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Binita Maharjan, [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), Shyam Sharan Shrestha, Stefania Sut, Ram Lal Swagat Shrestha, Stefano Dall’Acqua

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[Chemical characterization of Corydalis chaerophylla D.C. extracts and preliminary evaluation of their in vitro and in vivo biological properties](https://mdr.nims.go.jp/datasets/82504be7-a267-43ed-a419-4d2359c194eb)

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Chemical Characterization of Corydalis chaerophylla D.C. Extracts and Preliminary Evaluation of Their in Vitro and in Vivo Biological PropertiesChemical Characterization of Corydalis chaerophylla D.C.Extracts and Preliminary Evaluation of Their in Vitro and inVivo Biological PropertiesBinita Maharjan,[a] Lok Kumar Shrestha,[b, c] Jonathan P. Hill,[b] Katsuhiko Ariga,[b, d]Shyam Sharan Shrestha,[e] Stefania Sut,[f] Ram Lal Swagat Shrestha,*[a] andStefano Dall’Acqua*[f]Genus Corydalis is a rich source of isoquinoline alkaloidsreported to having potential bioactivities. Corydalis chaerophyllacollected from Nepal at an altitude of 2400–4800 m wasextracted using hexane, methanol and chloroform as solvents.The resulting hexane, methanol and chloroform extracts weresubjected to LC–DAD-MSn analysis to yield fifteen differentalkaloids. To assess any potential pharmacological properties,antimicrobial activity against two Gram-positive, two Gram-negative bacterial strains and one fungal strain was assessed,revealing significant inhibitive action of the methanol andchloroform extracts. Of the extracts obtained using chloroformcontained the highest content of phenolic compounds at113 mg GAE/g, while the highest total flavonoid content wasfound for the hexane extract with a value of 46.45 mg QE/g.The chloroform extract also exhibited a considerable antiox-idant activity at IC50 value, 261.5�3 μg/mL, for the DPPH assay.Conversely, the methanol extract exhibited the highest LC50value for Brine Shrimp cytotoxicity at 196�3 μg/mL being leastpotential for the test. The methanol extract was found to be themost active against α-amylase inhibition with an IC50 of 51.52�2 μg/mL. In an in vivo acute oral toxicity study against mice,methanol and chloroform extracts presented harmful effectswith 1000.36 mg/kg BW and 515 mg/kg BW for LD50, respec-tively. By analyzing all the results of the solvents used, thechloroform extract was found to be the most active, a featurethat will be used in future isolation procedures and otherpharmacological tests.IntroductionPlant derived natural products and chemicals obtained fromother natural sources as, bacteria, yeast, fungi, and marinespecies have been, since antiquity, the most important sourcesfor mankind for the development of medicines and remedies,and still in the recent years new bioactive agents have beendiscovered and brought to the clinical uses. Thus nature is aunique library of bioactive natural compounds and theirstructural variety, complexity, and propensity for displayingbiological activities explain their significant role as pharmaco-phore and lead molecule sources in medicinal chemistry andthereafter for the development of clinical medications.[1–3] Plantsare in many cases rich sources of natural products and theproduction of bioactive secondary metabolites is strictly relatedto numerous factors including plant genetics environmentalfactors. The Corydalis genus in particular has been prolific as asource of natural products. There are 470 species in the genusCorydalis worldwide, 57 of which are found in Nepal.[4] Thisgenus is abundant in isoquinoline alkaloids, which serve as achemical defense against different microorganisms andherbivores.[5,6] Although certain species in this genus have beenimplicated in poisoning cases involving cattle, many others arethought not to be poisonous. Corydalis species are extensivelyemployed in conventional Chinese medicine due to theirevident antibacterial, antiviral, and anticancer properties.[7–9] InAsian folk medicines, they are used to cure a variety ofdisorders,[10–12] and research has revealed positive activity[a] B. Maharjan, Dr. R. L. Swagat ShresthaDepartment of Chemistry, Amrit Campus, Tribhuvan University, 44613Kathmandu, NepalE-mail: swagatsta@gmail.com[b] Dr. L. Kumar Shrestha, Dr. J. P. Hill, Dr. K. ArigaResearch Center for Materials Nanoarchitectonics (MANA), National Institutefor Materials Science (NIMS), 1-1 Namiki, 305-0044 Tsukuba, Ibaraki, Japan[c] Dr. L. Kumar ShresthaDepartment of Materials Science, Faculty of Pure and Applied Sciences,University of Tsukuba, 1-1-1 Tennodai, 305-8573 Tsukuba, Ibaraki, Japan[d] Dr. K. ArigaGraduate School of Frontier Sciences, The University of Tokyo, 5-1-5Kashiwanoha, 277-8561 Kashiwa, Chiba, Japan[e] Dr. S. Sharan ShresthaHimalayan Research and Development Center, Nepal, 44613 Kathmandu,Nepal[f] Dr. S. Sut, Dr. S. Dall’AcquaDepartment of Pharmaceutical and Pharmacological Sciences, University ofPadova, Via Marzolo 5, 35121 Padova, ItalyE-mail: stefano.dallacqua@unipd.itSupporting information for this article is available on the WWW underhttps://doi.org/10.1002/cbdv.202301209© 2023 The Authors. 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 Mittwoch, 13.12.20232312 / 327099 [S. 263/274] 1Chem. Biodiversity 2023, 20, e202301209 (1 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGwww.cb.wiley.comdoi.org/10.1002/cbdv.202301209 Research Articlehttps://doi.org/10.1002/cbdv.202301209http://crossmark.crossref.org/dialog/?doi=10.1002%2Fcbdv.202301209&domain=pdf&date_stamp=2023-11-14against ulcers,[13] breast cancer,[14] and leishmaniasis,[15] andexcellent pharmacokinetic properties.[1] Corydalis is known tosynthesize sanguinarine, alongside a group of anti-inflammatorybenzylisoquinoline alkaloid (BIA) compounds referred to ascavidines. Examples of cavidines include apocavidine, cavidine,dehydroapocavidine, and dehydrocavidine.[16] The extract ofCorydalis edulis (CE), was found to induce insulin exocytosisthrough a signaling pathway that is dependent on proteinkinase C (PKC). Furthermore, CE was observed to specificallyactivate new protein kinase Cs (nPKCs) and atypical PKCs(aPKCs), while not affecting conventional PKCs (cPKCs), in HIT-T15 cells. CE exhibits promising capacity as a new therapeuticagent for the management of type 2 diabetes mellitus (T2DM)in human subjects.[17]Corydalis chaerophylla D.C. (C. chaerophylla), collected fromNepal, is a glabrous herb found in high-altitude regions ofNepal, India, and Pakistan, flourishing in moist, shaded environ-ments at an altitude of 2400–4800 m. It has been used to treatsyphilis, scrofula, dysentery, and diarrhoea.[14] In traditionalmedicinal use, the juice of C. chaerophylla is given for pepticulcers in doses of about 4 teaspoons, 3 times per day. Juice ofthe root (dosage: about 6 teaspoons, 3 times per day) is givenfor indigestion where the root juice is blended in equalamounts with the root juice of Cyathula capitate Moq.(N. Kuro).[18] Previous scientific studies of other Corydalis speciesled to the separation of numerous alkaloids that have proveneffective in improving immune function, and in inhibiting thegrowth of tumours, cancer-causing liver viruses, amoebae, andhepatitis.[19–21] However, a review of the literature has revealedthat little research has been performed specifically on C. chaer-ophylla. Considering the chemical composition one paperreported the isolation and structural elucidation, from C. chaer-ophylla the alkaloids chaerophylline, corypalmine, berberine,isocorypalmine, corydalmine and bicuculline have beenisolated.[22]To improve the knowledge on Nepalese C. chaerophylla andto further explore the potential usefulness as bioactive com-pound source of this plant here we report a comparativeanalysis of extracts of C. chaerophylla. The proposed approachincluded a simple spectrophotometrically based screening,liquid chromatography diode array and mass spectrometricdetection (LC–DAD-MS) profiling, TPC, TFC, antimicrobial, anti-oxidant, cytotoxicity, antidiabetic and in vivo acute oral toxicityanalysis. The present work will become the basis for the furtherbioassay-guided isolation of bioactive compounds (Figure 1).Results and DiscussionYields of Extracts and Phytochemical ScreeningThe yields of hexane, methanol and chloroform extracts werecalculated to be 0.14%, 13.12% and 2.20%, respectively.Phytochemical screening performed on the extracts revealedthat alkaloids, flavonoids, glycosides, steroids and terpenoidsare present in the hexane extract. The methanol extractcontained alkaloids, flavonoids, phenolic compounds, carbohy-drates, glycosides, steroids, tannins, terpenoids and saponins,while the chloroform extract contained alkaloids, flavonoids,phenolic compounds, carbohydrates, glycosides, steroids, tan-nins, and terpenoids, as shown in Table 1.Figure 1. Overview of Research process.Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 264/274] 1Chem. Biodiversity 2023, 20, e202301209 (2 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 LicenseAlkaloid composition studied by Liquid ChromatographyDiode-Array Detection and multiple stage mass spectrometry(LC–DAD-MSn)The extracts were analyzed combining the Diode-Array detec-tion (DAD) and multiple stage mass spectrometry (MSn) toobtain structural information on the eluted compounds. Thechromatograms obtained from the hexane, methanol andchloroform extracts showed numerous peaks with significantUV absorptions. Based on the UV spectra of the major peaks,the presence of different classes of alkaloids, can be at leastpreliminary established.[23] Peaks presenting UV max at 296 nmsuggest the presence of benzyltetrahydroisoquinoline deriva-tives, while protoberberinic alkaloids are characterized by theUV with maximum at 425 nm and 345 nm, and phthalideisoquinolinic by the maximum at 320 nm and 290 nm.Furthermore, in the last part of the chromatogram, peaksshowing maximum absorption at 325 nm and 283 nm supportthe presence of benzophenanthridine alkaloids. The Figures 2–4summarise that the main peaks of the three extracts are clearlysupporting the presence of different alkaloids on the basis oftheir UV spectra.Interpretation of MSn spectra, comparison with the relevantliterature, and the confirmation obtained with the injection ofreference compounds allowed the identification of fifteendifferent alkaloids (1–15) that were also quantified on the basisof the LC–DAD in the three extracts. Compounds and amountare summarised in Table 2, structures are reported in Figure 5and spectra are reported in supplementary materials. Fromqualitative point of view the use of multiple stage allowed theprofiling of the alkaloid content in the different extracts.Table 1. Phytochemical screening of extracts of C. chaerophylla.Phytochemicals Type of Tests Result in ExtractsH M CAlkaloids Dragendorff”s Test + + +Mayer’s Test + + +Wagner’s Test + + +Flavonoids Lead Acetate Test + + +Shinoda Test + + +Phenolic Compounds Ferric Chloride Test � + +Lead Acetate Test � + +Alkaline Reagent Test � + +Carbohydrates Molisch’s Test � + +Fehling’s Test � + +Benedict’s Test � + +Proteins Millon’s Test � � �Biuret Test � � �Glycosides Keller-Killiani Test + + +Legal’s Test + + +Steroids Salkowski’s Test + + +Tannins Ferric Chloride Test � + +Lead Acetate Test � + +Alkaline Reagent Test � + +Anthraquinones Anthraquinones Test � � �Terpenoids Chloroform test + + +Saponins Foam test � + �H: hexane, M: methanol, C: chloroform.Figure 2. LC–DAD chromatogram (280 nm) of the hexane fraction, the UV spectra ascribable to main classes of isoquinoline alkaloids are shown.Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 265/274] 1Chem. Biodiversity 2023, 20, e202301209 (3 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 magnocurarine (1) presence was established due to themolecular ion [M+H]+ at m/z 314 which due to the loss of thedimethylamino group lead to the ion at m/z 269 that furthergenerate the fragment at m/z 175 due to the loss ofhydroxybenzylic group. N� Me-tetrahydropalmatine (2) wasassigned on the basis of the molecular ion [M+H]+ at m/z 370and fragments due to methyl loss generating m/z 352 and thefragment at m/z 190 formed from the N-methyl-isoquinoliniummoiety. Similar fragmentation scheme can be observed for thetetrahydro columbamine (3) with molecular ion [M+H]+ at m/z342 generating in MS2 the species at m/z 178 ascribable to themethoxy-hydroxy-isoquinolinium ion. Sharing similar structureFigure 3. LC–DAD chromatogram (280 nm) of the methanol fraction, the UV spectra ascribable to main classes of isoquinoline alkaloids are shown.Figure 4. LC–DAD chromatogram (280 nm) of the methanol fraction, the UV spectra ascribable to main classes of isoquinoline alkaloids are shown.Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 266/274] 1Chem. Biodiversity 2023, 20, e202301209 (4 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 Licenseis the compound presenting molecular ion [M+H]+ at m/z 356which in MS2 form species at m/z 192, 177 and 148 correspond-ing to the formation of the N-methyl-methoxy-hydroxy-dihy-droisoquinolinium species and the corresponding loss of methylgroup and CO (m/z 148) and that was assigned to N� Me-tetrahydrocolumbamine (4). The compound presenting molec-ular ion [M+H]+ at m/z 324 that in MS2 showed the loss of 15and 17 Da leading to ion species at m/z 309 and 307respectively. The MS3 fragmentation led to the further loss of15 Da originating species at m/z 294 and 279. MS4 of the ion atm/z 294 generate fragments at m/z 269, 266 and 237. Thecompound can be assigned to a demethylene berberine (5) orto its isomer. Peak presenting m/z 368 was assigned tobicuculline (6) and the typical fragments of the phthalideisoquinoline are the ion at m/z 190 formed from the N-methyl-isoquinolinium moiety originated after the bond cleavage withphthalide ring. Fragmentation of the ion at m/z 190 show lossof the methyl group generating the ion at m/z 175. Theidentification was confirmed by co-injection of referencecompound. The compound (7) showed molecular ion [M+H]+at m/z 400 and a loss of water leading to m/z 382. Furtherfragments were revealed at m/z 337, 319 and 289 supportingthe presence of a protoberberine derivative bearing a hydroxylsubstituent and methoxylation. Structure hypothesis for com-pound 7 is presented. The benzophenantridin derivativeprotopine (8) m/z 354 was identified on the basis of thediagnostic fragments at m/z 336 due to water loss and the oneat m/z 188 and 204 corresponding to the structural cleavageand subsequent loss of water from the isoquinoline fragment. Asimilar derivative showing the same loss of water and intensefragment at m/z 190 presented molecular ion [M+H]+ at m/z356 and was assigned to hunnemanine (9) a benzophenan-tridine derivative.The protoberberine alkaloid jatrorrhizine (10) presentedmolecular ion [M+H]+ at m/z 338 was identified on the basis ofthe loss of the methyl groups generating m/z 323,324 and 307as well as due to the cleavages that separate the isoquinolineand the benzyl moieties generating small ions at m/z 190. Thefragmentation in the experimental condition was comparedwith authentic standard and retention time as well asfragmentation pattern were superimposable. Two more proto-berberine derivatives were observed. The alkaloid presentingmolecular ion [M+H]+ at m/z 368 showed loss of water and ofmethyl groups and it was assigned to berberastine (11). Thecompound presenting molecular ion [M+H]+ at m/z 352 wasassigned to palmatine (12) due to the loss of the methyl groupand the strong MS2 signal at m/z 336 and intense signals at 348and 307. The peak presenting molecular ion [M+H]+ at m/z366 and fragments at 348, 318 and 307 and 190 was tentativelyassigned to dehydrocorydaline (13). Two intense peaks athigher retention time were assigned to benzophenantridinicalkaloids namely sanguinarine (14) characterized by the molec-ular ion [M+H]+ at m/z 332 and fragment at 317 and intensefragment at 304 as reported in the literature and from theanalysis of reference compound. The second presenting molec-ular ion [M+H]+ at m/z 334 and similar pattern of fragmenta-tion but with intense signal in MS2 at m/z 319 and 304 wasassigned to dihydrosanguinarine (15).Thus, the Nepalese C. chaerophylla, presented variousclasses of alkaloids and it can be a valuable botanical source forthe extraction of those compounds.Table 2. Compounds observed in LC–DAD-MS of extracts of C. chaerophylla.S.N. Rt m/z Fragments Compound Extracts (mg/g) ReferenceH M C1. 6.2 314 298.0, 269.0, 237.0, 175.0, 137.0, 107.0 Magnocurarine – 0.12 0.08 [24]2. 6.8 370 352.0, 334.0, 320.0, 290.0, 190.0, 175.0, 149.0,131.0N� Me-tetrahydropalmatine 0.77 23.31 218.013. 8.53 340 178.0, 163.0 Tetrahydrocolumbamine 2.31 4.10 1.37 [25,26]4. 8.58 356 192.0, 177.0, 148.0 N� Me-tetrahydrocolumb-amine0.28 1.10 1.155. 8.65 324 309 Demethylene berberine 4.26 12.48 45.386. 8.84 368 307.0, 190.0 Bicuculline* 17.94 93.24 115.307. 9.04 400 382.0, 355.0, 337.0, 319.0, 279.0 Protoberberine derivative 0.05 4.14 8.288. 9.31 354 338.0, 190.0, 188.0, 149.0, 130 Protopine* 5.08 58.69 99.04 [24,27]9. 9.34 356 338.0, 190.0 Hunnemanine 0.58 64.29 64.4910. 9.4 338 323.0, 294.0, 307.0, 279.0 Jatrorrhizine* 22.04 116.04 63.16 [24,25]11. 9.5 368 338.0, 353.0, 321.0, 320.0, 307.0, 278.0 Berberastine 3.22 6.38 2.16 [24]12. 9.76 352 336.0, 321.0, 308.0, 292.0, 278.0, 292.0, 275.0 Palmatine 3.30 8.30 2.09 [25,26]13. 11.5 366 348.0, 323.0, 307.0, 190.0 Dehydrocorydaline 0.22 0.21 – [25]14. 15.8 332 317.0, 304.0, 274.0, 246.0 Sanguinarine* 1.88 2.52 0.36 [24,27]15. 16.3 334 319.0, 304.0, 275.0, 246.0, 261.0 Dihydrosanguinarine 54.32 1.19 2.06 [24]H: hexane, M: methanol, C: chloroform; compounds indicated with “*” were also confirmed by standard injection.Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 267/274] 1Chem. Biodiversity 2023, 20, e202301209 (5 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 LicenseFigure 5. Structures of the identified alkaloids.Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 268/274] 1Chem. Biodiversity 2023, 20, e202301209 (6 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 quantitative data revealed that the use of solvents withdifferent polarity can influence the composition of the obtainedextract. In particular the nonpolar hexane is less favorable forthe extraction of the whole alkaloids but in quite specific waycan be used to obtain fraction mostly presenting the morenonpolar compounds as dihydrosanguinarine. The methanoland chloroform extracts on the other hand present larger abilityto extract the different alkaloids and efficiently can extractjatrorrhizine, protopine and hunnemanine.Total Phenolic and Flavonoid Content AnalysisTotal phenolic content (TPC) was calculated as a milligram ofgallic acid equivalent using the gallic acid calibration curve, andtotal flavonoid content (TFC) per gram of dried material isexpressed as milligrams of quercetin equivalents.[28,29] As can beseen from Figure 6, the methanol and chloroform extractscontain similar concentrations of phenolic and flavonoidcompounds. Methanol extract has a TPC of 113.38 mg GAE/gand a TFC of 25.25 mg QE/g. Chloroform extract also has a TPC113.90 mg GAE/g with TFC 24.95 mg QE/g. However, hexanepresented a lower quantity of phenolic (TPC: 18.43 mg GAE/g)but a higher quantity of flavonoids (TFC: 46.45 mg QE/g).The antioxidant properties of plants are directly correlatedwith the phenolic and flavonoid content they possess. Thesesubstances function as reducing agents, providing hydrogenatoms, and possess the ability to scavenge free radicals.[30,31] Theplant extracts of C. chaerophylla exhibit a noteworthy abun-dance of phenolics and flavonoids, which are likely to play asubstantial role in the antioxidant properties. Due to theaforementioned characteristics, it is probable that this plantspecies has been employed in various traditional herbalremedies. The amount of phenols and flavonoids are alsoinfluenced by the polarity of the solvents employed during theextraction process.[32]Antimicrobial Screening AnalysisThe area around an antimicrobial disk where germs do notproliferate is known as the zone of inhibition.[33] To estimate theantimicrobial activity of plant extracts, the diameter of the zoneof inhibition (ZOI) formed on certain bacterial and fungalcultures was measured. Using the method described in thesucceeding section, the ability of various fractions of C. chaer-ophylla extracts to inhibit bacterial and fungal growth at a fixeddose (200 mg/mL) was evaluated. The results were representedin terms of the diameter of the zone of inhibition (table 3).Hexane, methanol, and chloroform extracts of C. chaerophyl-la were evaluated for their capacity to inhibit the growth of twoGram-positive bacteria, Bacillus subtilis ATCC 6051 and Staph-ylococcus aureus ATCC 6538P, and two Gram-negative bacteria,Escherichia coli ATCC 8739 and Klebsiella pneumonia ATCC700603, and a fungal culture, Candida albicans ATCC 2091. Thezone of inhibition (ZOI) was measured in centimeters.The antimicrobial properties of hexane, methanol andchloroform extracts at concentration of 200 mg/mL wereinvestigated and results are shown in Table 3. Methanol andchloroform extract showed comparable ZOI in all microbesused relative to the positive control Kanamycin 5 mg/mL.Whereas, the hexane extract showed antimicrobial activityagainst Staphylococcus aureus and Candida albicans only. Theobserved potent antimicrobial activity exhibited by the extractsfrom C. chaerophylla suggests that these plant extracts has thepotential to serve as valuable antimicrobial agents. Further-more, methanol and chloroform extracts may serve as apromising starting point for the isolation and identification ofexciting antimicrobial compounds.Figure 6. Total phenolic (TPC) and total flavonoid (TFC) content of the extracts of C. chaerophylla.Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 269/274] 1Chem. Biodiversity 2023, 20, e202301209 (7 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 LicenseAntioxidant Screening AnalysisThe DPPH results showing the percentage scavenged obtainedby the absorbance at 517 nm of each extract is shown inTable 4.The IC50 values of hexane, methanol, and chloroformextracts of C. chaerophylla were found to be 1586�3 μg/mL,758.3�3 μg/mL, and 261.5�3 μg/mL, respectively. Accordingto the data, hexane extract was shown to be the least effectiveat scavenging DPPH, methanol revealed moderate scavengingproperty whereas chloroform extract was found to be mosteffective. The observed DPPH radical scavenging activity can beattributed to the greater amount of phenolic compounds in themethanol and chloroform extracts compared to the hexaneextract. These phenolic compounds are known to inhibit orprevent the detrimental effects of oxidative stress.[34] Phenoliccompounds exhibit reducing and antioxidant propertiesthrough the hydrogen-donating ability of their hydroxylgroups.[35]In vivo Brine Shrimp Lethality AnalysisThe fact that the mortality rate of brine shrimp nauplii causedby C. chaerophylla increases with increasing concentrationsuggests that the hexane, methanol, and chloroform extractscontain cytotoxic agents that show dose dependent effect.The lethality concentration (LC50) of the three extracts,hexane, methanol and chloroform, were calculated as 112.3�3 μg/mL, 196.0�3 μg/mL and 153.9�3 μg/mL, respectively(Table 5). This result indicates that the hexane, methanol andchloroform extracts of C. chaerophylla are all active againstbrine shrimp lethality in vivo test. However, the hexane extractexhibited the lowest LC50 value for Brine Shrimp cytotoxicity at112.3 mg/mL hence, the most active. The lethality of the threeplant extracts against brine shrimps suggests that there isevidence of strong cytotoxic and perhaps anticancer constitu-ents within the plant. Since the data were obtained at extractconcentrations lower than 1000 μg/mL, all the extracts can beconsidered to be cytotoxic.[36]Antidiabetic AnalysisIn this study, the in vitro α-amylase inhibitory assay wasemployed to evaluate the antidiabetic properties of the extractsderived from C. chaerophylla.The hexane, methanol, and chloroform extract of C. chaer-ophylla were found to have an IC50 values of 1156�3 μg/mL,51.52�2 μg/mL, and 93.13�2 μg/mL, respectively (Table 6).From the data, methanol extract was found to have moresignificant activity for α-amylase inhibition than the otherextracts with hexane extract being least active. The enzyme α-amylase is accountable for the process of hydrolyzing the 1,4-glucosidic bond found in substances such as starch, glycogen,and oligosaccharides. One of the techniques employed in themanagement of diabetes involves the inhibition of specificenzymes responsible for facilitating the breakdown of starch,hence reducing glucose absorption[37] thereby preventingTable 3. Antimicrobial activity of extracts of C. chaerophylla.Bacterial/FungalstrainReferencecultureType Positive control(c+) cmNegative control(c� ) cmHexaneExtractZOI (cm)MethanolExtractZOI (cm)ChloroformExtractZOI (cm)Escherichia coli ATCC 8739 Gram� ve 2.6 0 0 2.5 1.8KlebseillapneumoniaeATCC 700603 Gram� ve 1.8 0 0 1.2 1.7Bacillus subtilis ATCC 6051 Gram+ve 2.6 0 0 1.2 1.7StaphylococcusaureusATCC 6538P Gram+ve 2.6 0 1.3 2.0 2.3Candida albicans ATCC 2091 Fungi 2.4 0 1.5 2.0 2.3Kanamycin was used as positive control at concentration of 5 mg/mL.Table 4. IC50 values for DPPH assay of different extracts at of C.chaerophylla.Extracts Concentration(μg/mL)PercentageScavengedIC50(μg/mL)Hexane Extract 4000 69.80 1586�32000 59.241000 37.35500 21.29250 14.36MethanolExtract1000 64.72 758.3�3500 54.02250 25.19125 8.7362.5 3.87ChloroformExtract1000 64.26 261.5�3500 57.13250 39.23125 26.3362.5 15.25Reference compounds used in the assay: Ascorbic acid IC50 12 μg/mL.Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 270/274] 1Chem. Biodiversity 2023, 20, e202301209 (8 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 Licensepostprandial hyperglycemia conditions. Further studies areneeded to assess the potential antidiabetic properties of theidentified alkaloids.In vivo Acute Oral Toxicity StudyThe median lethal doses (LD50) of the active ingredient inC. chaerophylla extracts were found to be >2000 mg/kg BW forhexane extract,[57–59] 1000.36 mg/kg BW for methanol extract,and 515 mg/kg BW for chloroform extract for mice underenvironmental conditions (Table 7).The data obtained reveal that hexane extract can beconsidered safe or less harmful, showing no in vivo toxicity. Incontrast, methanol extract is slightly hazardous, with chloroformextract being moderately hazardous. The different toxicities ofthe extracts are probably due to their variable compositions.ConclusionsThis work is focused on investigating phytochemicals fromC. chaerophylla to establish their relative in vitro and in vivobiological properties. Extraction of plant materials using hexane,methanol and chloroform to give extracts, and their phyto-chemical screening have been performed. LC–DAD-MSn of thethree extracts indicated the presence of fifteen differentalkaloids. Hexane extract contained high amount of dihydrosan-guinarine, the spectrophotometrical assays indicated a largerflavonoid component over phenolic content. In contrast,methanol and chloroform extracts present high amount ofJatrorrhizine and Bicuculline while the methanol one alsopresents high amount of N� Me-tetrahydropalmatine. Themethanol and chloroform extract in the spectrophotometricalmeasurements showed a more significant phenolic componentover flavonoids. Methanol and chloroform extracts exhibitedsignificant ZOI against all tested pathogens in antimicrobialassay. Methanol extract exhibited moderate results for DPPHscavenging while the chloroform extract is more active. Allthree extracts presented cytotoxic activity against Brine Shrimpdemonstrating >1000 μg/mL LC50. The methanol and chloro-form extracts can be considered as an important inhibitor of α-amylase in antidiabetic activity due to the measured IC50 values.Furthermore, the in vivo acute oral toxicity in mice revealed aharmful effect of methanol and chloroform extract whenswallowed.Experimental SectionCollection of the Plant MaterialsC. chaerophylla plants were collected in Phulchowki, Lalitpur, Nepal,at an altitude between 2400 and 2700 m (27.5711° N, 85.4056° E)Table 5. LC50 values for Brine Shrimp Lethality Assay of different extractsof C. chaerophylla.Extracts Concentration(μg/mL)PercentageMortalityLC50(μg/mL)Hexane Extract 1000 100 112.3�3500 100250 80125 56.6762.5 43.33Methanol Extract 1000 100 196.0�3500 96.67250 56.67125 46.6762.5 43.33ChloroformExtract1000 100 153.9�3500 100250 100125 43.3362.5 23.33Table 6. IC50 values for α-amylase inhibition of different extracts of C.chaerophylla.Extracts Concentration(μg/mL)Percentage α-amylaseinhibitionIC50(μg/mL)Hexane Ex-tract5000 70.31 1156�32500 51.171250 41.83625 31.48312.5 22.19MethanolExtract500 65.17 51.52�2250 60.19125 51.3062.5 39.34ChloroformExtract500 70.93 93.13�2250 62.04125 41.6562.5 38.61Acarbose was used as the standard and IC50 was 6.1�0.1 μg/mL.Table 7. Median Lethal Dose (LD50) of different extracts of C. chaerophylla.Extracts LD50(mg/kg BW)Hazard Statement RemarksHexane >2000 mg/kg BW May be harmful if swallowed No death at 2000 mg/kgMethanol 1000.36 mg/kg BW Harmful if swallowed Death with convulsionChloroform 515 mg/kg BW Harmful if swallowed Death with convulsionWiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 271/274] 1Chem. Biodiversity 2023, 20, e202301209 (9 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 identified by Mr. Ganga Datt Bhatt, Research Officer, NationalHerbarium and Plant Laboratories, Godawari, Lalitpur, Nepal(Voucher specimen 901).ExtractionThe plant materials so collected were cleaned, dried in the shadeand processed into a powder. The powdered C. chaerophylla plantmaterial was treated with hexane. The hexane extract was thenconcentrated using a rotary evaporator. The plant residue remainedafter the hexane extraction was extracted using methanol employ-ing cold percolation and the Soxhlet process. The resultingmethanol extract solution was concentrated using a rotaryevaporator. The crude methanol extract was then treated with 7%citric acid, neutralized with NH4OH, and extracted using chloroform.Phytochemical ScreeningThe extracts, namely hexane, methanol, and chloroform, weresubjected to analysis in order to determine the presence of variouschemical constituents including alkaloids, flavonoids, phenoliccompounds, carbohydrates, proteins, glycosides, steroids, tannins,anthraquinones, terpenoids, and saponins. This analysis wasconducted following the standard protocol as described inreferences.[38,39] The qualitative findings are represented by thesymbols (+) and (� ), denoting the presence and absence ofphytochemicals, respectively in Table 1.LC–DAD-MSnDried extracts obtained after solvent removal of the hexane,chloroform and methanol plant extracts, were exactly weighted(20 mg) and dissolved in methanol by sonication and then analysedby LC–DAD-MSn. An Agilent 1260 chromatograph equipped withautosampler and diode array (DAD) detector was used forchromatographic separation, while Varian Mass spectrometer (MS)Ion Trap, model MS500, was used operating in positive ion modewith electrospray (ESI) ion source. For the separation, agilent SBC184.6×50 mm (1.8 micron) was used. As eluent mixtures of threedifferent solvents in gradient mode was used, as reported in theTable 8.The flow rate was set to 750 μL/min. Diode array spectra wereacquired in the 200–600 nm range. After the column, the flow wassplit using a passive T junction allowing detection by DAD and MS.Spectra were acquired using the TDDS function of the instrumentthat allows the fragmentation of detected ionic species. Alkaloidswere detected in positive ion mode, phenolics were detected innegative ion mode. Berberine, protopine, and bicuculline wereused as reference alkaloids. Rutin, quercetin, catechin, epicatechin,chlorogenic acid and gallic acid were used as reference phenoliccompounds. All compounds were also used for quantitativepurposes by generating calibration curves.[24–27,40]Total Phenol Content AssayThe total concentration of phenolic compounds in the hexane,chloroform and methanol extracts was determined using the Folin-Ciocalteu method at 765 nm. Estimated total phenolic contents aregiven as Gallic acid equivalents per gram of dried extract.[29,41,42]Total Flavonoid Content AssayTotal flavonoid concentration in the extracts of C. chaerophylla wasascertained using the aluminium chloride colorimetric method at415 nm using methanol as control and Quercetin was used as astandard. The flavonoid concentration was calculated as mg ofequivalent quercetin per gram of dried material.[41,43]Antimicrobial ActivityPreparation of microbial culture mediaThe liquid broth (LB) media was prepared by dissolving 13 g of LBpowder (Himedia research Laboratories Pvt. Ltd., India) in 1 L ofwater. The mixture was autoclaved at 15 psi pressure at 121 °C for25 minutes. The sterilized media was cooled down to 40–50 °C,followed by transferring into sterilized 15 mL falcon tubes (5 mLeach). The prepared media was used to co-culture bacterial seedculture in each tube separately and was incubated for 24 hours.Preparation of MH media plates and antimicrobial assayThe Mueller -Hinton Agar (MHA) plates were prepared by dissolving39 g of MH agar powder (Himedia Research Laboratories Pvt. Ltd.,India) in 1 L of water. The mixture was autoclaved at 15 psi pressureat 121 °C for 25 minutes. The sterilized media was cooled down to40–50 °C, followed by transferring into Petri dishes (25 mL each).The prepared media plates were stored in refrigerator until used.The prepared media plates were labeled properly with samplenames and 150 μL liquid bacterial suspension were spread with thehelp of sterile cotton swab on the surface of media plates. The wellswere made on the surface of agar and the samples 200 mg/mL(100 μL) and standard kanamycin 5 mg/mL (10 μL) were loaded inthe prepared well. The media plates were then incubated for24 hours at 37 °C. The antimicrobial test results were observed after24 hours.[44–46]Antioxidant ActivityAntioxidant assays were performed against DPPH assay at 517 nmusing and methanol as a control.[47] A calibration curve was alsoconstructed. The IC50 value (Inhibitory concentration of 50%) is theeffective sample concentration that must be present for the sampleto scavenge 50% of the DPPH free radicals. Using the doseinhibition curve in the logarithm range, we were able to determinethe IC50 values by plotting the extract concentration vs. thecorresponding scavenging action.[48–50]Table 8. Ternary gradient.Min Water 1% formic acid Acetonitrile MeOH0 95 5 02.5 85 15 012 80 18 215.5 50 40 1019 20 70 1021 0 85 1523 0 85 1524 0 100 026 0 100 027 95 5 0Wiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 272/274] 1Chem. Biodiversity 2023, 20, e202301209 (10 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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 LicenseBrine Shrimp Lethality AssayLethality experiments using brine shrimp (Artemia salina, fairyshrimp, or sea monkeys) were carried out. The nauplii of brineshrimp were exposed for 24 hours to solutions containing varyingconcentration of each extracts from C. chaerophylla. The number ofmotile nauplii was used to determine efficacy of the three extract.The Brine Shrimp Lethality Assay indicates that tested extract areactive if the LC50 value is lower than 1000 μg/mL.[51–55]In vitro inhibition of α-amylaseIn this experiment, the 3,5-dinitrosalicylic acid (DNSA) techniquewas used to inhibit α-amylase. C. chaerophylla extracts were dilutedto a minimum concentration of 10% DMSO. The material wascombined with buffer, NaCl, and DMSO at a pH of 6.9 to provide awide range of concentrations. A 200 μL aliquot of this mixture wasthen added to the α-amylase solution and the mixture wasincubated for 10 minutes at 30 °C. After that, 200 μL of the 1%starch solution was added to each tube, and the tubes were left toset for 3 minutes. Addition of DNSA reagent (200 μL), detained thisprocess. The combined sample was heated in an 85–90 °C waterbath for 10 minutes. After the combined sample had cooled toroom temperature, it was diluted with distilled water (5 mL). A UVspectrophotometer was used to measure the sample‘s absorbanceat 540 nm and compared to the blank solution. Phosphate bufferpH 7.4 (200 μL), was used in place of the plant extract to produce ablank with 100% enzyme activity.[56] A plot of extract concentrationagainst percentage of α-amylase inhibition was constructed toobtain the IC50 values, which show the concentration at which α-amylase inhibition is 50%.In vivo assaysAll the experimental protocols were reviewed and approved withreference number 312079/80.In vivo Acute Oral Toxicity StudyBased on the Chemical Testing Guidelines for Acute Oral Toxicity,the OECD’s Acute Toxic Class Method 425 was applied. In theNatural Product Research Laboratory (NPRL), Thapathali, Nepalpharmacology lab, experiments were conducted on mice. Thesubjects were fasted for twelve hours prior to commencement ofthe experiment. The body weight of the mice was measured justprior to provision of the extracts. Two groups of animals wererandomly assigned. The first (control) group received physiologicalsaline, the second group received the extract through an orogastrictube at a dose of 2000 mg/kg of body weight, as reference Table 9was used to classify effects.Clinical observations of the subjects were conducted four times perday, with close attention paid to their behavior, general health,nasal mucosa, alterations to their skin and fur, respiratoryfrequency, somatomotor activity, and any potential occurrence ofsymptoms such as tremors, convulsions, diarrhoea, fatigue, salivat-ing, low response to stimuli, sleep, light sensitivity, and coma.Abdominal palpation was also performed. The experimental groupwas given 2000 mg/kg of seed oil following 48 hours of clinicalobservation, during which there were no indicators of harm. Thestatistical test “t-Test for Independent Groups” was implemented inSTATISTIC V. 7.0 for Windows; P values of 0.005% were consideredrelevant. The mice were cremated humanely at the conclusion ofthe experiment.[57–59]Author ContributionsConceptualization: B. Maharjan, R. L. Shrestha, S. Sut, S.Dall’Acqua; Lab Work: B. Maharjan, S. Sut, S. Dall’Acqua, J. P. Hill,K. Ariga; Manuscript preparation: B. Maharjan, S. Sut, S.Dall’Acqua and R.L. Shrestha; Review and Editing: S. S. Shrestha,R. L. Shrestha, J. P. Hill, K. Ariga; Supervision: S. Dall’Acqua andR. L. Shrestha.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).Conflict of InterestsThe authors declare no conflict of interest.Data Availability StatementThe data that support the findings of this study are availablefrom the corresponding author upon reasonable request.Keywords: Corydalis chaerophylla · extraction · chemicalanalysis · bioactivitiesTable 9. Classification of substances according to the guidelines of the Globally Harmonized system of classification and labeling of chemicals (GHS), thirdedition[57]Ranges (mg/kg) Category Classification Hazard statement>2000 mg/kg Category 5 Not classified May be harmful if swallowed>300�2000 mg/kg Category 4 Dangerous Harmful if swallowed>50�300 mg/kg Category 3 Toxic Toxic if swallowed>5�50 mg/kg Category 2 Very toxic Fatal if swallowed<5 mg/kg Category 1 Highly toxic Fatal if swallowedWiley VCH Mittwoch, 13.12.20232312 / 327099 [S. 273/274] 1Chem. Biodiversity 2023, 20, e202301209 (11 of 12) © 2023 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AGdoi.org/10.1002/cbdv.202301209 Research Article 16121880, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cbdv.202301209 by Cochrane Japan, Wiley Online Library on [20/12/2023]. 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