Limonexic Acid Research Articles

Magnetic ligand fishing protocol combined with HPLC-FT-ICR-MS for screening potential α-Glucosidase inhibitors from UCG and in silico analysis

Uremic Clearance Granule (UCG) is a traditional Chinese medicines (TCMs) that has been recognized as potentially effective treatment for diabetic nephropathy (DN). However, the chemical composition and the hypoglycemic ingredients of UCG remain unclear. In this experiment, a magnetic ligand fishing protocol combined with HPLC coupled Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR-MS) technology was developed to screen and identify α-Glucosidase inhibitors from UCG. First, constituents of UCG was identified by HPLC-FT-ICR-MS. Next, α-Glucosidase coated Fe3O4@SiO2@NH2 (Fe3O4@SiO2@NH2@α-Glucosidase) nano composites was synthesized. The essential parameters (concentration of Glutaraldehyde (GA), ratio of Fe3O4@SiO2@NH2 to enzyme, crosslinking time and immobilization time) were optimized to obtain maximum enzyme activity and the performance of immobilized enzyme was compared with that of free enzyme. The α-Glucosidase inhibitory activities were then evaluated using autodock and the 4-Nitrophenyl-α-D-glucopyranoside (pNPG) method. As a result, 142 compounds were identified were identified by comparing the retention time, molecular ions and fragmentation behaviors with the reference compounds or the in-house database. 20 possible α-Glucosidase activity inhibitory components were identified using HPLC-FT-ICR-MS. Molecular docking and inhibition studies showed that 4 compuunds including Limonexic acid, Sanggenol A, Glabrone, Matrine were more likely to stronger α-Glucosidase inhibitory activities than acarbose. In conclusion, the proposed approach, which combined highly specific screening with HPLC-FT-ICR-MS, provided a powerful platform for discovering bioactive components from multi-component and multi-target traditional Chinese medicines (TCMs).

Metabolic diversity shapes vegetation-enhanced methane oxidation in landfill covers: Multi-omics study of rhizosphere microorganisms

Vegetation root exudates have the ability to shape soil microbial community structures, thereby enhancing CH4 bio-oxidation capacity in landfill cover systems. In this study, the CH4 oxidation capacity of indigenous vegetation rhizosphere microorganisms within operational landfill covers in Chongqing, China, was investigated for the first time, with the objective of identifying suitable plant candidates for CH4 mitigation initiatives within landfill cover systems. Furthermore, a multi-omics methodology was employed to explore microbial community structures and metabolic variances within the rhizospheric environment of diverse vegetation types. The primary aim was to elucidate the fundamental factors contributing to divergent CH4 oxidation capacities observed in rhizosphere soils. The findings demonstrated that herbaceous vegetation predominated in landfill covers. Notably, Rumex acetosa exhibited the highest CH4 oxidation capacity in the rhizosphere soil, approximately 20 times greater than that in non-rhizosphere soil. Root exudates played a crucial role in inducing the colonization of CH4-oxidizing functional microorganisms in the rhizosphere, subsequently prompting the development of specific metabolic pathways. This process, in turn, enhanced the functional activity of the microorganisms while concurrently bolstering their tolerance to microbial pollutants. Consequently, the addition of substances like Limonexic acid strengthened the CH4 bio-oxidation process, thereby underscoring the suitability of Rumex acetosa and similar vegetation species as preferred choices for landfill cover vegetation restoration.

Phytochemical and anthelmintic investigation of Citrus aurantifolia Swingle seeds

Petroleum ether and acetone extracts of Citrus aurantifolia Swingle seeds were tested against Toxocara vitulorum (Ascaris), and Fasciola gicantica {larvae and Eggs), Trihconstronylus colubriformis{adult worms , larvae and Eggs), larvae of Trichinella spiralis , in addition to Eimeria oocyst .The results revealed variation in the lethal effect with different organisms. Petroleum ether(40-60) extract tested at 100% concentration showed 100 % mortality effect against all parasites except T. colubriformis. larvae (39. 6% mortality) .The larvicidal effect of acetone extract vary from T. colubriformis (adult worms) , F. gicantica (mature fluks) and T.spiralis larvae (LC 50 = 415,522,630 ppm ) respectively, but no effect was observed against ascaris (adult worms) or T. colubriformis (larvae). Acetone extract was effective against the eggs of T.vitulorum, F. gicantica and T. colubriformis (LC50 275, 155, 309 ppm) respectively. Also, an oocidal effect was observed against Eimeria (LC50 = 631 ppm) Phytochemical study of the bioactive extracts lead to identification of limonin and limonexic acid beside nine coumarins from acetone extract, the isolated compounds were identified by chemical and spectral analysis. The petroleum ether extract was saponified and analyzed by GC/Mass.

Protective effect of compounds from the flowers of Citrus aurantium L. var. amara Engl against carbon tetrachloride-induced hepatocyte injury

5-Hydroxy-6,7,3',4'-tetramethoxyflavone (HTF), limonexic acid (LA) are two compounds isolated from the flowers of Citrus aurantium L. var. amara Engl with various biological activities. This study was designed to investigate their protective effects against carbon tetrachloride (CCl4)-induced hepatocyte injury, using human hepatic cell line HL-7702 to determine the cell cytotoxicity, cell viability, levels of hepatic marker enzymes, malondialdehyde (MDA). Results showed that pretreatment with HTF, LA could significantly reverse CCl4-induced HL-7702 cell viability decrease, LA displayed a higher activity. HTF, LA also showed their capability of decreasing the CCl4-induced leakage of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), inhibiting the lipid peroxidation, HTF showed more significant activity. Given that HTF, LA were not toxic, it is concluded that HTF, LA could effectively protect hepatocyte against CCl4-induced injury.

Limonoids and their anti-proliferative and anti-aromatase properties in human breast cancer cells

Lemons are a widely used citrus crop and have shown several potential health benefits. In the present study, the mechanism and effectiveness of the anti-cancer and anti-aromatase properties of limonoids were investigated for the first time. Defatted lemon (Citrus lemon L. Burm) seed powder was extracted with ethyl acetate (EtOAc) and methanol (MeOH) for 16 h each, successively. These extracts were fractionated using 1D (silica) and 2D (ion exchange and SP-70 columns) column chromatography to obtain nine limonoids. The compounds were identified by TLC, HPLC, and LC-MS techniques. A panel of 9 purified limonoids, including limonin, nomilin, obacunone, limonexic acid (LNA), isolimonexic acid (ILNA), nomilinic acid glucoside (NAG), deacetyl nomilinic acid glucoside (DNAG), limonin glucoside (LG) and obacunone glucoside (OG) as well as 4 modified compounds such as limonin methoxime (LM), limonin oxime (LO), defuran limonin (DL), and defuran nomilin (DN), were screened for their cytotoxicity on estrogen receptor (ER)-positive (MCF-7) or ER-negative (MDA-MB-231) human breast cancer cells. We further tested the mechanism of the anti-proliferative activity of limonoids using an in vitro aromatase enzyme assay and western blot with anti-caspase-7. Among the tested limonoids, 11 limonoids exhibited cytotoxicity on MCF-7 whereas 8 limonoids showed cytotoxicity against the MDA-MB-231 cell lines. Although most of the limonoids showed anti-aromatase activity, the inhibition of proliferation was not related to the anti-aromatase activity. On the other hand, the anti-proliferative activity was significantly correlated with caspase-7 activation by limonoids. Our findings indicated that the citrus limonoids may have potential for the prevention of estrogen-responsive breast cancer (MCF-7) via caspase-7 dependent pathways.

Bioactivity evaluations of ingredients extracted from the flowers of Citrus aurantium L. var. amara Engl

Citrus aurantium L. var. amara Engl, a member of genus Citrus (Rutaceae), widespread in China, is used as folk medicine for the treatment of helping digest, phlegm, enteritidis, stomachic and other deceases. In the present research, silica gel column, Sephadex LH-20, mass spectrometer (MS) and nuclear magnetic resonance (NMR) were used to separate and identify the chemical compounds from the flowers of C. aurantium var. amara, and several bioactivity assays were used to evaluate their antioxidant, anti-inflammatory, anti-virus and antitumour activities. Two major compounds, 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (HTF) and limonexic acid (LA), were isolated and identified from C. aurantium var. amara for the first time. The results of the bioactivity assays showed that HTF and LA displayed significant antioxidant activities and showed significant inhibition effects on the B16 cell lines at a concentration range from 6.25 to 50μg/ml, and on the SMCC-7721 cell lines from 12.5 to 200μg/ml. The antitumour effect, anti-inflammatory activity and the inhibiting expression of HBsAg and HBeAg of 2.2.15 cells displayed the tendency in a concentration-dependent manner. These two compounds from C. aurantium var. amara could potentially be used as a promising natural agent in the pharmaceutical industries.

Methyl nomilinate from citrus can modulate cell cycle regulators to induce cytotoxicity in human colon cancer (SW480) cells in vitro

Limonoids are triterpenoids found in citrus and possess cancer preventive properties in in vitro and in vivo assays. Although several mechanisms for the chemopreventive properties of limonoids have been postulated, the specific mechanisms involved in the anti-cancer effects have not been explored. In the present study, limonoids, including methyl nomilinate, isoobacunoic acid, isolimonexic acid (ILNA), and limonexic acid (LNA), were purified, identified by LC–MS and NMR spectral data and evaluated for their biological effects on SW480 human colon adenocarcinoma cells. Methyl nomilinate was the most potent inhibitor of cell metabolic activity in MTT and EdU incorporation assays. These limonoids did not affect apoptotic markers such as caspase-3 and PARP, but methyl nomilinate treatment resulted in significant induction of G0/G1 cell cycle arrest. Furthermore, methyl nomilinate suppressed CDK4/6 and cyclin D3 and the expression of CDK inhibitors. Taken together, the results suggest inhibition of cell proliferation by methyl nomilinate occurs due to G1 cell cycle arrest, indicating that methyl nomilinate has potential as a chemopreventive agent.

Chemical constituents of Glycosmis parva and their anti-herpes simplex virus activities

The investigation of EtOAC of branches and leaves of Glycosmis parva Craib [1, 2] (Rutaceae) collected in Thailand led to the isolation of a new acridone alkaloid, glycosparvarine (1), three new sulfur-containing propanamide derivatives, (+)-S-deoxydihydroglyparvin (2), (+)-S-deoxytetrahydroglyparvin (3) [3] and (+)-tetrahydroglyparvin (4), together with nine known compounds (N-methylatalaphylline, N-methylcyclo-atalaphylline-A, glycofolinine, citramine, arborinine, limonin, a mixture of limonexic acid and isolimonexic acid, glyparvin-A and dihydroglyparvin). Antiviral activity evaluation of isolated compounds against herpes simplex virus (HSV) type 1 and 2 disclosed that glycosparvarine (1), (+)-tetrahydroglyparvin (4) and glycofolinine, showed moderate inhibitory activities with EC50 of 348, 229 and 151 µM, respectively. In addition, (+)-S-deoxydihydroglyparvin (2) exhibited activities with EC50 of 29.8 and 44.6 µM against HSV-1 and HSV-2, respectively.

Activities of extracts and compounds from Spiranthera odoratissima St. Hil. (Rutaceae) in leaf-cutting ants and their symbiotic fungus

The study of the Spiranthera odoratissima St. Hil (Rutaceae) branches extracts led to the isolation of the furoquinoline (dictamine, γ-fagarine and skimmianine) and 2-arylquinoli-4-one (1-methyl-2-phenylquinolin-4-one) alkaloids and limonoids (limonexic acid and limonin). The compounds 1-methyl-2-phenylquinolin-4-one and limonexic acid were isolated for the first time from the Spiranthera. These furoquinoline and 2-arylquinoli-4-one alkaloids and limonoids showed insecticidal and/or fungicidal activity in the nest of the Atta sexdens rubropilosa.

Bioactive Compounds from Sour Orange Inhibit Colon Cancer Cell Proliferation and Induce Cell Cycle Arrest

Epidemiological studies suggest that dietary limonoids and phytosterols offer protection from certain types of cancers. Potential cancer preventive constituents of sour orange (Citrus aurantium L.) were isolated and identified from ethyl acetate extract. The structures of the compounds were identified by one-dimensional (1H or 13C) and two-dimensional (1H-H and 1H-13C) nuclear magnetic resonance experiments as limonexic acid and beta-sitosterol glucoside (SG). The identified compounds were tested for the potential inhibition of human colon cancer cell (HT-29) proliferation, apoptosis, and also noncancerous cells (COS-1). Cell proliferation, arrest of the cell cycle, and induction of apoptosis were assessed by MTT assay, flow cytometry, and nuclear staining methods, respectively. The MTT assay indicated that both compounds exhibited significant inhibition at various concentrations. These compounds did not show any toxic effects on noncancerous cells. These compounds caused 4-5-fold increases in the counts of G2/M stage cells at 50 microM, indicating a potential role in cell cycle arrest. These findings support the hypothesis that limonoids and phytosterols are effective apoptosis-promoting agents and incorporation of enriched fractions of these compounds in the diet may serve to prevent colon cancer. To the best of our knowledge, this is the first report of the isolation, identification, and cell proliferation assay for limonexic acid and SG from sour orange.

Bioactive Compounds from Mexican Lime (Citrus aurantifolia) Juice Induce Apoptosis in Human Pancreatic Cells

Lime (Citrus aurantifolia Swingle) is one of the major citrus fruits and widely consumed, but there is limited evidence about its health-promoting properties. Hence, an investigation was conducted to understand the chemopreventive effects of lime juice on pancreatic cancer cells and the possible mechanism for induction of apoptosis using Panc-28 cells. Freeze-dried lime juice was extracted with different solvents, such as chloroform, acetone, MeOH, and MeOH/water (8:2). The chloroform extract showed the highest (85.4 and 90%) radical-scavenging activity by 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) methods at 624 microg/mL, whereas the MeOH/water extract showed the lowest (<20%) activity. The active components were identified by high-performance liquid chromatography (HPLC) using a C-18 column as rutin, neohesperidin, hesperidin, and hesperitin. Furthermore, the limonoids identified are limonexic acid, isolimonexic acid, and limonin. All of the extracts of lime juice inhibited Panc-28 cancer cell growth. The MeOH extract exhibited the maximum activity, with an IC50 value of 81.20 microg/mL after 72 h. The inhibition of Panc-28 cells was in the range of 73-89%, at 100 microg/mL at 96 h. The involvement of apoptosis in induction of cytotoxicity was confirmed by expression of Bax, Bcl-2, casapase-3, and p53. The results of the present study clearly indicate that antioxidant activity is proportionate to the content of flavonoids and proliferation inhibition ability is proportionate to the content of both flavonoids and limonoids.

Characterization of Citrus aurantifolia bioactive compounds and their inhibition of human pancreatic cancer cells through apoptosis

In the current study, we have isolated and purified five putative compounds from lime ( Citrus aurantifolia Swingle) seeds using Soxhlet extraction and chromatographic separation. The purity and structures of the isolated compounds were identified by TLC, HPLC and mass spectra as limonin, limonexic acid (LNA), isolimonexic acid (ILNA), β-sitosterol glucoside (SG) and limonin glucoside (LG). These putative compounds exhibited significant inhibition of Panc-28 cells with IC 50 values in the range of 18–42 µM in MTT assay after incubation for 72 h, which was confirmed by viable cell count. Further, induction of apoptosis was confirmed through annexin-FITC staining of the cells and expression of apoptosis related proteins was also studied to understand the mechanism. Expression of apoptosis related protein showed apoptosis through induction of p53 by, p21 dependent manner and cytochrome- c mediated caspases driven intrinsic pathway. Expression of apoptosis favoring proteins in cells treated with isolated compounds were in the order of ILNA > LNA > SG > Limonin > LG based on the expression ratio of bax/bcl2. These results were further supported through fluorescent probed microscopic images. To the best of our knowledge, this is the first report on isolation and identification of LNA, SG and LG from lime and their mechanism of inhibition of human pancreatic cancer cells.

Antinociceptive action of limonexic acid obtained from Raulinoa echinata

The antinociceptive effect of the limonexic acid isolate of Raulinoa echinata Cowan in four models of pain in mice is described. When evaluated against acetic acid-induced abdominal constrictions, limonexic acid (10, 30 and 60 mg kg(-1), i.p.) produced dose-related inhibition of the number of constrictions, with a mean ID50 value of 43 (2.3-79) micromol kg(-1), and was more potent than some standard drugs. In the formalin test, limonexic acid inhibited both the first and second phases of formalin-induced pain. Furthermore, the effect was more pronounced in the second phase, with a mean ID50 value of 13.66 (9.35-19.61) micromol kg(-1), and had a pharmacological profile that was similar to standard drugs such as acetaminophen and acetyl salicylic acid. Limonexic acid also produced dose-related inhibition of glutamate- and capsaicin-induced pain, with mean ID50 values of 11.67 (8.51-16.0) micromol kg(-1) and 47.17 (36.51-60.93) micromol kg(-1), respectively. The mechanism of action is not completely understood, but seems to involve direct interaction with the GABAergic and nitroxidergic pathways.

Leaf-cutting ants toxicity of limonexic acid and degraded limonoids from Raulinoa echinata.: X-ray structure of epoxy-fraxinellone

Phytochemical survey of roots extracts of the South Brazilian endemic plant Raulinoa echinata Cowan, Rutaceae led to the isolation of known degraded limonoids: fraxinellone, fraxinellonone and epoxy-fraxinellone. The latter was previously isolated also from the stems, but the relative configuration could not be solved by NMR experiments. This paper deals with the X-ray diffraction analysis of epoxy-fraxinellone and its toxicity to leaf-cutting ants, describes the isolation of fraxinellonone and fraxinellone for the first time in Raulinoa. Epoxy-fraxinellone showed no toxicity to the leaf-cutting ants (Atta sexdens rubropilosa). The limonoid limonexic acid, isolated from stems, presented high toxicity to the leaf-cutting ants, diminishing considerably their longevity.

Limonoids from the endemic Brazilian species Raulinoa echinata.

Phytochemical survey of stems and leaves of the South Brazilian endemic Raulinoa echinata Cowan, Rutaceae led to the isolation of five limonoid derivatives: the widespread limonin, limonexic acid, kihadalactone B, a methoxylated limonexic acid derivative and a degraded limonoid structurally related to fraxinellone. The two latter compounds have been isolated for the first time. These compounds displayed weak inhibitory activity when assayed in vitro against trypomastigote forms of Trypanosoma cruzi. In this paper, the isolation, structure elucidation and bioactivity of these compounds are reported.

Limonoids and alkaloids of the root bark of Dictamnus angustifolius

Abstract Six limonoids, limonin, limonin diosphenol, limonexic acid, obacunone, fraxinellone and fraxinellonone; nine alkaloids, dictangustine-A, iso- γ -fagarine, dictamine, γ -fagarine, skimmianine, isodictamine, isomaculosidine, N -methylflindersine and preskimmianine; five coumarins, angustifolin, umbelliferone, herniarin, scopoletin and scoparone; three steroids, sitosterol, stigmast-4-ene-3,6-dione and stigmast-4-ene-3-one, together with dictafolin-A and dictafolin-B were isolated from the root bark of Dictamnus angustifolius . Their structures were elucidated by spectral analysis. Among them, dictangustrine-A, iso- γ -fagarine, dictafolin-A and dictafolin-B are reported for the first time from a natural source.

Alkaloids and limonoids of Tetradium trichotomum: Chemotaxonomic significance

An examination of the stem and root bark of Tetradium trichotomum (Rutaceae) has yielded the alkaloids α-allocryptopine and dictamnine and the limonoids limonin, limonexic acid and calodendrolide. The presence of the 1-benzyltetrahydroisoquinoline-derived alkaloid α-allocryptopine is further chemical evidence supporting the argument that Tetradium is closely allied to Zanthoxylum and Phellodendron.

Citrus bitter principles—II : Application of NMR to structural and stereochemical problems

The NMR spectra of limonin, its congeners, and some of their derivatives are discussed. The results confirm the structures assigned to citrolin, limonexic acid, and many other degradation products of limonin. Stereochemical assignments are made to the 1-position in nomilin and the 17-position in obacunone on the basis of theire NMR spectra. This furfurylic proton at C 17 in limonin is compared with a similar protonin columbin. Evidence for an anisotropic shielding effect of the epoxy group is discussed.