New amide and diterpene alkaloids with anticholinesterase activity from Delphinium cyphoplectrum roots.

BACKGROUND: The cholinergic hypothesis posits a robust correlation between the onset of Alzheimer's disease and a pronounced deficit in acetylcholine, a pivotal neurotransmitter crucial for the central cholinergic nervous system's function, pivotal for memory and learning. Diterpene alkaloids exhibit intricate and distinctive chemical structures that facilitate their passage through the blood-brain barrier. Moreover, their potent pharmacological attributes render them promising candidates for addressing central nervous system disorders. OBJECTIVES: This investigation aims to scrutinize the alkaloidal composition of Delphinium cyphoplectrum (Ranunculaceae) roots, further exploring their anticholinesterase inhibitory activity and mode of inhibition. METHOD: Innovative chromatography techniques were repetitively employed to purify the alkaloids. Acetylcholinesterase (AChE) inhibition assays were conducted using Ellman's tests. The mode of inhibition was meticulously characterized through Michaelis-Menten, and Lineweaver-Burk plots. Conducting molecular docking studies, we employed the AUTO DOCK 4.2 software package. RESULTS: Eight alkaloids were identified including five C19-diterpene alkaloids (6,14,16,18-tetramethoxy-1,7,8-trihydroxy-4-methylaconitane (1), 6,16,18-trimethoxy-1,7,8,14-tetrahydroxy-4-methylaconitane (2), 6,8,16,18-tetramethoxy-1,7,14-trihydroxy-4-methylaconitane (3), 6,14,16-trimethoxy-1,7,8,18-tetrahydroxy-4-methylaconitane (4), and 14-O-acetyl-8,16-dimethoxy-1,6,7,18-tetrahydroxy-4-methylaconitane (5)), an epoxy C18-diterpene alkaloid (6,8,16-trimethoxy-1,7,14-trihydroxy-3,4-epoxyaconitane (6)), a known (pyrrolidin-2-one (7) and an undescribed amide alkaloid (1-(2'-hydroxylethylamine)-3,5,5,-trimethyl-1,5-dihydro-2H-pyrrol-2-one (8). All diterpene alkaloids underwent assessment for acetylcholinesterase (AChE) inhibition assay and displayed noteworthy AChE activity, surpassing that of the reference drug (with IC50 values of 13.7, 21.8, 23.4, 28.2, 40.4, and 23.9 for compounds 1-6, respectively, in comparison to 98.4 for Rivastigmine). Analysis of Michaelis-Menten and Lineweaver-Burk plots represents an uncompetitive mode of inhibition for compound 1 on AChE. Notably, computational docking simulations indicated that all diterpene alkaloids were accommodated within the same enzymatic cleft as the reference ligand, and displaying superior free binding energy values (from - 10.32 to -8.59 Kcal.mol-1) in contrast to Rivastigmine (-6.31 Kcal.mol-1). CONCLUSION: The phytochemical analysis conducted on the roots of Delphinium cyphoplectrum yielded the identification of eight alkaloidal compounds including one C18-diterpene, five C19-diterpene, one pyrrolidine and one amide alkaloids. AChE inhibition assay and molecular simulations unveiled remarkable significant potency attributed to the C19-diterpene alkaloids by the order of 1 > 2 > 3,6 > 4 > 5. Presence of hydroxyl group on C-1, C-7, C-8, C-14, and C-18 increased the effect. The best in vitro activity was recorded for compound 1 able to bind to Asp72 in the narrow region of PAS, while interacting by pi-sigma with Phe330 at the hydrophobic region of the gorge involving the acyl and choline binding site. This observation underscores the substantial promise of this category of natural products in the realm of drug discovery for Alzheimer's Disease, offering a compelling avenue for further research and therapeutic development.

Brunonianines A-C, C20-diterpenoid alkaloids with cyano group from Delphinium brunonianum Royle.

Cyano tends to have better biological activity, but it is rarely reported in natural products, especially in the C20-diterpene alkaloids. Herein, three unprecedented C20-diterpenoid alkaloids, brunonianines A-C (1-3), possessing rare cyano functional group as well as an atisine backbone constructed from a phenethyl substituent and a tetrahydropyran ring, along with four C19-alkaloids (4-7) and one amide alkaloids (8), were isolated from the whole plant of Delphinium brunonianum Royle. Compounds 1-3 are also the first atisine type diterpenoid alkaloids with cyano group obtained from nature. The structures of the previously undescribed compounds were elucidated by HR-ESI-MS, 1D/2D NMR spectroscopic data and electronic circular dichroism calculations and single-crystal X-ray diffraction. Reasonable speculations have also been made regarding the biogenic synthetic pathways of compounds 1-3. In addition, the inhibitory activity of all compounds was also tested against four tumor lines: A549, Caco-2, H460 and Skov-3, where compound 2 (IC50 2.20 ± 0.21 µM) showed better inhibitory activity against Skov-3 cells than the hydroxycamptothecin. Using flow cytometry, cell staining, migration and invasion analysis, and Western blot, compound 2 was found to arrest cells in the G2/M phase and was able to effectively inhibit cell motility to achieve potent anti-tumor effects. In addition, compound 2 can effectively induce apoptosis by activating the Bax/Bcl-2/Caspase-3 signaling pathway.

Diterpenoid Alkaloids from Delphinium ajacis and Their Anti-inflammatory Activity.

Three novel diterpenoid alkaloids, comprising two C19 -diterpenoid alkaloids (1 and 2) and one C20 -diterpenoid alkaloid (3), were isolated from Delphinium ajacis, alongside the six known compounds (4-9). Their structures were elucidated by spectroscopic methods (MS, UV, IR, 1D and 2D NMR) and chemical properties. Simultaneously, the anti-inflammatory properties of all compounds (1-9) was conducted, focusing on nitric oxide (NO) production in LPS-induced BV-2 cells. The results indicated compounds 1-3, 7, and 8 have potential anti-inflammatory activity.

Kamaonensine A-G: Lycaconitine-type C19-diterpenoid alkaloids with anti-inflammatory activities from Delphinium kamaonense Huth.

Delphinium kamaonense Huth is a sort of folkloric plant resource which is cultivated and planted with great ornamental and medicinal values. In this work, seven undescribed lycaconitine-type C19-diterpenoid alkaloids, especially a rare skeleton with -CH=N and N-oxide moieties, along with ten known compounds, were isolated from D. kamaonense, of which the structures were determined by various spectroscopic data, combined with calculated electronic circular dichroism (ECD) and single-crystal X-ray diffraction analysis. In vitro nitric oxide inhibitory activities assay of these compounds indicated that lycaconitine-type C19-diterpenoid alkaloids had significant anti-inflammatory inhibitory activities, with kamaonensine E being the most potent (0.9 ± 0.2 µM) stronger than positive (9.0 ± 1.3 µM). In the network pharmacology studies, binding three key targets mitogen-activated protein kinase 8 (MAPK8), mitogen-activated protein kinase 14 (MAPK14), and heat shock protein HSP 90-alpha (HSP90α), the anti-inflammatory mechanism might be related to MAPK signaling pathways. Furthermore, the molecular docking results revealed that the uncommon amides and methylenedioxy groups might be the most two promising pharmacophores for lycaconitine-type C19-diterpenoid alkaloids.

Diterpenoid alkaloids from Delphinium trichophorum.

Three new hetisine type C20-diterpenoid alkaloids, named as trichophorines A-C (1-3), were isolated from Delphinium trichophorum, together with nine known alkaloids (4-12). Their structures were elucidated on the basis of spectroscopic data (1D, 2D NMR, single-crystal X-ray, and HR-ESI-MS). All compounds were evaluated for the inhibitory activities against LPS induced NO production in RAW 264.7 macrophage cells, and none of them showed considerable inhibitory activity.

Two New C19 -Diterpenoid Alkaloids from Delphinium shawurense.

Shawurenine C (1a) and D (1b), a new pair of regioisomeric C19 -diterpenoid alkaloids, and five known C19 -diterpenoid alkaloids (2-6) were isolated from the aerial part of Delphinium shawurense W. T. Wang. The chemical structures of new compounds were established based on spectroscopic analyses: HR-ESI-MS, and 1D, 2D NMR spectroscopic data. The anti-inflammatory and cytotoxic activities of these diterpenoid alkaloids were also evaluated.

A new diterpenoid alkaloid from Delphinium gyalanum C. Marquand & Airy Shaw.

A new C19-diterpenoid alkaloid named gyalanutine A (1) and fourteen known compounds 2-15 were isolated from the plant of Delphinium gyalanum C. Marquand & Airy Shaw. Compound 1 displayed an unusual lycoctonine-type C19-diterpenoid alkaloid skeleton with the cleavage of N-C19 and C7-C17 bonds, and the construction of the N-C7 bond. Structures were identified by multiple spectroscopic analyses including 1 D, 2 D NMR, IR and HR-ESI-MS. Compounds were tested for acetylcholinesterase inhibitory and anti-inflammatory activity.

Isolation, crystal structure, DFT calculation and molecular docking of uncinatine-A isolated from Delphinium uncinatum.

The main objective of our present research work was to explore molecular insight for potentially active new acetylcholinesterase inhibitor from the aerial parts of Delphinium uncinatum. New norditerpenoid alkaloids, uncinatine-A, was isolated from the basic alkaloidal fraction of D. uncinatum, based on bioactivity guided isolation. The structure of uncinatine-A was determined through latest spectroscopic techniques including single X-Ray diffraction technique. The structural data and electronic properties of uncinatine-A was also calculated by Density Functional Theory (DFT) using B3LYP/6-31þ G (p) basis set. The isolated natural product was evaluated for their acetyl cholinesterase inhibitory potential in dose dependent protocol (62.5-1000 µg/mL), followed by molecular docking studies. Significant competitive type inhibition activity (IC50 = 207.73 ± 0.3) was shown by isolated natural norditerpenoid against cholinesterase targets in comparison with standard drugs available in the market such as galanthamine. The molecular docking results showed that isolated natural product was well accommodated by AChE in the active site with docking scores -11.0326. This is the first report indicating uncinatine-A as a potent acetylcholinesterase inhibitor and can be used as a target drug in cerebral dementia and Alzheimer diseases.

In Vitro and In Silico Investigation of Diterpenoid Alkaloids Isolated from Delphinium chitralense.

This study reports the isolation of three new C20 diterpenoid alkaloids, Chitralinine A-C (1-3) from the aerial parts of Delphinium chitralense. Their structures were established on the basis of latest spectral techniques and single crystal X-rays crystallographic studies of chitralinine A described basic skeleton of these compounds. All the isolated Compounds (1-3) showed strong, competitive type inhibition against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in comparison to standard allanzanthane and galanthamine however, chitralinine-C remained the most potent with IC50 value of 11.64 ± 0.08 µM against AChE, and 24.31 ± 0.33 µM against BChE, respectively. The molecular docking reflected a binding free energy of -16.400 K Cal-mol-1 for chitralinine-C, having strong interactions with active site residues, TYR334, ASP72, SER122, and SER200. The overall findings suggest that these new diterpenoid alkaloids could serve as lead drugs against dementia-related diseases including Alzheimer's disease.

Three New Compounds from Delphinium kamaonense Hunth and Their in†Vitro Cytotoxic and Potential Antioxidant Activities.

A new amide (1), two new phenylpropanoid derivatives (2, 3), along with three new natural products, including three nitrogen chirality compounds, N-(3-methoxy-1,3-dioxopropyl)-D-phenylalanine methyl ester (4), N-(3-methoxy-1,3-dioxopropyl)-L-phenylalanine methyl ester (5), and N-acetyl-L-phenylalanine methyl ester (6), as well as dimethyl (2R,3R)-2-hydroxy-3-(((E)-3-(4-hydroxyphenyl)acryloyl)oxy)succinate (7) and dimethyl (S,E)-2-((3-(4-hydroxy-3-methoxyphenyl)acryloyl)oxy)succinate (8) were isolated from Delphinium kamaonense Hunth. Their structures were elucidated by extensive analysis of 1D and 2D NMR, and HR-ESI-MS experiments, and the absolute configurations were determined by comparative analysis of specific optical rotation. Compound 1 exhibited a moderate cytotoxicity effect against Hep-3B cancer cell lines (IC50 41.39±0.13 µM) and an excellent antioxidant activity (IC50 0.527±0.06 µM in ABTS assay, and 1.235±0.09 µM in DPPH assay, respectively), which was superior to vitamin C in ABTS (IC50 1.670±0.07 µM) and DPPH (IC50 19.10±0.40 µM) methods.

Polysubstituted Cyclopentene Benzamides and Dianthramide Alkaloids from Delphinium anthriscifolium Hance.

Thirteen new benzamide alkaloids, delphiniumines A-M (1-13), together with one known analogue (14), were isolated from Delphinium anthriscifolium Hance. All of the structures were determined by spectroscopic and spectrometric analyses. Absolute configuration for 1 was established using experimental and calculated ECD data, as well as by X-ray crystallography analysis. Compound 1 possesses a previously undescribed polysubstituted cyclopentene carbon framework. Compound 2 was isolated as an artifact from 1 during the extraction process. Compound 7 is glycosylated with a ß-D-glucose unit. Compound 13 bears a chlorine substituent. At a concentration of 10 µM, compounds 6, 8, and 10-12 suppressed LPS-induced NO production in RAW264.7 cells with inhibition rates ranging from 40.3% to 78.8%.

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

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

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

Antifungal Activity of New Diterpenoid Alkaloids Isolated by Different Chromatographic Methods from Delphinium peregrinum L. var. eriocarpum Boiss.

This paper aimed to investigate the potential antifungal influences of new alkaloids from Delphinium peregrinum L. var. eriocarpum Boiss. New Diterpenoid alkaloids Delcarpum (1), Hydrodavisine (4) and known alkaloids Peregrine (2), Delphitisine (3) were isolated by different chromatographic methods from the aerial parts of D. Peregrinum eriocarpum Boiss, which grows in Syria. The structures of alkaloids were proposed based on 1D NMR spectroscopy 1H-NMR, 13C-NMR, DEPT-135, DEPT-90, 2D NMR spectroscopy DQF-COSY, HMQC, EI-Ms mass spectrum, and IR spectroscopic measurements. The antifungal activity of the isolated alkaloids was evaluated against different dermatophyte fungal isolates compared with fluconazole. In the case of Peregrine (2) the minimum inhibitory concentrations(MICs) recorded 128-256, 32-64, and 32 for Epidermophyton floccosum, Microsporum canis, and Trichophyton rubrum, respectively, compared to 32-64, 16, and 32 µg/mL in the case of fluconazole, respectively. The MICs recorded on application of the four alkaloids mixture were 64, 32, and 16 in the case of E. floccosum, M. canis, and T. rubrum, respectively, which were significantly lower than that measured for each of the individual alkaloid and were compatible for fluconazole. In conclusion, MICs of the tested alkaloids showed a variable potential effect on the investigated fungal isolates. Peregrine (2) was the most effective alkaloid, however, the application of the mixture of alkaloids induced significant synergistic activity that was more pronounced than the application of individual ones.

Alkaloids of Delphinium grandiflorum and their implication to H2O2-induced cardiomyocytes injury.

Three new diterpenoid alkaloids (1-3), and eight known alkaloids (4-11) were isolated from the aerial parts of Delphinium grandiflorum. Grandifline A (1) represents an unprecedented diterpenoid alkaloid ring system featuring a C-7NC17 hemiaminal moiety and a lactone fragment through the linkage of C-17OC19 unit. And we named this newly-discovered class of rearranged C19-diterpenoid alkaloid scaffold as grandiflodines (B-12). Grandifline B (2) is the first naturally-occurring 7,17-secolycoctonine diterpenoid alkaloid with a C-7OC17 unit forming a hemiacetal. Their structures were elucidated via spectroscopic data and single-crystal X-ray diffraction analysis. The protective effects of compounds 1-11 on H2O2-induced cardiomyocytes injury were assayed. And compounds 6 and 10 showed significant protective effects, with IC50 values of 1.881 ± 0.680 µM and 1.904 ± 0.750 µM, respectively. Further, compound 6 could reduce oxidative damage by inhibiting cell death via the AMPK/AKT/mTOR signaling pathway in H2O2-induced H9C2 cells.

Phytochemical analysis of crude extract of Delphinium brunonianum and its effect on hypertension and metabolic perturbations in fructose fed rats.

The present study aims at phytochemical profiling and valuating the effect of crude extract of Delphinium brunonianum on fructose mediated rise in blood pressure and metabolic abnormalities in rats. Therefore, rats were fed on fructose (10%w/v) for 6 weeks. Rats in treatment groups received amlodipine 250, 500 and 1000 mg/kg of DB-Cr separately in concurrent to fructose. Various parameters of metabolic perturbations were assessed at the end of study. Further, DB-Cr was analyzed using LC-MS technique. DB-Cr exerted remarkable antihypertensive effect whereas, sympathetic hyperactivity and hyperinsulinemia in these rats was significantly blunted, further, endothelium functionality was successfully restored. LC-MS analysis of DB-Cr revealed the presence of a variety of chemical constituents (41) including quinic acid, scopolin, gingerol, Robinetin 3-rutinoside, KAPA and maleic acid. In conclusion, D. brunonianum possess the potential to combat the fructose mediated hypertension and metabolic perturbations, which may partially be due to its chemical constituents.

Phytochemical analysis and reappraisal of diuretic activity of Delphinium brunonianum Royle and its mode of action in experimental rats.

The aim of this study was the evaluation of diuretic potential of Delphinium brunonianum. Acute diuretic effect in rats was evaluated 8 h after administration of various doses of crude extract, fractions and hydrochlorthiazide. While, prolonged effect of butanolic fraction was assessed after 7days of oral administration in rats. Thereafter, involvement of different pathways in diuretic activity was also appraised. Furthermore, polyphenolic contents in butanolic fraction were assessed using HPLC/UV-VIS technique. All doses of extract and fractions induced a prominent increase in urine and Na+ excretion with no effect on excretion of K+. Prior administration of indomethacin and atropine considerably avoided the diuretic effect of butanolic fraction. Regarding the quantitative chemical analysis the polyphenolic contents were recorded as 28.78 µg/mg. Thus results of present investigation suggested that Delphinium brunonianum possess remarkable diuretic potential.

Delphatisine D and Chrysotrichumine A, two new diterpenoid alkaloids from Delphinium chrysotrichum.

Chemical investigations of the aerial parts of Tibetan folk medicine Delphinium chrysotrichum resulted in the isolation of two new diterpenoid alkaloids, delphatisine D (1) and chrysotrichumine A (2), together with ten known diterpenoid alkaloids (3-12) and 3ß,6α-dihydroxysclareolida (13). Delphatisine D (1) is a rare atisine-type alkaloid from genus Delphinium and is the C-15 epimer of spiramine C which bears an internal carbinolamine ether linkage (N-C-O-C) between C-7 and C-20. Chrysotrichumine A (2) is a rare natural C19-diterpenoid alkaloid possessing a nitrone group between C-17 and C-19. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compound 13 is a sesquiterpene first isolated from this genus. Compounds 1, 3-7 and 13 were investigated for cytotoxicity against human breast cancer cell lines of MCF-7 and MDA-MB-23, none of them presented significant activity except compound 7 which exhibited slight activity at 100 µM against MDA-MB-231, but did not reach the EC50.

The effect of Delphinium denudatum (Jadwar) on fatigue: A randomized double blind placebo-controlled clinical trial.

OBJECTIVES: Fatigue is a common problem in modern-day life. The aim of this study was to evaluate the effect of Delphinium denudatum (Jadwar) on fatigue. METHODS: This study was a randomized double-blind placebo-controlled clinical trial between healthy normal university students. In each group, participants were given one capsule of either WEACURE® (containing 500 mg of Jadwar root powder) or placebo for 15 consecutive days. Multidimensional Fatigue Inventory (MFI) questionnaire was used before and after the intervention to evaluate different aspects of fatigue. RESULTS: A total number of 64 participants completed the study. Data analysis showed decrease in the scores of all five domains of fatigue in Jadwar group (13.31 ±â€¯3.05-7.75 ±â€¯2.66, 12.31 ±â€¯3.55-7.63 ±â€¯2.62, 12.22 ±â€¯4.26-6.97 ±â€¯2.06, 11.56 ±â€¯4.21 to 7.28 ±â€¯2.37, 12.91 ±â€¯3.09-7.34 ±â€¯2.13 in general fatigue, physical fatigue, reduced activity, reduced motivation, and mental fatigue domains, respectively) which was statistically significant (P value<0.0001). This situation was significantly superior to the placebo group. Prescribed dosage of WEACURE® capsule was well tolerated. CONCLUSION: As a complementary tonic agent, Jadwar have a potential to reduce fatigue in normal population. However, objective evaluation of its anti-fatigue effect should be further evaluated.

New amide alkaloids from Delphinium brunonianum.

Five new amide alkaloids, named delamide A-E (1-5), along with five known ones, methyl-N-(3-carboxy-2-methylpropanoyl) anthranilate (6), benzoic acid, 2-[(1-oxodecyl) amino]-methylester (7), puberline (8), benzoic acid, 2-[(4-methoxy-2-methyl-1, 4-dioxobutyl) amino]-methylester (9) and benzoic acid, 2-[(4-methoxy-3-methyl-1, 4-dioxobutyl) amino]-methylester (10) were isolated from the extract of Delphinium brunonianum. Their structures were elucidated by extensive spectroscopic analyses (including 1D-, 2D-NMR, and HR-ESI-MS). 1-10 were also evaluated for their acetylcholinesterase (AChE) inhibiting activity by the Ellman's method. Delamide A (1) showed highly selective AChE inhibition activity. The kinetic analysis revealed that 1 was a mixed-type reversible inhibitor of AChE.