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Binita Maharjan, Ilaria Rossi, Stefania Sut, Timila Shrestha, [Lok Kumar Shrestha](https://orcid.org/0000-0003-2680-6291), [Jonathan P. Hill](https://orcid.org/0000-0002-4229-5842), [Katsuhiko Ariga](https://orcid.org/0000-0002-2445-2955), Veronica Benetazzo, Maria Pia Adorni, Bianca Papotti, Shyam Sharan Shrestha, Ram Lal Swagat Shrestha, Nicola Ferri, Stefano Dall'Acqua

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[Bioactive Alkaloids from Nepalese <i>Corydalis chaerophylla</i> D.C. Acting on the Regulation of PCSK9 and LDL‐R <i>In Vitro</i>](https://mdr.nims.go.jp/datasets/cb0927bd-d371-4fd6-a1b1-befaba53ea4a)

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Bioactive Alkaloids from Nepalese Corydalis chaerophylla D.C. Acting on the Regulation of PCSK9 and LDL‐R In VitroBioactive Alkaloids from Nepalese Corydalis chaerophyllaD.C. Acting on the Regulation of PCSK9 and LDL-R In VitroBinita Maharjan,[a] Ilaria Rossi,[b] Stefania Sut,[b] Timila Shrestha,[a] Lok Kumar Shrestha,[c, d]Jonathan P. Hill,[c] Katsuhiko Ariga,[c, e] Veronica Benetazzo,[b] Maria Pia Adorni,[f]Bianca Papotti,[g] Shyam Sharan Shrestha,[h] Ram Lal Swagat Shrestha,*[a] Nicola Ferri,[i, j] andStefano Dall’Acqua*[b]Four new alkaloids Chaeronepaline-A (1), Chaeronepaline-B (2),Chaeronepaline-C (3), and Chaeronepaline-D (4) were isolatedfrom Corydalis chaerophylla D.C. collected from Nepal and theirstructures were elucidated by spectroscopic data, 1D, 2D NMRand mass spectrometry. The structures were established as3,12- Dimethoxy-5,6-dihydroisoquinolino [2,1-b] isoquinolin- 7-ium- 2, 9- diol (1), 7-Methyl-2,3 : 11,12-bis(methylenedioxy)-7,13a-secoberbin-13–14-epoxide (2), 7- methyl-5, 6, 7, 8-tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-in-dane-isoquinoline (3) and 7- methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquino-line-N-oxide (4). The new alkaloids were tested in humanhepatoma cell line to assess their ability to modulate theexpression of low-density lipoprotein receptor (LDL� R), ofproprotein convertase subtilisin/kexin 9 (PCSK9) and to affectcellular cholesterol biosynthesis with the aim to evaluate theirpotential hypocholesterolemic effect. Results indicated thatcompounds 2 and 3 upregulate the LDLR, and inhibited thecholesterol biosynthesis with compound 2, which also reducedthe secretion of PCSK9 by Huh7 cells. These in vitro dataindicated a potential hypocholesterolemic effect of compound2 that requires further in vivo validation.1. IntroductionThe genus Corydalis, comprising over 470 species in Eurasia andNorth America, is indigenous to the temperate Northern Hemi-sphere. Among them, fifty-seven species can be found in Nepal.These plants, characterised by their vibrant and attractiveflowers, are classified as both annual and perennial herbaceousplants. They produce significant number of isoquinolinealkaloids,[1–4] which exhibit diverse biological properties.[4,5]Traditionally, some Corydalis species have been widely em-ployed in China, Korea, Japan, and other Eastern Asian nationsto treat gastric and duodenal ulcers, dysmenorrhoea, rheuma-tism and cardiac arrhythmia disease.[2–4,6] The phytochemicalanalysis of Corydalis plant extracts resulted in the isolation ofmore than 100 isoquinoline alkaloids from this genus.[4] Some ofthese alkaloids have been studied as possible treatment ofserious diseases such as cancer, Alzheimer’s disease, andmicrobial infections. Corydalis alkaloids present good drug-likeproperties demonstrated by the numerous reported biologicalactivities.[4] Various alkaloids derived from Corydalis have beenstudied for their in vitro metabolic effects,[7] for hepatoprotec-tive properties,[8] as urease activity inhibitors,[9] asleishmanicidal,[10] analgesic,[11] apoptosis inducers,[12] as cytotoxicagents on human cancerous cell lines[13] and as cholesterolcontrolling agents.[14]Corydalis chaerophylla D.C. is a glabrous herb found in high-altitude areas of Nepal, India, and Pakistan. It survives in wet,shadowy conditions at elevations ranging from 2400–4800 a.l.s.[a] B. Maharjan, T. Shrestha, R. L. S. ShresthaDepartment of Chemistry, Amrit Campus, Tribhuvan University, Kathmandu44600, NepalE-mail: swagatstha@gmail.com[b] I. Rossi, S. Sut, V. Benetazzo, S. Dall’AcquaDepartment of Pharmaceutical and Pharmacological Sciences, University ofPadova, Padova, ItalyE-mail: stefano.dallacqua@unipd.it[c] L. K. Shrestha, J. P. Hill, K. ArigaResearch Center for Materials Nano Architectonics (MANA), NationalInstitute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan[d] L. K. ShresthaDepartment of Materials Science, Faculty of Pure and Applied Sciences,University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573 Japan[e] K. ArigaGraduate School of Frontier Sciences, The University of Tokyo, 5-1-5Kashiwanoha, Kashiwa, Chiba 277-8561, Japan[f] M. P. AdorniDepartment of Medicine and Surgery, University of Parma, Parma, Italy[g] B. PapottiDepartment of Food and Drug, University of Parma, Parma, Italy[h] S. S. ShresthaHimalayan Research and Development Center Nepal, Kathmandu, Nepal[i] N. FerriDepartment of Medicine, University of Padova, Padova, Italy[j] N. FerriVeneto Institute of Molecular Medicine (VIMM), Padova, ItalySupporting information for this article is available on the WWW underhttps://doi.org/10.1002/cbdv.202401388© 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AG.This is an open access article under the terms of the Creative CommonsAttribution Non-Commercial License, which permits use, distribution andreproduction in any medium, provided the original work is properly citedand is not used for commercial purposes.Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 242/252] 1Chem. Biodiversity 2024, 21, e202401388 (1 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGwww.cb.wiley.comdoi.org/10.1002/cbdv.202401388 Research Articlehttps://doi.org/10.1002/cbdv.202401388http://crossmark.crossref.org/dialog/?doi=10.1002%2Fcbdv.202401388&domain=pdf&date_stamp=2024-10-28C. chaerophylla was used as traditional medicine in Nepal forpeptic ulcers in doses of about four teaspoons, three times perday. The administration of a dosage of around six teaspoons,three times per day, of the root juice is recommended for thetreatment of dyspepsia. This therapeutic approach involvesblending the root juice with an equal amount of the root juicederived from Cyathula capitates Moq. (N. Kuro).[15] Previousinvestigations reported the isolation from C. chaerophyllacollected in Nepal, of a new alkaloid called chaerophylline, aswell as (� )-corypalmine, berberine, (� )-isocorypalmine, (� )-corydalmine, and (+)-bicuculline.[16] In a recent paper our groupalso considered Nepalese C. chaerophylla showing the presenceof fifteen different alkaloids, with N� Me-tetrahydropalmatine,bicuculline, protopine, hunnemanine and jatrorrhizine as mostabundant derivatives.[17]In a previous study, our research group considered a seriesof alkaloids isolated from plants as potential bioactive com-pounds targeted on the key enzyme involved in cholesterolmetabolism, namely the low-density lipoprotein receptor(LDL� R) and the proprotein convertase subtilisin/kexin 9(PCSK9). In that work, we observed significant induction ofLDLR, similar to the hydroxy-methyl glutaryl Coenzyme A (HMG-CoA) inhibitor, simvastatin, berberine, californidine and gova-niadine. Californidine and berberine reduced the expression ofPCSK9, while govaniadine, similar to simvastatin, induced thePCSK9 expression. Additionally, it was shown that all of thetested compounds reduced the total cholesterol level in thehepatocytes.[14]Focusing on the discovery of novel bioactivities associatedwith alkaloids, with a particular emphasis on the biodiversity ofthe Nepalese plant kingdom, starting from our previousinvestigation on alkaloid content of this plant[17] we consideredC. chaerophylla, as potential source of bioactive compoundsbeing the Nepalese population of this plant, up to now, only inlimited part investigated.2. Result and Discussion2.1. Structural Elucidation of New Isolated Compounds 1–4The compound (1) was analysed by mass spectrometry andshowed molecular ion [M + H]+ at m/z 324, and fragmentationshowed the loss of methyl group leading to ion at m/z 309.From the HR-MS data, the molecular formula was calculated asC19H18NO4.The 1H-NMR showed the presence of four singlets at δ 9.30,8.03, 7.39 and 6.93, each integrating for one proton. Further-more, two singlets integrating three protons each wereobserved at δ 3.94 and 3.90, suggesting the presence of twomethoxy groups. Two ortho coupling doublets (J=8.0) wereobserved at δ 7.58 and 6.94, supporting the presence of anaromatic ring, and two triplets at δ 4.63 and 3.16, showingaliphatic coupling (J=6.2), each integrating for two protonswere observed. The HSQC-DEPT spectrum allowed to observeall the non-quaternary positions and revealed that the com-pound possesses six aromatic CH, two aliphatic CH2, onebenzylic (δH 3.16-δC 27.3; C-5) and one N-linked (δH 4.64-δC 54.2;C-6). Also, the two methoxyl positions were confirmed. Thecomplete structure of the compound was obtained by combin-ing the data of HMBC, COSY and NOESY spectra, structure andmain observed diagnostic NMR correlations are reported inFigures 1 and 2. The singlet at δH 7.39 (H-1) showed HMBCcorrelation with C-4a (δC 126.2), C-3 (δC 149.3) and C-14 (δC134.6), while the other at δH 6.93 (H-4) showed HMBC with C-2(δC 146.6) supporting the presence of an electron attractivegroup, C-14a (δC 120.2) and C-5 (δC 27.3). The methoxyl group atδH 3.94 presents HMBC correlation with C-3, confirming thepresence of 3-methoxyl substitution. The HMBC observed fromH-5 (δH 3.16) with C-4 (δC 110.0), C-14a, and C-4a support thedirect linkage between positions 4a and 5. The COSY couplingfrom H-5 to H-6 and the diagnostic HMBC correlation from H-6with C-4a and C-14 allowed to establish the presence of a 3,4-dihydroquinoline moiety. Further diagnostic HMBC from H-6 isobserved with C-8 (δC145.9). From the H-8 (δH 9.30), HMBCcorrelations are observed with C-14 confirming the presence ofthe six-member ring of 3,4-dihydroisoquinoline, C-12a (δC 132.4)and C-9 (δC 162.0) this latter supporting the presence of ahydroxyl group. The H-13 present long range HMBC correlationswith C-8a (δC 120.1) and C-12 (δC 149.7) this latter correlation isin common with the singlet at δH 3.90, supporting the presenceof a 12-linked methoxy group. Two ortho coupling doubletswere finally assigned to positions H-10 and H-11, and theypresent long range correlations with C-8a, C-12 and C-9 and C-12a, respectively. NOESY correlation confirmed the methoxyla-tion position shown by the cross peak between H-16 and H-4and H-15 with H-13 H-11. All the obtained data allowed toestablish for the compound 1 the structure of 3,12-Dimethoxy-Figure 1. Structures of compounds 1–4.Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 243/252] 1Chem. Biodiversity 2024, 21, e202401388 (2 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License5,6-dihydroisoquinolino[2,1-b]isoquinolin-7-ium-2,9-diol andnamed chaeronepaline-A. As per our knowledge, this is the firstreport of the isolation of this compound from natural sources.The compound (2) presented molecular ion [M + H]+ at m/z354 and the HR-MS data allowed to obtain the molecularformula of C20H20NO5. The 1H-NMR spectrum showed foursinglets in the deshielded region, two at δH 7.14, 6.79,integrating for one proton each and two ortho coupleddoublets (J=7.0) at δH 6.86 and 6.81 integrating one protoneach. The signals support the presence of two aromatic rings,the spectrum in the aromatic is similar to the one published forprotopine.[19] Singlets at δH 6.02, 6.03 integrating two protonseach indicate the presence of two methylenedioxy groups.In the aliphatic region, broad signals at δH 4.48 (2H), 3.86(1H), 3.85 (1H), 3.77 (1H), 3.51 (2H) and 3.22 (2H) suggested thepresence of aliphatic N-linked methylene and methyne bearingelectron attractive group. Broad signals ascribable to methylenehave been described for protopine suggesting that thecompound (2) may be belonging to this family of alkaloids.[19,20]Another signal suggesting the structure of protopine-typealkaloid is the singlet integrating for three protons at δH 2.91,ascribed to a N-linked methyl group. Compound appear quiteunstable and further purification lead to alteration of pattern ofsignals in the 1H-NMR so we decided to perform the structureelucidation on the partially purified compound. The HSQC-DEPTrevealed the presence of four aromatic CH at δH 7.14-δC 115.9(C-1), δH 6.79-δC 108.9 (C-4), δH 6.86-δC 122.9 (C-9), δH 6.81-δC108.6 (C-10), two methylene dioxy groups at δH 6.00-δC 102.0 (C-17), δH 6.00-δC 102.0 (C-16). In the aliphatic region, two partiallyoverlapped CH were observed at δH3.85- δC 56.5 (C-13 and C-14), due to their chemical shifts they were assigned as benzylicCH bearing an epoxide group. Other significant signals wereone benzylic CH2 at δH 3.25-δC 24.6 (C-5) and two N-linked CH2at δH 4.48-δC 53.7 (C-6) one at 3.77–3.51 broad signals δC 55.0 (C-8). N-linked methyl group was observed at δH 2.91-δC 41.3 (C-15). The structure was elucidated combining the data obtainedfrom HSQC-DEPT, HMBC, COSY and NOESY. The HMBC observedfrom H-1 with carbon resonances at δC 148.8 (C-3), 124.5 (C-4a)and 56.5 (C-14) as well as the diagnostic long range correlationobserved from H-4 with carbon at δC 148.7 (C-2), 127.8 (C-14a).NOESY correlation was observed from H-4 with δH 3.25 (H-5) anfrom H-1 with δH 3.86–3.85 (H-14 and H-13). Furthermore, thelong range correlation observed from H-5 with C-4 and C-14a aswell as from H-14 with C-4a suggest that the compoundpresent protopine type moiety. NOESY correlations were alsoobserved from H-6 with H-5 and N-Methyl group as well asfrom H-8 with N-Methyl group. Diagnostic HMBC correlationwas observed from H-13 with C-12 (δC 148.1) confirming theepoxide position in 13–14. Furthermore, the long rangecorrelations from H-9 with C-8, C-11, C-12a allowed to establishthe position of the second methylenedioxy group. Completestructure assignments are reported in Tables 1 and 2. Thestructure of the compound was assigned to derivative ofprotopine bearing the 13–14-epoxy group instead of the ketofunction in position 14. Compound can be nominated as 7-Methyl-2,3 : 11,12-bis(methylenedioxy)-7,13a-secoberbin-13–14-epoxide and we here propose the name chaeronepaline-B. TheMSn fragmentation support the proposed assignment in fact wecan observe in MS2 the intense fragment at m/z 188 and 149.The compound presents optical activity and the CD spectrumrevealed a negative effect at 205 and 215 nm, but with theisolated amount was not possible to assign absolute config-uration.The MS of the compound (3) presented molecular ion at m/z [M + H]+ 370. The molecular formula deduced from the HR-MS data was C20H20NO6. The H-NMR revealed the presence offour singlets at δH 6.89, 6.87, 6.68 and 6.30, the first integratingfor two protons and the other three integrating for one proton.Further signals are detected as singlets at δH 6.00 and 5.81, bothintegrating for two protons. Finally, two more singlets, integrat-ing one proton each, are observed at δH 5.53 and 5.34. in thealiphatic region, three multiplets were detected at δH 3.68, 3.62and 2.97, the first two integrating one proton each and thethird integrating for two protons. One singlet suggesting thepresence of one N-linked methyl group was detected at δH 2.84(3H). Complete structure assignment was obtained by combin-ing HSQC-DEPT, HMBC, COSY and NOESY data. One tetrahydroisoquinoline moiety was supported by the two CH observed atδH 6.30-δC 109.1 (C-1) and δH 6.68-δC 108.2 (C-4) and by therelevant long range HMBC correlations observed from H-1 withδC 128.3 (C-4a), δC 147.4 (C-3) and a quaternary carbonresonance at δC 81.6 (C-8) as well as by the one observed fromH-4 with δC 145.3 (C-2), δC 120.0 (C-8a) and δC 21.5 (C-5). Theother correlations allowing the identification of the dehydropyperidine ring were observed from H-6 with C-4a, C-8 and theδC 37.9 (C-17), this latter assigned to the N-linked methyl group.Figure 2. Key HMBC(red arrows), 1H-1H COSY (bold) correlation of compounds 1–4.Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 244/252] 1Chem. Biodiversity 2024, 21, e202401388 (3 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons LicenseThe singlets at δH 5.53 and 5.34 were assigned to oxygen-bearing CH due to the shift of their carbons that, were δC 76.3and 77.8, respectively. From these signals assigned to positionsH-14 and H-9 diagnostic HMBC were observed with C-8 and C-8a, supporting the direct linkage of these CH with thetetrahydroquinoline moiety. The chemical shift of the quater-nary position C-8 suggests the presence of a spiro derivatives.[21]The protons H-14 and H-9 present a COSY correlation,suggesting a “w” type long range coupling. From the H-9 andH-14, identical and numerous long range HMBC correlationscan be observed and are depicted in the Figure 2. In particular,long range correlations were observed from the H-9 and C-14and from H-14 and C-9, supporting the linkage of the two CH toone quaternary carbon (C-8). Furthermore, from H-14, two bondcorrelations are observed with C-8 and C-14a. Three bondcorrelations were observed with C-8a, C-9a and C-13, but alsofour bond correlations were observed with C-10 and C-12, andfive bond correlations were observed with C-11. The samecorrelations were also observed from H-9. These HMBCcorrelations suggest the presence of a planar and rigid portionof the compound. Due to the presence of the H-9 and H-14 aswell as the singlets ascribed to H-10 and H-13 and themethylenedioxy H-16, the second portion of the molecule wasassigned to an indane derivative bearing a methylene dioxysubstituent and sharing one carbon of the pentacyclic hydro-carburic cycle with the isoquinoline moiety. NOESY correlationsallowed to establish relative stereochemistry, and we imposedthe alpha configuration to H-14. The H-14 showed NOESY withsignal at δH 3.62 that on the basis of our hypothesis can beascribed to H-6α, while H-9 presents NOESY correlation withproton at δH 3.68 that consequently can be ascribed to H-6β.Both H-9 and H-14 show NOESY correlation with the N-methylgroup.The structure of the compound is a spiroindane isoquinolinecharacterised by the presence of two methylenedioxy substitu-ents, one for each aromatic ring. The structure is formed by atetrahydro isoquinoline moiety fused with an oxygenatedindane, and the basic carbon skeleton is like ochrobirine,[22] butwith a different position of the methylenedioxy substituent inthe indane portion. The compound (3) is characterized as 7-methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline and named chaeronepa-line-C.The compound present optical activity (+ 38), and the CDspectrum showed two negative cotton effects, one at 215 nmand one at 295, while a positive effect was recorded at 270 nm.The literature[23] assigned absolute configuration for ochrobirinesince opposite cotton effects in CD spectrum recorded inmethanol. On the basis of our data, we can assume thatcompound 3 presented opposite absolute configuration at thecarbons 6, 9 and 14 of ochrobirine. Complete assignment ofabsolute stereochemistry could be confirmed only after asym-metric synthesis.The MS of compound (4) presented molecular ion at [M +H]+ 386, with relevant fragments at m/z 370, suggesting that itcontained a further oxygen atom compared to the previousderivative. The molecular formula deduced from the HR-MSdata was C20H20NO7. H-NMR presented similarity to compound 3with an almost superimposable spectrum related to the indaneportion and small differences in the chemical shift of position 1and 4 and 9 and 14. In contrast, significant differences wereobserved in the chemical shift of the N-methyl that appeareddeshielded with δH 2.97. The analysis of 2D spectra allowed toestablish the same spiroindane isoquinoline moiety and com-pound was characterised as the N-Oxide derivative of 3 as 7-methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline-N-oxide and named chaer-onepaline-D. Compound 4 presented the same behaviour as 3at circular dichroism and presented optical rotation activity, asfor the compound 3 we imposed H-14 as alpha. NOESYcorrelations are observed from H-14 with H-6α, while from H-9with H-6 β and from H-9 and H-14 with the N-Methyl group. Onthe basis of the literature, we assume that compound 4presented opposite absolute configuration at the carbons 6, 9and 14 compared to ochrobirine. In any case, the completeassignment of absolute stereochemistry could be confirmedonly after asymmetric synthesis Figure 1.2.2. LC–MSn of the Isolated AlkaloidsThe behaviour of the new alkaloids was evaluated using liquidchromatography with multiple stage mass spectrometry (LC–MSn) in positive ion mode, allowing the observation of relevantion species useful for structural elucidation and for the develop-ment of further analytical methods.Compound 1 presented a charged nitrogen in the structure;in the spectrum, it was observed as [M]+ ions at m/z 324 inpositive ion mode. When CID was performed in an ion trap, the[M]+ ion produced the prominent product at m/z 309, due tothe loss of methyl radical (See supplementary material S36).This product ion was further subjected to MS3 analysis leadingto the ion at m/z 294 corresponding to the loss of a furthermethyl radical, confirming the presence of two methoxylgroups in the structure. Ion at m/z 294 was subjected to MS4,which afforded the product ion at m/z 266, corresponding to aloss of 28 Da.Compound 2 presented peculiar behaviour in mass spec-trometry. When ionised in positive mode, it presented ion [M +H]+ at m/z 354. When CID was performed, ion at m/z 354produced ions at m/z 336(2a), 206 (2b), 188 (2c) and 149 (2d)m/z corresponding to sequential loss of 18, 148, 166, 206 Da(Figure S37). The ion m/z 188 and 149 are diagnostic forprotopine-type alkaloids.[24]Compound 3 presented ion [M + H]+ at m/z 370 in positiveion mode. When CID was performed, ion at m/z 370 producedions at m/z 352, corresponding to a loss of 18 Da (Figure S38).In MS3, ion at m/z 352 afforded the product ion at m/z 190corresponding to the formation of methylenedioxy 1,2-dehydromethylisoquinoline ion. This latter was subjected to CIDfragmentation leading to the formation of 131, 149 and 175,corresponding to 1,2-dehydroisoquinoline, a species that origi-nated due to nitrogen loss (uneven ion) and a methylenedioxy1,2 dehydro isoquinoline.Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 245/252] 1Chem. Biodiversity 2024, 21, e202401388 (4 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons LicenseCompound 4 presented ion [M + H]+ at m/z 386 in positiveion mode, with a difference of 16 Da compared to compound 3,corresponding to the N oxide derivative. When CID wasperformed, the [M + H]+ ion produced the prominent productat m/z 368, which was formed by the loss of 18 Da. In MS3, ionat m/z 368 afforded the product ion at m/z 206, correspondingto the formation of methylenedioxy 1,2-dehydromethylisoqui-noline N oxide ion. This latter undergo to CID fragmentationleading the formation of 191 corresponding to methylenedioxy1,2 dehydro isoquinoline N oxide ion, which was formed by theloss of methyl radical (Figure S39).The observed behaviour in mass spectrometry allowed toidentify these compounds with the observation of diagnosticfragments and can be a useful strategy for the further study ofthe presence of such derivatives also in other Corydalis species.2.3. Bioactivity of the Isolated CompoundsThese four compounds were tested for their possible action onmain cholesterol players, such as LDL� R and PCSK9. We firstdetermined the cytotoxicity activity in human hepatocyte-derived cancer cell line Huh7 after a concentration-dependentexposure to the compounds for 72 hours in a cultured mediumwith 10 % fetal bovine serum (FBS). The SRB assay revealed thatonly compounds 2 and 3 elicited a significant cytotoxic actionat 50 μM and 100 μM, respectively (Figure 3).We next performed a series of experiments aimed atevaluating the effect of the four alkaloids on LDL� R and PCSK9intracellular expression by Western blot analysis of total proteinextracts. For these experiments Huh7 cells were incubated for72 hours with 100 μM of compound 4, 50 μM of compound 1and 3, and 25 μM of compound 2. The HMG-CoA reductaseinhibitor simvastatin was utilized as positive control, as well asberberine, an isoquinoline alkaloid, with well-known inhibitoryeffect on PCSK9 transcription. As expected, simvastatin signifi-cantly induced the expression of both LDLR and PCSK9 by 8.3�2.4 and 4.9�2.3 fold, respectively, while berberine showed aminor effect on the LDL� R and a strong inhibition on PCSK9(� 75 %) (Figure 4). Compound 2 and 3 determined a significantincrease of the LDL� R expression by 3.0�1.2 fold and 2.4�0.4fold, respectively (Figure 4). On the contrary compounds 1 and4 did not show any significant effect. Compound 3 appeared toinduce the expression of PCSK9, although the difference wasnot statistically significant. Taken together, only compounds 2Figure 3. Cytotoxicity assay of alkaloids from Nepalese C. chaerophylla D.C. The cytotoxic effect was determined by SRB assay after 72 hours incubation ofHuh7 with 6.25, 12.5, 25, 50 or 100 μM of compounds. Data are expressed as mean�SD of three independent experiments. **p<0.01; ***p<0.001 vsuntreated control by Student’s t-test.Figure 4. Effect of alkaloids from Nepalese C. chaerophylla D.C. on LDL� R and PCSK9 expression in Huh7 cell line. Cells were incubated with simvastatin 5 μMor compound 4 (100 μM), compound 1 and 3 (both at 50 μM), and compound 2 (25 μM) for 72 h. A) Representative western blotting analysis for theexpression of LDL� R and PCSK9 upon treatments. GAPDH was used as loading control; B) Densitometric analysis of LDL� R/GAPDH ratio; C) Densitometricanalysis of PCSK9/GAPDH ratio. Data are presented as mean�SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs control by Student’sT-test. Simva: simvastatin.Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 246/252] 1Chem. Biodiversity 2024, 21, e202401388 (5 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licenseand 3 showed a positive effect on LDL� R expression suggestinga possible hypocholesterolemic activity.To further investigate the potential hypocholesterolemiceffect of these new alkaloids, we determined their action on thesecretion of PCSK9 from Huh7 cell line. Berberine was utilised aspositive control and, as previously described,[25] reduced PCSK9extracellular levels by 40.5 % (Figure 5). All the compoundsshowed, to a different extent, a reduction in PCSK9 secretion,with compounds 1 and 2 that had the most potent andeffective activity.These data indicate that compounds 2 and 3 may havesome potential hypocholesterolemic effect by determining anupregulation of the LDL� R (Figure 4), however compound 2seems to act differently from 3 with an additive inhibitoryaction on the secretion of PCSK9 (Figure 5). Thus, compound 2seems to have a similar mechanism of action of berberine thatwas shown to inhibit PCSK9 transcription, and, as a conse-quence, to lower its intracellular and extracellular (secretion)levels.[26]We then determined their effect of cholesterol biosynthesisin hepatoma HepG2 cell line. As expected, simvastatin andberberine significantly reduced cholesterol biosynthesis (Fig-ure 6). Although to a lower extent than simvastatin andberberine, all the alkaloids partially inhibited cholesterol biosyn-thesis, with compounds 2 that showed the most potent activity(Figure 6).These data indicated that these alkaloids, especially 2,showed a similar mechanism of action than berberine, by bothreducing the expression of PCSK9 and cholesterol biosynthesis,effects that determined a significant increase of the LDL� Rexpression. Further consideration can be done considering thechemical structure, in fact compound 2 is a protopine derivativethat differs from afromentioned alkaloid due to the presence, inposition 13–14 of epoxy group, while in protopine the position14 is occupied by a keto group and position 13 is a CH2. Thisdifference strongly influence the bioactivity of the compound infact in our previous paper we observed that 50 μM protopinestrongly downregulated both the LDLR and PCSK9 in the samecellular model.[14]Finally, we tested the activity of C. chaerophylla D.C. derivedcompounds on lipid accumulation in Huh7 cell line. As shownin Figure 7, after the treatments with the compounds, there wasno significant variation in the quantity of neutral lipids,specifically triglycerides and cholesterol esters, except afterincubation with compounds 2 and 4. In this instance, asignificant reduction in lipid accumulation was observed with 2resembling the effect of simvastatin rather than berberine while4 seems to have a more berberine-like lipid accumulation.Although compound 2 has previously exhibited a berberine-likemechanism of action by reducing PCSK9 secretion and choles-terol biosynthesis with an induction of LDL� R expression, thisassay revealed a protective effect on liver lipid accumulation. Itis, thus, tempting to speculate that there could be a potentialadditional effect on the triglyceride synthesis pathway.Figure 5. Effect of alkaloids from Nepalese C. chaerophylla D.C. on PCSK9secreted by Huh7 cell line. Cells were incubated with berberine 10 μM,simvastatin 5 μM or compound 4 (10 μM), compound 1 and 3 (both at5 μM), and compound 2 (2.5 μM) for 72 h. At the end of the incubation, theconditioned media were collected, and PCSK9 levels were determined byELISA assay. Data are presented as mean�SD of three independentexperiments. *p<0.05 vs control by Student’s T-test. Berb: berberine.Figure 6. Effect of alkaloids from Nepalese C. chaerophylla D.C. on cholesterol biosynthesis in HepG2 cell line. Cells were incubated with simvastatin 5 μM,berberine 20 μM, or compounds at the indicated concentrations for 48 h. At the end of the incubation, cells were incubated with 14C-acetate in MEMcontaining 0.4 % FCS. After 24 h the 14C-acetate incorporation into cholesterol was determined. Data are presented as mean�SD of three independentdeterminations. **p<0.01; ***p<0.001 vs control by Student’s T-test. Simva: simvastatin; Berb: berberine.Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 247/252] 1Chem. Biodiversity 2024, 21, e202401388 (6 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License3. ConclusionsThe Nepalese C. chaerophylla D.C. was subjected to detailedphytochemical analysis. Four new alkaloids were isolated, andtheir structures were established using spectroscopic techni-ques. Compounds 1 is a protoberberine type compound withtwo methoxyl functions in position 3 and 12 and with twohydroxyl groups in position 2 and 9. The compound 2 is aprotopine derivative with epoxy group. Compounds 3 is aspiroindane benzoquinoline and compound 4 is its N-oxidederivative. The new compounds were assayed on key proteinsinvolved in cholesterol metabolism and 2 expressed the mostpromising activity by inhibiting cholesterol biosynthesis, intra-cellular lipid accumulation, and PCSK9 secretion with asignificant increase in LDL� R expression. However, the hypo-cholesterolemic effect of compound 2 still needs to bedetermined in in vivo experimental models.4. Experimental Section4.1. General Experimental ProcedureAll NMR spectra were recorded on Bruker 400 MHz spectrometersoperating at 400.11 MHz for 1H and 100 MHz for 13C. 1H-NMR,13CNMR, COSY, HMBC, HSQC and NOESY experiments were acquiredusing standard Bruker pulses sequences optimising values of p1, d1and mixing times. Mass spectrometry and fragmentations wereobtained on a Varian 500MS Ion trap, while the HR-ESI-MS spectrawere obtained from Waters Xevo G2 QTof. IR spectra were recordedon a Perkin Elmer FTIR spectrometer. Jasco digital 2000 polarimeterwas used for the measurement of optical rotation power, whileJasco J-2000 circular dichroism was used for CD measurements.Column chromatography was performed on silica-gel 40 mesh. Pre-coated silica-gel 60 F254 (0.25 mm) plates were used for TLC. Thepurity of isolated compounds was checked by HPLC using anAgilent 1260 system equipped with a diode array and using anAgilent C18 XDB column (3×150 mm, 3.5 μm) eluting in isocraticmode acetonitrile/water 70/30.4.2. Plant MaterialThe complete botanical specimen of Corydalis chaerophylla D.C.was procured from Phulchowki, Lalitpur, Nepal, at an elevationranging from 2400–2700 metres. The identification of the specimenwas conducted, and a voucher specimen (901) was deposited byMr. Ganga Datt Bhatt, a Research Officer at the National Herbariumand Plant Laboratories located in Godawari, Lalitpur, Nepal.4.3. Extraction and IsolationThe air dried powdered whole plant (10 kg) of C. chaerophylla wascold percolated with hexane (20 L). The hexane fraction wasseparated, and the plant residue was extracted with methanolusing 15 L of solvent. The methanol extract was concentrated onthe rotary evaporator under a vacuum, and 900 g methanol extractFigure 7. Accumulation of neutral lipids after C. chaerophylla D.C. treatments on Huh7 cell line. Cells were incubated with the positive controls and thecompounds at the indicated concentrations for 72 h. At the end of the incubation, neutral lipids were stained with Oil Red-O. Data are presented asmean�SD of three independent determinations. The upper panel showed representative images of Oil Red O staining (n = 6) *p<0.05; **p<0.01;****p<0.0001 vs control by Student’s T-test. Simva: simvastatin; Berb: berberine.Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 248/252] 1Chem. Biodiversity 2024, 21, e202401388 (7 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensewas obtained. The methanol extract was stirred with a 7 % citricacid and filtered. The filtrate was neutralized with NH3 solution andextracted with chloroform. The chloroform extract (120 g) wassubjected to column chromatography over a silica gel column(70 cm×10 cm) by using an ethyl acetate/hexane (0.5/95.5) solventsystem. Then elution was obtained increasing the percentage ofethyl acetate/hexane (5/95) and (10/90). Fractions were checkedusing TLC and based on the detected spots three main fractionswere obtained pooling the collected fractions namely fraction A(16 g), B (22 g) and C (24 g). After eluent system was changed usingchloroform/methanol (90/10), and two fractions, D (9 g) and E(32 g), were obtained pooling the collected fractions. From fractionE significant spots were observed in TLC and was. subjected tosilica gel column chromatography (20 cm×5 cm) using a dichloro-methane (DCM)/methanol (95/5) solvent system with a few dropsof NH3 solution with increasing polarity. A total of 11 sets offractions (E1-E11) were collected, which yielded four new com-pounds. Compound (1) (60 mg) from E9 fraction and compound (2)(30 mg) from E6 fraction yielded from the solvent system DCM/Methanol with a few drops of NH3 solution in the ratio of 8 : 2.Similarly, compound (3) (600 mg) from E3 fraction and compound(4) (500 mg) from E4 fraction were yielded from 9 : 1 DCM/Methanolwith few drops of NH3 solution.Chaeronepaline-A3,12-Dimethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinolin-7-ium-2,9-diol (1) C19H18NO4, dark red powder. [α]D + 15.2(c 0.050, MeOH);UV max 228 274 350 444 nm; IR (KBr) νmax 3372 2922 1609 15131454 1277 1236 1214 cm� 1;1H-NMR (400 MHz, MeOD) and 13 C-NMR(100 MHz, CDCl3) data, see Tables 1 and 2; HR-ESI-MS m/z 324.1238[M]+, calcd for C19H18NO4+,324.1236.Chaeronepaline-B7-Methyl-2,3 : 11,12-bis(methylenedioxy)-7,13a-secoberbin-13-14-ep-oxide (2) C20H20NO5, white amorphous solid. [α]D� 5.1 (c 0.050,MeOH); UV max 240 288 nm; IR (KBr) νmax 3416 2907 1612 14721362 1236 1037 cm� 1 ;1H-NMR (400 MHz, MeOD) and 13 C-NMR(100 MHz, CDCl3) data, see Tables 1 and 2; HR-ESI-MS m/z 354.0980[M + H� 2H]+, calcd for C20H20NO5, 354.0977.Chaeronepaline-C7-methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline (3) C20H20NO6, yellow powder.[α]D + 38.17 (c 0.050, MeOH); UV max 228 242 288 nm; IR (KBr) νmax3416 2892 1620 1476 1391 1236 1037 cm� 1;1H-NMR (400 MHz,MeOD) and 13 C-NMR (100 MHz, CDCl3) data, see Tables 1 and 2; HR-ESI-MS m/z 370.1289 [M + H]+, calcd for C20H20NO6, 370.1290.Chaeronepaline-D7-methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline-N-oxide (4) C20H20NO7, yellowpowder. [α]D + 58.7 (c 0.050, MeOH); UV max 224 242 290 nm; IR(KBr) νmax 3409 2922 2649 1620 1480 1380 1236 1037 cm� 1;1H-NMR (400 MHz, MeOD) and 13 C-NMR (100 MHz, CDCl3) data, seeTables 1 and 2; HR-ESI-MS m/z 386.1240 [M + H]+, calcd forC20H20NO7+, 386.1239.Table 1. 13C NMR Data for compounds 1–4 (δ in ppm, 100 MHz, in MeOD4), *overlapped signals.Position 1 2 3 41 111.1 CH 115.9 CH 109.1 CH 108.8 CH2 146.6 C 148.7 C 145.3 C 144.9 C3 149.3 C 148.8 C 147.4 C 147.4 C4 110.0 CH 108.8 CH 108.2 CH 107.7 CH4a 126.2 C 124.5 C 128.3 C 127.9 C5 27.3 CH2 24.6 CH2 21.5 CH2 21.5 CH26 54.2 CH2 53.7 CH2 47.2 CH2 47.0 CH27 – – – –8 145.9 CH 55.0 CH2 81.6 C 82.4 C8a 120.1 C 127.8 C 120.0 C 118.1 C9 162.0 C 122.9 CH 77.8 CH 77.4 CH9a – – 120.3 C 120.8 C10 107.9 CH 108.6 CH 108.7 CH 108.8 CH11 123.3 CH 148.2 C 149.1 C 148.9 C12 149.7 C 148.1 C 143.5 C 142.8 C12a 132.4 C 124.0 C – –13 117.9 CH 56.5 CH* 115.8 CH 115.1 CH14 134.6 C 56.5 CH* 76.3 CH 75.9 CH14a 120.2 C 127.8 C 135.9 C 135.1 C15 55.6 CH3 41.3 CH3 100.7 CH2 101.4 CH216 55.2 CH3 102.0 CH2 101.7 CH2 102.4 CH217 – 102.0 CH2 37.9 CH3 36.9 CH3Wiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 249/252] 1Chem. Biodiversity 2024, 21, e202401388 (8 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License4.4. Reagents Used for In Vitro ExperimentsEagle’s minimum essential medium (MEM), trypsin- EDTA, penicillin,streptomycin, sodium pyruvate, L-glutamine, nonessential aminoacid solution and fetal bovine serum (FBS) were purchased fromEuroClone (Milan, Italy) as well as all plastic supplies. C. chaerophyllacompounds were diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to a final concentration of 0.08 μM. Simvastatin (Merck,Sharp, and Dohme Research Laboratories, Kenilworth, NJ, USA) wasdissolved to a stock concentration of 50 mM in 0.1 M NaOH, andthe pH was adjusted to 7.2 according to the manufacturer’sinstructions. The solution was then sterilised by filtration. Berberinechloride (cod. B3251, Sigma-Aldrich) was dissolved to a finalconcentration of 80 mM in DMSO.4.5. Cell CulturesHuman hepatic cancer cells (Huh7 and HepG2) were cultured inMEM supplemented with 10 % Fetal Bovine Serum (FBS), 1 % L-glutamine 200 mM, 1 % sodium pyruvate 100×, 1 % nonessentialamino acids 100×, and 1 % penicillin/streptomycin solution(10,000 U/mL and 10 mg/mL, respectively), at 37 °C in a humidifiedatmosphere of 5 % CO2 and 95 % air. For the experiments, cellswere incubated with the indicated final concentrations in MEM/10 % FBS. The final concentration of solvent (DMSO) did not exceed0.5 % v/v, and the same amount was added to all of theexperimental points in each assay.4.6. Cell Viability AssayCells were seeded in MEM/10 % FBS in a 96-well tray at a cellulardensity of 8000 cells/well. The day after, treatments were added(four experimental points for each compound: 50 μM, 25 μM,12.5 μM and 6.25 μM) for 72 h, after which the cell viability wasevaluated by the sulforhodamine B assay (SRB) according to apreviously published protocol.[18]4.7. Western Blot AnalysisHuh7 cells were seeded in MEM/10 % FBS in 6-well trays at thecellular density of 300,000 cells/well. The day after, the medium wasreplaced with the compounds at the indicated concentrations inDMEM/10 % FBS. After 72 h of incubation, intracellular proteincontent was extracted in lysis buffer (50 mM Tris pH 7.5, 150 mMNaCl, and 1 % Nonidet-P40, containing 1 % v/v of protease andphosphatase inhibitor cocktails). Protein samples (25 μg) and amolecular mass marker (Bio-Rad, Hercules, CA, USA) were separatedusing 4–20 % SDS-PAGE (Bio-Rad) under denaturing and reducingconditions. The protein samples were then transferred to a nitro-cellulose membrane using the Trans-Blot® Turbo™ Transfer System(Bio-Rad), and nonspecific binding sites were blocked with a 5 %non-fat dried milk tris-buffered tween 20 (TBS� T20) solution, withagitation for 60 min at room temperature. The blots were incubatedovernight at 4 °C with a diluted solution (5 % non-fat dried milk) ofanti-LDLR (rabbit polyclonal antibody, GeneTex GTX132860; dilution1 : 1000), anti-PCSK9 (rabbit polyclonal antibody, GeneTexGTX129859; dilution 1 : 1000), anti-GAPDH (rabbit polyclonal anti-body, GeneTex GTX100118; dilution 1 : 10000). The membranesTable 2. 1H-NMR Data for compounds 1–4 (δ in ppm, 400 MHz, in MeOD4).Position 1 2 3 41 7.39, s, 1H 7.14, s, 1H 6.30, s, 1H 6.30, s, 1H2 – – – –3 – – – –4 6.93, s, 1H 6.79, s, 1H 6.68, s, 1H 6.71, s, 1H4a – – – –5 3.16, t, 2H, J=6.2 3.25, m, 2H 2.97, m, 2H 3.08, m, 2H6 4.64, t, 2H, J=6.2 4.48, m, 2H 3.62 3.68, m, 2H 3.80 3.74, m, 2H7 – – – –8 9.30, s, 1H 3.77 3.51, m, 2H – –8a – – – –9 – 6.86, d, 1H, J=7.0 5.34, s, 1H 5.39, s, 1H9a – – – –10 6.94, d, 1H, J=8.0 6.81, d, 1H, J=7.0 6.87, s, 1H 6.91, s, 1H11 7.58, d, 1H, J=8.0 – – –12 – – – –12a – – – –13 8.03, s, 1H 3.85 m* 6.89, s, 1H 6.90, s, 1H14 – 3.86 m* 5.53, s, 1H 5.58, s, 1H14a – – – –15 3.90, s, 3H, OCH3 2.91, s, 3H 5.81, s, 2H 5.85, s, 2H16 3.94, s, 3H, OCH3 6.00, s, 2H 6.00, s, 2H 6.02, s, 2H17 – 6.00, s, 2H 2.84, s, 3H 2.97, s, 3HWiley VCH Donnerstag, 12.12.20242412 / 374575 [S. 250/252] 1Chem. Biodiversity 2024, 21, e202401388 (9 of 11) © 2024 The Author(s). Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202401388 Research Article 16121880, 2024, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202401388 by National Institute For, Wiley Online Library on [16/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensewere washed with TBS� T20 and exposed for 90 min at roomtemperature to a diluted solution (5 % non-fat dried milk) of thesecondary antibodies (peroxidase-conjugate goat anti-rabbit, Jack-son ImmunoResearch, dilution 1 : 5000, cod. 111–036-045). Immu-noreactive bands were detected by exposing the membranes toClarityTM Western Enhanced Chemi Luminescence (ECL) chemilumi-nescent substrates (Bio-Rad) for 5 min, and images were acquiredwith an Uvitec Alliance Q9 (Uvitec, Cambridge, UK). The densito-metric readings were evaluated using Image LabTM software (Bio-Rad).4.8. Cholesterol Biosynthesis AssayHepG2 cells were seeded in MEM supplemented with 10 % FBS in12-well trays with a density of 1×10 6 cells/well. After 24 hours, cellswere treated with the compounds at the indicated concentrationsdissolved in DMEM supplemented with 0.2 % (w/v) Bovine SerumAlbumin (BSA; Merck, Darmstadt, Germany) for 48 hours. Subse-quently, cells were incubated for an additional 24 hours with MEMsupplemented with 2 μCi/mL [2–14C] acetate (Perkin-Elmer, MA, USA),0.4 % (v/v) FBS, 0.5 % v/v of a solution of sodium acetate [8 mg/ml]together with the compounds, that were thus incubated for a totalof 72 hours. Cell monolayers were lysed with 0.1 M NaOH overnightat 4 °C; 105 cpm/sample of [1,2–3H] cholesterol (Perkin-Elmer, MA, USA)were then added as an internal reference to each cell lysate andsaponification was performed at 60 °C for 1 hour in alcoholic KOH.Lipid extraction was carried out using low-boiling point PetroleumEther; thin-layer chromatography (TLC) was performed usingpetroleum ether 40°–60 °C/diethyl ether/acetic acid (70 : 30 : 1) asmobile phase to separate cholesterol from the other cellular sterolsand to allow the quantification of radioactivity derived from theincorporation of [2–14C] acetate through liquid scintillation counting.Cellular cholesterol biosynthesis was expressed as cpm per milli-gram of protein for each cell lysate, measured through thebicinconic acid (BCA) assay (Thermofisher, MA, USA) following themanufacturer’s instructions (PMID: 36293049).4.9. Neutral Lipid Staining with Oil Red-OTo perform Oil Red-O staining, Huh7 cells were seeded 50,000 cells/well in 24-well plates with sterile microscope cover glasses 10 mmØ (VWR international). After 24 hours, the medium was replaced byfresh MEM/10 % FBS containing the described treatments. 72 hourslater, cells were rinsed with PBS and fixed in 2 % formaldehyde for10 min and washed with PBS without calcium and magnesium.After a short rinse in 20 % isopropanol, Oil Red-O (Sigma-Aldrich,Cod. O0625) solution was added. Oil Red-O stock solution is 0.5 %w/v in isopropanol, diluted in distilled water 6 : 4 and filtered twicewith 0.220 μm PVDF and PES filters prior to use. The staining is leftfor 20 minutes, then rinsed with 20 % isopropanol and tap waterrespectively, for 1–2 min. Nuclei were stained with DAPI solution inPBS (Sigma-Aldrich, cod. D9542), then rinsed in tap water and twicein distilled water.The slides or the coverslips were mounted with Fluoromount™Aqueous Mounting Medium (Sigma-Aldrich, Cod. F4680). Imageswere obtained with Leica DMRE mounting Leica camera with Leica541 517 HC zoom and Leica Application Suite X Software. Oil RedO-stained areas were quantified using ImageJ (v.1.52 h, NIH) andnormalised with nuclei count.4.10. Statistical AnalysisData are expressed as mean� standard deviation. Differencesbetween the two groups were analysed via Student’s t-test analysis(GraphPad, San Diego, CA, USA). P-values lower than 0.05 wereconsidered statistically significant.Author ContributionsConceptualisation: B.M., R.L.S., S.S., S.D.A., N.F.; Data curation:B.M., I.R., V.B., S.S., N.F.; Formal analysis: B.M., R.L.S., I.R., V.B., S.S.,S.D.A., B.P., M.P.A.; Funding acquisition: R.L.S., N.F., S.D.A.;Investigation and methodology: I.R., V.B., S.S. ; Project admin-istration: R.L.S., N.F., S.D.A.; Supervision: S.D.A, N.F., R.L.S.;Visualisation: B.M., I.R., V.B., S.S.S.; Roles/Writing - original draft:S.S., B.M., R.L.S., S.D.A; Writing - review & editing: B.M., S.S.S.,S.S., S.D.A., B.P., M.P.A., I.R., R.L.S., L.K.S., J.P.H., K.A.AcknowledgementsB. Maharjan acknowledges University Grants Commission (UGC),Sanothimi, Bhaktapur, Nepal, for awarding research grantsunder PhD Fellowship and Research Support (PhD-75/76-S&T-6)Open Access publishing facilitated by Università degli Studi diPadova, as part of the Wiley - CRUI-CARE agreement.Conflict of InterestsThe authors declare no conflicts of interest.Data Availability StatementData will be made available on request.Keywords: Isoquinoline alkaloids · Corydalis chaerophylla D.C. ·PCSK9 · LDL� R[1] W. Majak, Y. Bai, M. H. Benn, Biochem. Syst. Ecol. 2003, 31, 649–651.[2] H. L. Li, W. D. Zhang, R. H. Liu, C. Zhang, T. Han, X. W. Wang, X. L. Wang,J. B. Zhu, C. L. Chen, J. Chromatogr. B 2006, 831, 140–146.[3] Z. Z. Ma, W. Xu, N. H. Jensen, B. L. Roth, L. Y. Liu-Chen, D. Y. W. Lee,Molecules 2008, 13, 2303–2312.[4] M. Iranshahy, R. J. Quinn, M. Iranshahi, RSC Adv. 2014, 4, 15900–15913.[5] S. Bhambhani, K. R. Kondhare, A. P. Giri, Molecules 2021, 26, DOI:10.3390/molecules26113374.[6] P. 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See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttps://doi.org/10.1155/2018/3171348https://doi.org/10.1007/s43450-020-00066-whttps://doi.org/10.1007/s43450-020-00066-whttps://doi.org/10.1016/j.bioorg.2022.105686https://doi.org/10.1080/14786410801996390https://doi.org/10.1080/14786410801996390https://doi.org/10.1002/cbdv.202301209https://doi.org/10.1002/cbdv.202301209https://doi.org/10.1093/jnci/82.13.1107https://doi.org/10.1016/j.ijpddr.2014.07.001https://doi.org/10.1055/s-2008-1074952https://doi.org/10.1139/v69-593https://doi.org/10.5478/MSL.2013.4.4.79https://doi.org/10.1016/j.numecd.2019.06.001https://doi.org/10.1016/j.atherosclerosis.2008.02.004 Bioactive Alkaloids from Nepalese Corydalis chaerophylla D.C. Acting on the Regulation of PCSK9 and LDL-R In Vitro 1. Introduction 2. Result and Discussion 2.1. Structural Elucidation of New Isolated Compounds 1–4 2.2. LC–MSⁿ of the Isolated Alkaloids 2.3. Bioactivity of the Isolated Compounds 3. Conclusions 4. Experimental Section 4.1. General Experimental Procedure 4.2. Plant Material 4.3. Extraction and Isolation Chaeronepaline-A Chaeronepaline-B Chaeronepaline-C Chaeronepaline-D 4.4. Reagents Used for In Vitro Experiments 4.5. Cell Cultures 4.6. Cell Viability Assay 4.7. Western Blot Analysis 4.8. Cholesterol Biosynthesis Assay 4.9. Neutral Lipid Staining with Oil Red-O 4.10. Statistical Analysis Author Contributions Acknowledgements Conflict of Interests Data Availability Statement