Chemical Evaluation of Liquidambar styraciflua L. Fruits Extracts and Their Potential as Anticancer Drugs.

Liquidambar styraciflua L. is an aromatic species, popularly used in traditional Chinese medicine to treat diarrhea, dysentery, coughs, and skin sores. The present study was designed to investigate the chemical composition and biological potential of extracts obtained from the fruits of this plant. For the chemical evaluation, it was used mainly liquid and gas chromatography, plus NMR, and colorimetric methods. The aqueous extract (EA) originated two other fractions: an aqueous (P-EA) and an ethanolic (S-EA). The three extracts were composed of proteins, phenolic compounds, and carbohydrates in different proportions. The analyses showed that the polysaccharide extract (P-EA) contained pectic polysaccharides, such as acetylated and methyl esterified homogalacturonans together with arabinogalactan, while the fraction S-EA presented phenolic acids and terpenes such as gallic acid, protocathecuic acid, liquidambaric acid, combretastatin, and atractyloside A. EA, P-EA, and S-EA showed antioxidant activity, with IC50 values of 4.64 µg/mL, 16.45 µg/mL, and 3.67 µg/mL, respectively. The cytotoxicity followed the sequence S-EA > EA > P-EA, demonstrating that the toxic compounds were separated from the non-toxic ones by ethanol precipitation. While the fraction S-EA is very toxic to any cell line, the fraction P-EA is a promising candidate for studies against cancer due to its high toxicity to tumoral cells and low toxicity to normal cells.

Identification of the anti-breast cancer targets of triterpenoids in Liquidambaris Fructus and the hints for its traditional applications.

BACKGROUND: Liquidambaris Fructus is the infructescences of Liquidambar formosana Hance and it has been used to treat some breast disease in Traditional Chinese Medicine. In the previous study we found the anti-breast cancer effect of triterpenoid in Liquidambaris Fructus. This study is a further investigation of the triterpenoids in Liquidambaris Fructus and aims to identify their anti-breast cancer targets, meanwhile, to estimate the rationality of the traditional applications of Liquidambaris Fructus. METHODS: Triterpenoids in Liquidambaris Fructus were isolated and their structures were identified by NMR spectrums. Potential targets of these triterpenoids were predicted using a reverse pharmacophore mapping strategy. Associations between these targets and the therapeutic targets of breast cancer were analyzed by constructing protein-protein interaction network, and targets played important roles in the network were identified using Molecular Complex Detection method. Binding affinity between the targets and triterpenoids was studied using molecular docking method. Gene ontology enrichment analysis was conducted to reveal the biological process and signaling pathways that the identified targets were involved in. RESULTS: Thirteen triterpenoids were identified and 6 of them were the first time isolated from Liquidambaris Fructus. Predicted ADME properties revealed a good druggability of these triterpenoids. We identified 18 protein targets which were closely related to breast cancer progression, especially triple-negative, basal-like or advanced stage breast cancers. The triterpenoids could bind with these targets as their inhibitors: hydrophobic skeleton is a favorable factor for them to stabilize at binding site and polar C17- or C3- substituent was necessary for binding. GO enrichment analysis indicated that inhibition of protein tyrosine kinases autophosphorylation might be the primary mechanism for the anti-breast cancer effect of the triterpenoids, and ErbB4 and EGFR were the most relevant targets. CONCLUSIONS: The study revealed that triterpenoids from Liquidambaris Fructus might exert anti-breast cancer effect by directly inhibit multiple protein targets and signaling pathways, especially ErbB4 and EGFR and related pathways. This study also brings up another hint that the traditional applications of Liquidambaris Fructus on hypogalactia should be reassessed systematically because it might suppress rather than promote lactation by inhibiting the activity of ErbB4.

Triterpenoids from Liquidambar Fructus induced cell apoptosis via a PI3K-AKT related signal pathway in SMMC7721 cancer cells.

A previously undescribed taraxerene-type triterpenoid possessing a class of rare natural taraxerene triterpenoid possessing skeleton with 14, 28-lactone, two undescribed oleane-type triterpenoids, and twenty-five known triterpenoids were isolated from Liquidambar formosana (Hamamelidaceae). The structures of undescribed compounds were determined on the basis of 1D and 2D NMR spectroscopic, HR-ESI-MS, and X-ray crystallographic data analysis. Among the isolates, ursolic acid, 3,6-dion-20(29)-lupen-28-oic acid, and 3-oxo-12α-hydroxyoleanan-28,13ß-olide induced a significant apoptosis in SMMC7721 cells in the flow cytometer experiment with apoptosis rates of 94.5%, 57.3% and 89.9% at 8.0 µM, respectively, exhibiting near equivalent apoptosis-inducing abilities to that positive drug taxol (apoptotic rate of 71.2% at 1.4 µM). Mechanism studies suggested that these three compounds could regulate the mitochondrial pathway by up-regulating the expressions of pro-apoptotic factors (Bad and Bax) and activating caspase-3 and caspase-9 to induce apoptosis. Further studies indicated that the pro-apoptotic effects of these three compounds were associated with PI3K-AKT pathway inhibition. Taken together, these studies provide evidence that triterpenoids from L. Fructus are promising candidates for the hepatocellular carcinoma therapy.

Furanocoumarin A: A Novel Anticancer Agent on Human Lung Cancer A549 Cells from Fructus liquidambaris.

BACKGROUND AND OBJECTIVE: The fruit of Fructus liquidambaris, which is recently being used for cancer treatment, has a history to be used as a traditional medicine in China for thousands of years. MATERIALS AND METHODS: Ten kg of dried F. liquidambaris was obtained with 70% alcohol-water solution under reflux for three times. The condensed extract was obtained from petroleum ether, ethyl acetate and N-butyl alcohol, respectively. Ethyl acetate extract was subjected to silica gel column, Sephadex LH-20, ODS column chromatography and RP-HPLC column chromatography to yield a new compound (1). The structure was identified through intensive analysis of NMR and MS spectra. The antitumor mechanism of the furanocoumarin A on human lung cancer A549 cells was confirmed by detecting the apoptosis-related proteins. RESULTS: Furanocoumarin A (1), a novel furanocoumarin constituent was isolated and identified from F. Liquidambaris. The IC50 value of furanocoumarin A on A549 cell lines was 65.28±5.36µM obtained by the method of MTT. The compound could induce the apoptosis of A549 cells by inducing 21.5% early apoptosis and 32.4% late apoptosis at the concentration of 60µmol/L. Western blot analysis indicated that protein expressions of p53, caspase 3 and Bax increased in a dose-dependent manner between the concentrations from 40 to 80µM. The protein expression of Bcl-2 decreased the concentration of 60 and 80µM. The ratio of Bcl-2 to Bax was inversely proportional to the dose concentration. CONCLUSION: Furanocoumarin A could be a novel anticancer agent from herbal medicine.

Composition, in vitro Cytotoxicity, Anti-mildew and Anti-wood-decay Fungal Activities of the Fruit Essential Oil of Liquidambarformosana from Taiwan.

This study investigated the chemical composition, in vitro cytotoxicity, anti-mildew, and anti-wood-decay fungal activities of the essential oil isolated from the fruit of Liquidainbar formosana from Taiwan. The essential oil from the fresh fruit was isolated using hydrodistillation in a Clevenger-type apparatus, and characterized by GC-FID and GC-MS. A total of 45 compounds were identified, representing 98.5% of the essential oil. The main components identified were a-pinene (16.8%), ß-caryophyllene (10.1%), τ-muurolol (8.3%), τ-cadinol (7.6%), ß-pinene (6.7%), and sabinene (5.7%). The essential oil exhibited cytotoxic activity against human oral, liver, and lung cancer cells. The active source compounds were ß-caryophyllene, τ-cadinol, and τ-muurolol. The fruit essential oil was shown to have excellent anti-mildew and anti-wood-decay fungal activities, the active compounds being evaluated as τ-cadinol and τ-muurolol.

Facile and fast removal of oil through porous carbon spheres derived from the fruit of Liquidambar formosana.

Porous carbon spheres with a diameter of 1-2 cm were prepared via a simple carbonization of the fruit of Liquidambar formosana. After carbonization, the spherical structure and inner finger-like pores were maintained with high resistance to impact. Due to the porous structure and the hydrophobic nature, the carbonized fruit of Liquidambar formosana can float on the water surface and show a super-fast oil or organic solvent sorption ability (sorption saturation can be achieved within 1-2 min). Moreover, about 99% of adsorbed oil can be easily removed from spheres via organic solvent such as ethanol or hexane, which shows good recyclability of samples. In general, considering the low-cost and abundance of raw material collected from nature and the facile synthetic process (only by carbonization), the centimeter-sized porous spheres via the carbonization of fruit of Liquidambar formosana are very promising to be used for the application of oil or organic solvent spill cleanup.

Effect of the extract from leaves of Liquidambar formosana Hance on S180 cells.

We examined the effects of the extract from leaves of Liquidambar formosana Hance on S180 cells and screened for antitumor active sites in the plant. Solvent extraction was conducted to prepare extracts from the leaves of L. formosana Hance and conduct preliminary separation, an MTT assay to determine the effect of leaf extract on the proliferation of S180 cells, and inverted microscopy to observe the effect of chloroform extract on the morphology of S180 cells. Double-staining (Annexin V/propidium iodide) with flow cytometry was conducted to determine the effect of the chloroform extract on S180 cell apoptosis. At some concentrations, the different extracts from the leaves of L. formosana Hance dose-dependently inhibited the proliferation of S180 cells. Among all extracts, the chloroform extract showed the strongest inhibitory effect on S180 cell proliferation. The IC50 values for the chloroform extract, ethyl acetate extract, n-butanol extract, and water layer were 0.238, 0.471, 0.844, and 0.411 mg/mL, respectively. We observed cell shrinkage, volume reduction, and varying sizes by inverted microscopy. Additionally, with increasing drug concentration, the number of cells decreased and debris increased. The cells showed typical apoptotic morphological changes. The chloroform extract induced the apoptosis of S180 cells in a dose-dependent manner. Different extracts from the leaves of L. formosana Hance inhibited the proliferation of S180 cells, and the chloroform extract was the main antitumor component. This extract from the leaves of L. formosana Hance inhibited the proliferation of S180 cells in part by inducing apoptosis.

Essential oil from leaves of Liquidambar formosana ameliorates inflammatory response in lipopolysaccharide-activated mouse macrophages.

The essential oil from Liquidambar formosana leaves (EOLF) was demonstrated to exhibit anti-inflammatory activity in mouse macrophages. EOLF reduced nitrite oxide generation, secretion levels of tumor necrosis factor-alpha and interleukin-6, and expression levels of prointerleukin-beta, inducible nitric oxide synthase, and cyclooxygenase-2 in lipopolysaccharide (LPS)-activated mouse macrophages. EOLF also reduced NLRP3 inflammasome-derived interleukin-1beta secretion. The underlying mechanisms for the EOLF-mediated anti-inflammatory activity were (1) reduction of LPS-induced reactive oxygen species generation; (2) reduction of LPS-induced activation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 MAP kinase; (3) reduction of LPS-induced nuclear factor-kappaBeta activation. Furthermore, 25 compounds were identified in the EOLF using GC-FID and GC-MS and the major compounds were terpinen-4-ol (32.0%), beta-pinene (18.0%), gamma-terpinene (13.8%), and alpha-terpinene (9.7%). We found that LPS-induced nitrite oxide generation was inhibited significantly by terpinen-4-ol. Our results indicated that EOLF has anti-inflammatory activity and may provide a molecular rationale for future therapeutic interventions in immune modulation.

Variations of the chemical composition and bioactivity of essential oils from leaves and stems of Liquidambar styraciflua (Altingiaceae).

OBJECTIVES: This study aimed to evaluate the variations of the chemical composition and bioactivity of essential oils of Liquidambar styraciflua L. (Altingiaceae) collected in different seasons. METHODS: The oils were analysed by GLC/FID and GLC/MS. The antioxidant activity was investigated by diphenylpicrylhydrazyl (DPPH) and superoxide anion radical scavenging assays and the deoxyribose degradation assay. Inhibition of both 5-lipoxygenase (5-LOX) and prostaglandin E2 (PGE2) production in hepatic cancer (HepG-2) cells were used to assess the anti-inflammatory activity. The cytotoxic activity was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. KEY FINDINGS: Altogether, 64 volatile secondary metabolites were identified. The major components of the leaf oil were d-limonene, α-pinene and ß-pinene, and of the stem oil were germacrine D, α-cadinol, d-limonene, α-pinene, and ß-pinene. Leaf and stem oils collected in spring could reduce DPPH● (IC50 = 3.17 and 2.19 mg/ml) and prevent the degradation of the deoxyribose sugar (IC50 = 17.55 and 14.29 µg/ml). The stem oil exhibited a higher inhibition of both 5-LOX and PGE2 than the leaf oil. The cytotoxic activity of leaf and stem oils was low in cancer cell lines (IC50 = 136.27 and 119.78 µg/ml in cervical cancer (HeLa) cells). CONCLUSIONS: Essential oils of L. styraciflua exhibited an interesting anti-inflammatory activity with low cytotoxicity, supporting its traditional use to treat inflammation.

Effect of acid, steam explosion, and size reduction pretreatments on bio-oil production from sweetgum, switchgrass, and corn stover.

Bio-oil produced from biomass by fast pyrolysis has the potential to be a valuable substitute for fossil fuels. In a recent work on pinewood, we found that pretreatment alters the structure and chemical composition of biomass, which influence fast pyrolysis. In this study, we evaluated dilute acid, steam explosion, and size reduction pretreatments on sweetgum, switchgrass, and corn stover feedstocks. Bio-oils were produced from untreated and pretreated feedstocks in an auger reactor at 450 °C. The bio-oil's physical properties of pH, water content, acid value, density, and viscosity were measured. The chemical characteristics of the bio-oils were determined by gas chromatography-mass spectrometry. The results showed that bio-oil yield and composition were influenced by the pretreatment method and feedstock type. Bio-oil yields of 52, 33, and 35 wt% were obtained from medium-sized (0.68-1.532 mm) untreated sweetgum, switchgrass, and corn stover, respectively, which were higher than the yields from other sizes. Bio-oil yields of 56, 46, and 51 wt% were obtained from 1% H(2)SO(4)-treated medium-sized sweetgum, switchgrass, and corn stover, respectively, which were higher than the yields from untreated and steam explosion treatments.

Pentacyclic triterpenes from the resin of Liquidambar formosana.

Two new pentacyclic triterpenes and eight known pentacyclic triterpenes were isolated from the petroleum ether extract of Liquidambaris Resina. The structural elucidation of these compounds was determined by spectroscopic data interpretation. Their cytotoxic and antiplatelet aggregation activities were examined to find potent cytotoxic and antiplatelet aggregation compounds from natural resources. The results showed that 3-keto oleane triterpenes had strong cytotoxicity against MDA-MB-435S cancer cells and that 28-carboxyl oleane triterpenes possessed significant inhibition of platelet aggregation induced by ADP.

Cancer chemopreventive activity of lupane- and oleanane-type triterpenoids from the cones of Liquidamber styraciflua.

In a search for cancer chemopreventive agents from natural sources, four lupane-type and seven oleanane-type triterpenoids, and ten synthetic analogs were screened as potential anti-tumor promoters by using the in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) activation assay. Among them, 25-acetoxy-3alpha-hydroxyolean-12-en-28-oic acid (1) and 3beta,25-epoxy-3alpha-hydroxylup-20(29)-en-28-oic acid (2) were examined for anti-tumor promoting activity in a two-stage carcinogenesis assay on mouse skin with 7,12-dimethylbenz[a]anthracene (DMBA) and TPA as promoter. 25-Acetoxy-3alpha-hydroxyolean-12-en-28-oic acid (1) and 3beta,25-epoxy-3alpha-hydroxylup-20(29)-en-28-oic acid (2) showed moderate inhibitory activities.