml/kg vehicle and were repeatedly vortexed between gavages to maintain consistent dosing. The mice were orally gavaged with their respective treatment solution at 9 : 00 a. m. and 5 : 00 p. m. using a syringe and an 18-gauge ball-tipped feeding needle. No detectable sign of irritation was observed from drug or vehicle administration. The animals were treated with the respective therapeutic agent twice per day, orally, for 4 weeks. The vehicle-
The preclinical in vivo data were analyzed using SigmaPlot (version 11.0). Statistical significance between groups was calculated by means of single factor analysis of variance followed by a paired t-test. P values less or equal to 0.05 (p ≤ 0.05) were considered significant. When the normality test failed, for nonparametric analysis, data were subjected to Mann-Whitney sum ranks for the t-test and Kruskal-Wallis one-way analysis of variance on ranks for ANOVA.
Supporting information 1
H and 13C NMR spectra of compound 7 and the feed intake of the mice treated for 4 weeks with Alli and AL‑EE are available as Supporting Information.
Acknowledgements !
This research was supported by a Grant from the Ministry of Food and Agriculture, Forestry and Fisheries (110109-03-24-HD110), Republic of Korea.
Conflict of Interest !
The authors declare no conflict of interest.
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Statistical analysis Enzymatic activity assays
Original Papers
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Authors
Ping Jiao 1, Julie Tseng-Crank 1, Brandon Corneliusen 1, Mesfin Yimam 1, Mandee Hodges 1, Mei Hong 1, Catherine Maurseth 1, Misun Oh 2, Hyunjin Kim 2, Min Chu 1, 2, Qi Jia 1, 2
Affiliations
1 2
Key words " Atractylodes lancea l " Asteraceae l " pancreatic lipase l " lipase inhibitor l " atractylodin l " polyacetylenes l " DIO mice l
Unigen USA, Seattle, WA, USA Unigen Korea, Cheonam Si, Chungnam, South Korea
Abstract !
The ethanol extract of Atractylodes lancea rhizome displayed significant lipase inhibition with an IC50 value of 9.06 µg/mL in a human pancreatic lipase assay from high-throughput screening. Bioassay-guided isolation led to the identification of one new polyacetylene, syn-(5E,11E)-3-acetoxy4-O-(3-methylbutanoyl)-1,5,11-tridecatriene7,9-diyne-3,4-diol (7), along with six known compounds (1–6). The structure of compound 7 was determined based on the analysis of NMR and MS data. Among these seven lipase inhibitors, the major compound atractylodin (1) showed the highest lipase inhibitory activity (IC50 = 39.12 µM). The antiobesity effect of the ethanol extract of Atractylodes lancea rhizome was evaluated in a high-fat diet-induced obesity mice model at daily dosages of 250 mg/kg and 500 mg/
Introduction ! received revised accepted
Dec. 17, 2013 March 6, 2014 March 10, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368354 Published online March 31, 2014 Planta Med 2014; 80: 577–582 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Ping Jiao Unigen USA 3005 1st Avenue Seattle, WA 98121 USA Phone: + 1 36 04 86 82 00 Fax: + 1 20 64 41 55 17 [email protected]
Obesity is becoming one of the most serious global health problems, with prevalence at an increasing rate both in adults and children. Obesity can increase the risk factors of many diseases such as diabetes mellitus type 2, high blood pressure, high blood cholesterol, high triglyceride levels, and some forms of cancer [1–3]. Despite a significant amount of research done in this field, obesity still remains a tough medical challenge. Therefore, efforts for the development of antiobesity drugs and therapeutic interventions have been continued intensively. Clearly defined core lipid metabolic pathways involving well-characterized enzymes provide potential opportunities and rich sources of targets for therapeutic interventions and obesity treatments [4, 5]. Pancreatic lipase has been validated as one of the important targets in the lipid metabolic process [6].
kg body weight for 4 weeks, and treatment with this extract demonstrated a moderate efficacy at the 500 mg/kg dose level.
Abbreviations !
AL‑EE: DGGMR: DIO: HFD: hPL: OE: SAR:
ethanol extract of Atractylodes lancea rhizome 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin)-ester diet-induced obesity high-fat diet human pancreatic lipase organic extract structure-activity relationship
Supporting information available online at http://www.thieme-connect.de/products
Pancreatic lipase is considered the primary enzyme involved in the hydrolysis of fat molecules in the digestive system and the release of monoglycerides and free fatty acids. By targeting pancreatic lipase, the approach is to specifically inhibit triglyceride digestion, thus reducing the dietary fat absorption and calorie intake from fat. Orlistat, a competitive pancreatic lipase inhibitor, is marketed as a long-term obesity treatment drug. It is a derivative of a potent natural pancreatic lipase inhibitor, lipstatin, isolated from the bacterium Streptomcyces toxytricini. Orlistat decreases the dietary fat absorption through the gastrointestinal system by inhibiting lipase activities, predominantly pancreatic lipase, to prevent weight gain and to induce weight loss [7, 8]. The most reported side effect of Orlistat is oily spotting/loose stool (sometimes referred to as treatment effects). The success of Orlistat has driven the development of lipase inhibitors from natural sources [9–12]. Pancreatic lipase inhibitors reported from
Jiao P et al. Lipase Inhibition and …
Planta Med 2014; 80: 577–582
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Lipase Inhibition and Antiobesity Effect of Atractylodes lancea
Original Papers
Fig. 1 Chemical structures of compounds 1–6 from the rhizome of Atractylodes lancea.
plants are mainly classified into three class groups, saponins, polyphenols, and terpenoids [6]. There is always a need and requirements for novel natural lipase inhibitors with better efficacy and less side effects. In order to discover natural lipase inhibitors, a high-throughput hPL inhibition assay was developed by utilizing recombinant hPL. A total of 8563 plant extracts from our in-house phytologic library collection were screened against hPL. The OE of the Chinese traditional herbal medicine Atractylodes lancea (Thunb.) DC. (Asteraceae) showed a relatively high lipase inhibition in this screening. Six known lipase inhibitors and one new compound were isolated and identified from this extract via bioassayguided purification. AL‑EE was prepared and evaluated in a high-fat DIO animal model for the prevention of weight gain effects. To the best of our knowledge, this is the first report of pancreatic lipase inhibitory activity from the A. lancea rhizome extract.
Results and Discussion !
A. lancea OE showed 84.77 % inhibition on hPL at the 120 µg/mL level in the primary screening. Bioassay-guided isolation led to the discovery of seven polyacetylenic compounds with pancreatic lipase inhibitory activity. Among all seven compounds, atractylodin (1) was the most active lipase inhibitor and compound 7 was identified as a novel polyacetylenic compound. A. lancea rhizome is one of the commonly used Chinese traditional herbs, also known as cangzhu in Chinese. Cang refers to the dark color of the outer skin layer of the rhizome. Zhu was recorded in Shennong Bencao Jing, one of the earliest Chinese dispensatory, with the description as being boiled with water, nourishing, free from hunger, and extending oneʼs life span [13]. It was included in many ancient formulas and is still being used in a lot of decoctions today. The essential oil is claimed to be the major active ingredient for this herbal material. The main function is for the treatment of indigestion and stomach disorders which occurs by strengthening the spleen, supplementing Qi, and removing dampness [13, 14]. A. lancea rhizome is rich in volatile oil with the presence of atractylodin as the main characteristic compound. During our primary screening, lipase activity was found in A. lancea OE extract with 84.77 % inhibition at a concentration of 120 µg/mL. To clarify the active components in the organic ex-
Jiao P et al. Lipase Inhibition and … Planta Med 2014; 80: 577–582
tract, bioassay-guided isolation was carried out, leading to the isolation of a mixture of atractylodin and β-eudesmol in a ratio of 2 : 1 based on NMR and LC‑MS analyses, with a high lipase inhibition (IC50 = 11.35 µg/mL). Further purification confirmed that atractylodin (1), the major compound in the crude extract, was the active compound responsible for the lipase inhibition. To further study the chemical profile of A. lancea, an additional five minor known polyacetylenic compounds and one related new compound were isolated and identified from AL‑EE. The structures of the five known compounds, including 1-(2-furanyl)-7-nonene-3,5-diyne-1,2-diacetate (2), (3Z,5E,11E)-3,5,11tridecatriene-7,9-diyne-1-O-acetate (3), (3Z,5E,11E)-3,5,11-tridecatriene-7,9-diyne-1,2-diacetate (4), 4,6,12-tetradecatriene8,10-diyne-1,3-diacetate (5), and (5E,11E)-1,5,11-tridecatriene7,9-diyne-3,4-diacetate (6), were established based on the analysis and comparison of NMR and MS data with literature reports " Fig. 1). [15–19] (l New compound 7 was recognized as a new member of the " Fig. 2). polyacetylene class by analysis of UV and NMR data (l Compound 7 showed maximal UV absorptions at wavelengths of 311, 291, 276, 260, and 231 nm, indicating the presence of the same ene-diyne-ene chromophore as observed in compound 6. The molecular weight 328 was determined by GC‑MS. The molecular formula C20H24O4 (9 degrees of unsaturation) was assigned for 7 based on the analysis of NMR and HR‑ESI‑MS data. The 1H NMR spectrum showed a terminal methyl group at 1.83 ppm coupling with two trans-configured olefinic protons at 6.34 and 5.58 ppm, which is consistent with all the other six acetylenes. Two terminal vinylic protons at 5.34 and 5.31 ppm were coupled with another olefinic proton at 5.72 ppm with coupling constants of 17.36 Hz and 10.76 Hz, respectively. The geometry of the C5–C6 double bond at 5.82 and 6.13 ppm was assigned as E on the basis of the coupling constant of 15.89 Hz. 1H-1H COSY spectrum together with chemical shift data revealed a spin sys" Fig. 3. Only one singlet methyl peak tem C1–C6 as shown in l was observed at 2.09 ppm, indicating the presence of one acetoxy group. The HMBC correlation of H-3 to the carboxyl carbon at 169.61 ppm enabled the connection of the acetate to C-3. One isovalerate unit was identified based on the analysis of 13C and 1 H, and COSY NMR spectra. Similarly, HMBC correlations of H-4 to the carboxyl carbon C-1′ allowed the assignment of this isovalerate unit attached to C4. Isovalerate polyacetylenes were also reported from A. chinensis and A. macrocephala [20, 21].
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Fig. 2 Chemical structure of isolated compound 7.
Fig. 3
Key HMBC and COSY correlations of compound 7.
Table 1 Pancreatic lipase inhibitory activity of A. lancea extracts and compounds.
Orlistat AL‑EE OE1-F12 Atractylodin (1) Compound 2 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7
IC50 (µg/mL)
IC50 (µM)
0.035 9.06 11.35 7.12 14.2 9.1 22.3 16.7 23.9 15.6
0.07 – – 39.12 47.33 39.91 77.97 55.67 83.57 47.56
In addition, atractyldoin (1) and its analogues could serve as a chemical platform for synthetic chemists to conduct SAR studies in order to search for desirable antiobesity drug candidates.
Materials and Methods !
Plant material The dried rhizome of A. lancea was collected from Xiangyang, Hubei, China. The plant material was identified by professor ShouYuen Zhao from Si-Chuan Chinese Traditional Medicine Research Institutes. A voucher of specimen A. lancea (P00541) was deposited at the plant library of Unigen, Seattle, WA, USA. The recollected A. lancea rhizome was obtained from an herbal store in Seattle, characterized, and confirmed in comparison with the original voucher specimen.
Apparatus and chemicals used for the phytochemical study The UV and MS data were collected from an LC‑MS/PDA system, MS M-8000 (Hitachi) and PDA L-4500A (Hitachi), and GC‑MS instrument 6890 N (Agilent Technologies). HR‑ESI‑MS was recorded on an LTQ-Orbitrap high-resolution mass spectrometer (Thermo Scientific). The proton and carbon NMR spectra were recorded on a VNMRS-500 spectrometer (Agilent Technologies). The extracts were produced by Dionex accelerated solvent extractor ASE 300 (Thermo Scientific). Isolation was carried on on a Hitachi high-throughput purification system HTP D7000 (Hita-
Jiao P et al. Lipase Inhibition and …
Planta Med 2014; 80: 577–582
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Although the absolute configurations of C-3 and C-4 of compound 7 could not be identified, a syn-configuration was assigned based on a relatively larger 3J coupling constant of 6.3 Hz. According to a literature report, the coupling constants of the vicinal protons in this type of compound were observed in the range of 5–8 Hz in the syn (or threo) form, while smaller coupling constants around 4 Hz were observed for the anti (or erythro) configuration [17]. Thus, the structure of 7 was determined as syn-(5E,11E)-3-acetoxy-4-O-(3-methylbutanoyl)-1,5,11-trideca" Fig. 2). triene-7,9-diyne-3,4-diol (l The pancreatic lipase inhibitory effects were evaluated by mea" Table 1). suring the IC50 values for individual compounds (l Among those seven compounds, atractylodin (1) substantially in" Fig. 4). hibited pancreatic lipase with an IC50 value of 39.12 µM (l The content of atractylodin of the ethanol extract was quantified " Fig. 5. as 11.16 % by weight using HPLC analysis as shown in l Due to its high content in the ethanol extract, atractylodin definitely contributed to the lipase inhibitory activity of the material. Compounds 2-6 showed similar or relatively weaker lipase inhibition compared with atractylodin. The inhibitory potency of the new compound 7 was determined with an IC50 value at 47.56 µM. Enyne compounds from marine sponges and algae have also been reported as lipase inhibitors [22, 23]. Although it is difficult to make a conclusion on the SAR based on the current activity of the polyacetylenic compounds isolated from A. lancea, this enediyne-ene skeleton could be a good template for lipase inhibitor drug design. The antiobesity effect of AL‑EE with an IC50 value at 9.06 µg/mL " Fig. 4) was evaluated in a high-fat diet-induced obesity mice (l model at daily dosages of 250 mg/kg and 500 mg/kg body weight. A progressive increase in body weight gain was observed when the mice were provided with a 60% kcal HFD for 4 weeks. The known pancreatic lipase inhibitor Orlistat-treated group showed statistically significant reductions in body weight gain as of day 7 during the treatment in compared to the vehicle-treated HFD " Fig. 6). Similarly, a trend in reduction in body weight group (l gain was also observed for mice treated with AL‑EE, with statistical significance on days 21 and 24 for the 500 mg/kg dosing group. When compared with the vehicle-treated HFD mice, these tendencies in reducing body weight gain for mice treated with AL‑EE at a dose of 500 mg/kg were found to have p values of 0.06, 0.08, and 0.09 on days 10, 14, and 17 after oral treatment, respectively. Both AL‑EE treatment groups at 500 mg/kg and 250 mg/kg showed the tendency to prevent the HFD-induced increases in the body weight of mice. We have successfully developed a reliable and robust highthroughput hPL inhibition assay. Our results reveal for the first time that AL‑EE showed hPL inhibitory activity, with atractylodin (1) as the major active compound. AL‑EE also prevented body weight gain in a high-fat diet mice model in comparison to the HFD controlled group. This extract could be considered a potential active ingredient for obesity and associated lipid dysfunction.
Original Papers
chi) and HPLC L-6200A (Hitachi). Orlistat (purity ≥ 98 %) was purchased from Sigma-Aldrich.
Plant extraction and isolation
Fig. 4 Human pancreatic lipase inhibition of the ethanol extract of A. lancea and compound 1 (atractylodin). Orlistat was used as a positive control.
Fig. 5 HPLC chromatogram of Atractylodes lancea ethanol extract at a wavelength of 320 nm. (Color figure available online only.)
Fig. 6 Effects of Atractylodes lancea rhizome ethanol extract on body weight in mice fed a high-fat diet for 28 days. Each point represents the mean ± SEM, and values significantly different from the controls are indicated by * p < 0.05.
Jiao P et al. Lipase Inhibition and … Planta Med 2014; 80: 577–582
A total of 59.4 grams of dried rhizome of A. lancea powder were loaded into two 100 mL stainless steel tubes and extracted twice with an organic solvent mixture (methylene chloride/methanol in a ratio of 1 : 1) under heat (80 °C) and pressure (1500 psi). The extract solution was automatically filtered and collected. The combined OE solution was evaporated with a rotary evaporator to give crude OE (14.1 g, 23.7 %). The A. lancea OE (13 g) was subjected to two silica gel columns (Biotage SNAP cartridge KP‑Sil 100 g) in parallel and eluted with a gradient solvent system of 25 % EtOAc in hexanes for 5 min, then increased to 100% EtOAc in 25 min, followed by introducing MeOH from 50% to 100 % MeOH in 20 min, and ended in washing with 100% MeOH for 16 min at a flow rate of 5 mL/min using a Hitachi high-throughput purification L7260 system. Eighty-eight fractions were collected for each column and combined to 10 fractions. Fraction 1 (600 mg) was further processed by a 10-µm Luna C18 column (30 × 250 mm, Phenomex) on an HPLC system L6200A eluted with 80% MeOH/H2O for 10 min, and increased to 100 % MeOH in 20 min at a flow rate of 10 mL/min with a UV wavelength of 250 nm to afford 12 fractions/compounds. Fraction 12 (tR = 35.12 min) was identified as a mixture of atractylodin (1) and β-eudesmol in a ratio of 2 : 1 (OE1-F12, 76.49 mg) based on NMR and LC‑MS analyses. Further bioassay-guided isolation and purification were carried out on the ethanol extract in order to get pure atractylodin and other analogues due to its complexity. The recollected A. lancea rhizome material (1.15 kg) was extracted with ethanol by reflux to give an ethanol extract (AL‑EE, 205.2 g, 17.8 %). The ethanol extract (44.1 g) was partitioned between hexanes and H2O. The hexane fraction (6.1 g) was isolated by a silica gel column (Biotage SNAP cartridge KP‑Sil 100 g) with a gradient elution by hexanes/ EtOAc/MeOH on the HPLC system L6200A at a flow rate of 5 mL/ min to yield 12 fractions. Atractylodin (1) (1.2 g) was obtained from fraction 6 (~ 500–600 mL). Fractions 7, 8, and 10 (~ 600– 700, 700–800, 1000–1100 mL) were further isolated by RP-HPLC to give compounds 2–6. Compound 7 (8.8 mg) was isolated by a 10-µm Luna C18 RP-HPLC column (30 × 250 mm, Phenomex) eluted with 70 % MeOH/H2O for 5 min, and increased to 100 % MeOH in 30 min, and 100 % MeOH for another 15 min at a flow rate of 10 mL/min, with a UV wavelength of 235 nm and tR at 31.67 min by injection of 114 mg of fraction 8. Syn-(5E,11E)-3-acetoxy-4-O-(3-methylbutanoyl)-1,5,11-tridecatriene-7,9-diyne-3,4-diol (7): colorless solid; UV (MeOH) λmax 311, 291, 276, 260, 231 nm; GC‑MS m/z 328 [M]+; HR‑ESI‑MS m/ z 351.15 836 [M + Na]+ (calcd. for C20H24O4Na: 351.15 668); 1H NMR (500 MHz, CDCl3) δ ppm 0.96 (6H, d, J = 6.60 Hz, H-4′, H-5′), 1.83 (3H, dd, J = 6.85, 1.50 Hz, H-13), 2.09 (3H, s, OCOCH3), 2.10 (1H, m, H-3′), 2.22 (2H, d, J = 6.85 Hz, H-2′), 5.31 (1H, d, J = 10.76 Hz, H-1a), 5.34 (1H, d, J = 17.36 Hz, H-1b), 5.38 (1H, t, J = 6.34 Hz, H-3), 5.47 (1H, t, J = 6.80 Hz, H-4), 5.58 (1H, d, J = 15.41 Hz, H-11), 5.72 (1H, ddd, J = 17.12, 10.64, 6.24 Hz, H-2), 5.82 (1H, d, J = 16.14 Hz, H-6), 6.13 (1H, dd, J = 15.89, 6.60 Hz, H5), 6.34 (1H, dq, J = 15.91, 6.85 Hz, H-12); 13C NMR (126 MHz, CDCl3) δ ppm 18.95 (CH3, C-13), 20.92 (CH3, OCOCH3), 22.35 (2CH3, C-4′, C-5′), 25.69 (CH, C-3′), 43.29 (CH2, C-2′), 71.91 (C, C9), 72.90 (CH, C-4), 74.03 (CH, C-3), 76.18 (C, C-8), 77.74 (C, C-7), 81.42 (C, C-10), 109.70 (CH, C-11), 113.40 (CH, C-6), 119.87 (CH2,
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C-1), 131.58 (CH, C-2), 139.09 (CH, C-5), 144.18 (CH, C-12), 169.61 (C, OCOCH3), 171.62 (C, C-1′)
Quantification of atractylodin (1) in the ethanol extract of Atractylodes lancea rhizome HPLC analysis of AL‑EE was performed to quantify atractylodin (1) content using a Hitachi HPLC D-7000 system with a 10-µm Luna RP‑C18 column (4.6 × 250 mm, Phenomenex) and diode array detector. The column was eluted at a condition of 70 % MeOH in H2O to 100% MeOH in 15 min, and 100 % MeOH for 8 min. This ethanol extract was shown to contain 11.16 % atractylodin " Fig. 5). (l
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treated animals received 0.5 % methylcellulose only. Regular rodent diet-fed (T2018, Harlan Teklad Diets) nonobese mice were included as a control in this study. The animals were maintained in a temperature controlled room (22.2 °C) on a 12-hour lightdark cycle. Body weight measurements were taken twice a week. All animal experiments were conducted according to institutional guidelines congruent with the guide for the care and use of laboratory animals. All procedures performed were in compliance with the Animal Welfare Act and US Department of Agriculture regulations (IACUC approval date: 01/06/2011; protocol No.: UAS-071205).
The coding region of hPL cDNA (Invitrogen) was cloned into the pcDNA3.1 plasmid vector for mammalian cell transfection, driven by the CMV promoter to create recombinant hPL. A native signal peptide sequence at the N-terminus ensured a proper secretion of hPL from the transfected 293 cells into the culture medium. The hPL protein produced excellent enzyme activity using a commercially available triglyceride lipase assay kit (Randox Laboratories), with DGGMR as the colorimetric substrate. A porcine co-lipase, which is necessary for pancreatic lipase activity, was supplied as part of the substrate and buffer formulation. A lipase inhibitory test was established utilizing recombinant hPL and a commercially available substrate (DGGMR, Nuclin Diagnostics). Orlistat (Sigma-Aldrich, purity ≥ 98 %) was used as a positive control in the assay at 2.4 µM. The reaction mixture containing the test sample was no greater than 3 % DMSO (20 µL) and the enzyme solution (100 µL, containing assay buffer at pH 8.4) was incubated for 10 minutes. The colorimetric substrate (50 µL) was added and the reactions were incubated at 37 °C for 30 min. Each sample was tested in triplicate and the 580 nm optical density (OD580) was read for each sample. IC50 values were determined using the same method with serial dilutions. All IC50 values were determined using SigmaPlot (Version 11.0) by a four parameter logistic nonlinear regression model with the sample absorbencies over the log concentrations of the test samples. The lipase inhibition activity was evaluated for compounds 1 (HPLC purity ≥ 98%), 2 (HPLC purity ≥ 95 %), 3 (HPLC purity ≥ 90%), 4 (HPLC purity ≥ 90%), 5 (HPLC purity ≥ 90 %), 6 (HPLC purity ≥ 80 %), and new compound 7 (HPLC purity ≥ 98 %) by calculating their IC50 values.
Animals and induction of obesity A HFD-induced obesity animal model was developed and used for the evaluation of potential antiobesity therapeutic agents. Male C57BL/6J mice were purchased from Jackson Laboratories at the age of 8 weeks with a body weight of 25.6–34.2 g. One week after an acclimation period, the animals were provided with high-fat (60% kcal) rodent pellets (D12492, Research Diets, Inc.) and water ad libitum for 8 weeks. Once the induction of obesity was confirmed (with a 40.9 % increase in body weight gain), the mice were randomized to treatment groups of Orlistat (Alli, GSK, 30 mg/kg), AL‑EE (250 mg/kg or 500 mg/kg), or vehicle with 9 animals in each group. The test compounds were suspended in 10 ml/kg vehicle and were repeatedly vortexed between gavages to maintain consistent dosing. The mice were orally gavaged with their respective treatment solution at 9 : 00 a. m. and 5 : 00 p. m. using a syringe and an 18-gauge ball-tipped feeding needle. No detectable sign of irritation was observed from drug or vehicle administration. The animals were treated with the respective therapeutic agent twice per day, orally, for 4 weeks. The vehicle-
The preclinical in vivo data were analyzed using SigmaPlot (version 11.0). Statistical significance between groups was calculated by means of single factor analysis of variance followed by a paired t-test. P values less or equal to 0.05 (p ≤ 0.05) were considered significant. When the normality test failed, for nonparametric analysis, data were subjected to Mann-Whitney sum ranks for the t-test and Kruskal-Wallis one-way analysis of variance on ranks for ANOVA.
Supporting information 1
H and 13C NMR spectra of compound 7 and the feed intake of the mice treated for 4 weeks with Alli and AL‑EE are available as Supporting Information.
Acknowledgements !
This research was supported by a Grant from the Ministry of Food and Agriculture, Forestry and Fisheries (110109-03-24-HD110), Republic of Korea.
Conflict of Interest !
The authors declare no conflict of interest.
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Original Papers
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