Biotransformation of Cynomorium flavan-3-ols in dairy sheep and their effects on inflammation and liver growth retardation.

The stems of Cynomorium songaricum are used in traditional Chinese medicine as a tonic and also used locally as a food material and livestock feed. It is known that some of the falvan-3-ol monomers and dimers that entered the milk of dairy sheep fed with C. songaricum stems are biotransformation products of the original flavan-3-ol polymers in C. songaricum stems. This study was performed to investigate the biotransformation process of the flavan-3-ols in dairy sheep and to evaluate the bioactivities. The results showed that procyanidin A2 and epicatechin could be released from the polymeric flavan-3-ols of C. songaricum through rumen microbial metabolism. On traumatic and lipopolysaccharide (LPS)-induced inflammation models of Tg (mpx: EGFP) zebrafish larvae and LPS-induced liver injury models of Tg (fabp10a: DsRed) zebrafish larvae, the milk from sheep fed with C. songaricum stems showed stronger anti-inflammatory and hepatoprotective activities compared to blank milk. The absorbed chemical constituents of C. songaricum stems and the metabolites also exhibited anti-inflammatory and hepatoprotective activities, with the dimeric flavan-3-ols being more effective than the monomers. The milk, the absorbed chemical constituents of C. songaricum stems, and the metabolites alleviated the increased level of reactive oxygen species induced by LPS in zebrafish larvae. PRACTICAL APPLICATION: This study found that C. songaricum stems as livestock feed could produce milk that has a beneficial impact on consumer and livestock health in terms of anti-inflammation and hepatoprotection.

Identification and bioactivity evaluation of ring-opening and lactone forms of enterolactone from sheep fed flaxseed cake.

The previously undescribed lactone ring-opening enterolactone and its sulphate were purified along with the lactone counterparts from the urine of dairy sheep fed flaxseed cake. The structures were determined by NMR and MS analyses. The ring-opening and lactone forms underwent mutual transformation with changes in pH and milk could protect the lactone form. Enterolactone exhibited more effective anti-proliferation activity on MDA-MB-231 breast cancer cells than its ring-opening counterpart, while the ring-opening enterolactone demonstrated more effective anti-osteoporosis activity than the lactone form. The results indicated the potential for targeting biological functions through pH and medium manipulation.

Anti-osteoporosis effects of mammalian lignans and their precursors from flaxseed and safflower seed using zebrafish model.

Secoisolariciresinol diglucoside (SDG) and tracheloside (TCL) are the main lignan components of flaxseed cake and safflower seed cake, which are by-products of oil extraction. Both SDG and TCL are metabolized into mammalian lignan enterolactone (EL) with the involvement of intestinal bacteria. In this research, we evaluated the anti-osteoporosis effects of SDG and the in vivo metabolites EL and enterodiol (ED) prepared in our previous work, as well as the newly isolated chemical constituents from safflower seed, including TCL, the lactone ring opening product of TCL (OTCL) and two alkaloids on the alloxan-induced zebrafish model. All the compounds showed significant anti-osteoporosis effects at 80 µM, with p < 0.05 for EL and p < 0.001 for other compounds compared with the model. SDG and TCL showed the most significant and concentration-dependent effects, with p < 0.001 compared with model at 20 µM. The alkaloids, N-coumaroylserotonin glucoside and N-feruloylserotonin glucoside, also showed anti-osteoporosis at 20 µM with p < 0.01, whereas EL, ED, and OTCL showed no significant effects. Quantitative real-time polymerase chain reaction revealed that SDG and TCL upregulated the expression of osteogenic genes Runx2, SP7, OPG, Col1a1a, Alp, ON, OPN, and OCN in alloxan-treated zebrafish. The in vivo metabolite of lignans, EL, showed significant anti-inflammatory effect (p < 0.01) at 20 µM, which might also help to combat osteoporosis and other complications caused by excessive immune response in the body. The results provided scientific data for using the oil extraction by-products as sources of anti-osteoporosis compounds. PRACTICAL APPLICATION: This study found that lignans in flaxseed cake and safflower seed cake exhibited anti-osteoporosis effects by upregulating the expression of osteogenic genes, making the oil extraction by-products sources of anti-osteoporosis compounds.

Chemical Constituents of Cyperus esculentus Leaves and the Protective Effect against Agricultural Fungicide-Induced Hepatotoxicity.

Cyperus esculentus is cultivated as a crop plant due to its edible and oily tubers (tiger nut). However, little is known about the phytochemicals and bioeffects of the leaves. This study was conducted to identify and quantify the chemical constituents of C. esculentus leaves and evaluate their bioactivities. By liquid chromatography-mass spectrometry, 30 compounds including flavan-3-ols, caffeic acid derivatives, and flavones, were identified from the leaves. The quantitative analysis revealed that gallocatechin (8), procyanidin B1 (15), catechin (16), chlorogenic acid (19), orientin (30), and luteolin 7-O-glucuronide (31) are the major chemical constituents of C. esculentus leaves. The contents of these six chemical constituents in the leaves collected in September in Hohhot, China, reached to 1460.85±7.66, 10178.77±302.65, 1048.35±17.37, 1722.15±26.13, 5318.62±277.16, and 1526.54±11.95 µg, respectively, in one gram of the dried leaves. The leaf extract (CELE) showed strong antioxidant activity in vitro, with compounds 8, 15, and 19 contributing the most. CELE showed significant protection against the agricultural fungicide tebuconazole-induced developmental toxicity and hepatotoxicity in zebrafish.

Effects and mechanisms of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide from Safflower on endothelial injury in vitro and on thrombosis in vivo.

Background: The florets of Carthamus tinctorius L. (Safflower) is an important traditional medicine for promoting blood circulation and removing blood stasis. However, its bioactive compounds and mechanism of action need further clarification. Objective: This study aims to investigate the effect and possible mechanism of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide (HGG) from Safflower on endothelial injury in vitro, and to verify its anti-thrombotic activity in vivo. Methods: The endothelial injury on human umbilical vein endothelial cells (HUVECs) was induced by oxygen-glucose deprivation followed by reoxygenation (OGD/R). The effect of HGG on the proliferation of HUVECs under OGD/R was evaluated by MTT, LDH release, Hoechst-33342 staining, and Annexin V-FITC apoptosis assay. RNA-seq, RT-qPCR, Enzyme-linked immunosorbent assay and Western blot experiments were performed to uncover the molecular mechanism. The anti-thrombotic effect of HGG in vivo was evaluated using phenylhydrazine (PHZ)-induced zebrafish thrombosis model. Results: HGG significantly protected OGD/R induced endothelial injury, and decreased HUVECs apoptosis by regulating expressions of hypoxia inducible factor-1 alpha (HIF-1α) and nuclear factor kappa B (NF-κB) at both transcriptome and protein levels. Moreover, HGG reversed the mRNA expression of pro-inflammatory cytokines including IL-1ß, IL-6, and TNF-α, and reduced the release of IL-6 after OGD/R. In addition, HGG exhibited protective effects against PHZ-induced zebrafish thrombosis and improved blood circulation. Conclusion: HGG regulates the expression of HIF-1α and NF-κB, protects OGD/R induced endothelial dysfunction in vitro and has anti-thrombotic activity in PHZ-induced thrombosis in vivo.

Quantification and purification of procyanidin B1 from food byproducts.

The flavan-3-ols of 10 primarily plant food byproducts, including Muscat Hamburg grape seed, hawthorn sarcocarp, litchi pericarp, cocoa bean, peanut skin, lotus seedpod, Xinyang Maojian green tea, Cinnamomi cortex, Sargentodoxa cuneata stem, and Cyperus esculentus, leaves were analyzed. Ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry was used for the analysis. Cyperus esculentus leaves contained a high amount of procyanidin B1 (198.9 mg/100 g), second only to Muscat Hamburg grape seed (292.9 mg/100 g). Unlike grape seed that contained several procyanidin B isomers with very similar retention times, C. esculentus leaves contained primarily procyanidin B1 with few isomers. Procyanidin B1 was enriched in the ethyl acetate fraction of a 70% methanol extract of C. esculentus leaves and purified at 95% purity by two runs of open column chromatography. Direct chromatography of the plant extract on octadecylsilane and Sephadex LH20 open columns improved the yield of the resultant leaf procyanidin B1 (95% purity) to 0.21‰. The present research demonstrated that the leaves of C. esculentus, byproducts of tigernut, are ideal plant sources for isolating and providing high-purity procyanidin B1. PRACTICAL APPLICATION: Procyanidin B1 has a broad range of health benefits. Cyperus esculentus is cultivated in many countries with nearly 6190 square hectares (hm2 ) in the Spanish Mediterranean region in 2020-2021 and over 16,700 hm2 in China in recent years, primarily for its tubers. The byproducts, the leaves of C. esculentus, contain high levels of procyanidin B1, with few isomers that interfere with its isolation and purification. Thus, the leaves of this plant provide a viable source for preparing high-purity procyanidin B1.

Identification and bioactivity evaluation of flavan-3-ols in the milk of dairy sheep fed Cynomorium songaricum.

Cynomorium songaricum is a traditional medicine and also a food material that is eaten raw or processed as tea or beverages. As a featured plant in semi-desert grasslands, C. songaricum is also eaten by the cattle and sheep in the area. This research study fed dairy sheep C. songaricum to determine the flavan-3-ols in sheep milk. Catechin (Cat), epicatechin (Epi), procyanidin A1 (A1), procyanidin A2 (A2), and procyanidin B1 (B1) were detected in sheep milk with the concentration being Epi > A2 > Cat > B1 > A1 at 24 h after the administration of C. songaricum. Neither A1 nor A2 were detected in the methanol extract of C. songaricum. Cysteine degradation of the plant revealed that in addition to Epi, A2 was the extending unit of the polymeric flavan-3-ols in C. songaricum, indicating that A2 is released digestively from the polymers and enters the milk. Procyanidin B-1 was converted to A1 on incubation in raw but not heated milk, indicating that the A1 in milk is the enzymatically transformed product of B1. Accelerated oxidation showed that the flavan-3-ols, B1, Cat, and Epi significantly protects the unsaturated triacyglycerols in the milk from oxidation. The flavan-3-ol could slow down the oxidation of glutathione and the latter may play an important role in preventing the milk triglycerides from oxidation. Flavan-3-ols are polyphenols with many health benefits. The present research revealed the antioxidant activities of flavan-3-ols that could be absorbed to sheep milk, adding new evidences for the values of these flavan-3-ols and for the milk.

Combination of Ephedra sinica stems and Terminalia chebula fruits produces new ephedrine derivatives in vivo that diminish the permeability to BBB while retaining airway dilation and hepatoprotective effects.

ETHNOPHARMACOLOGICAL RELEVANCE: The stems of Ephedra sinica and the fruits of Terminalia chebula are combined using in traditional Mongolian medicine formula "Gurigumu-7" for liver diseases. E. sinica stems contains ephedrine with broncho-dilatory activity. However, ephedrine can pass through the blood-brain barrier (BBB) and excite the central nervous system (CNS) to cause insomnia and restlessness. AIM OF THE STUDY: The present study was to investigate the structures and bioactivities of new compounds formed in vivo after co-administration of E. sinica stems and T. chebula fruits. MATERIALS AND METHODS: Pharmacokinetic investigation was carried out in rats. A parallel artificial membrane permeability measurement system was used to determine BBB permeability. Ex vivo experiments using tracheal rings of guinea pig was performed to examine the tracheal relaxation effect. In vivo hepatoprotective tests were carried out in Tg (fabp10a: dsRed) liver transgenic zebrafish. The fluorescent probe, 2,7-dichlorodihydrofluorescein diacetate, was used to measure reactive oxygen species, and UHPLC-MS was used to determine glutathione concentrations after derivatization with N-ethylmaleimide. RESULTS: New ephedrine derivatives (1 and 2) formed in vivo and reached their maximum serum concentrations at 0.5 h after administration of the two herbal drugs. Compounds 1 and 2 showed lower BBB permeability than ephedrine, suggesting that they have less adverse effects on the CNS. Compounds 1 and 2 relaxed the tracheal rings and had strong hepatoprotective effect on transgenic zebrafish with liver specific expression of RFP. Compounds 1 and 2 significantly reduced the level of reactive oxygen species while increasing that of glutathione in thioacetamide-treated zebrafish, which might be the hepatoprotective mechanism. CONCLUSION: These results provided evidences that the chemical constituents in various herbal drugs in a medicinal formula can interact to generate new compounds with fewer side effects and increased or additive bioactivity.

Hydroxysafflor yellows alleviate thrombosis and acetaminophen-induced toxicity in vivo by enhancing blood circulation and poison excretion.

BACKGROUND: Hydroxysafflor yellow A (HSYA) from the flower of Carthamus tinctorius (Safflower) has been reported to have various pharmacological effects. However, little is known about the bioactivities of other chemical constituents in Safflower and the relationship between enhancement of blood circulation and hepatoprotection by HSYA. PURPOSE: The present research was to evaluate the antithrombotic and hepatoprotective activities of HSYA and C, examine their mechanisms of actions, including influence on the excretion velocity of acetaminophen, and the relationship between the antithrombotic, hepatoprotective, and other bioactivities. METHODS: The hepatoprotective activities were examined by acetaminophen (APAP)-induced zebrafish toxicity and carbon tetrachloride (CCl4)-induced mouse liver injury. The concentrations of APAP in zebrafish and APAP that was excreted to the culture media were quantified by UHPLC-MS. The anti-thrombosis effect of HSYA and C were examined by the phenylhydrazine (PHZ)-induced zebrafish thrombosis. RESULTS: HSYA and HSYC showed robust protection on APAP-induced toxicity and PHZ-induced thrombosis. The hepatoprotective effects of HSYA and C were more potent than that of the positive control, acetylcysteine (61.7% and 58.0%, respectively, vs. 56.9% at 100 µM) and their antithrombosis effects were more robust than aspirin (95.1% and 86.2% vs. 52.7% at 100 µM). HSYA and C enhanced blood circulation, rescued APAP-treated zebrafish from morphological abnormalities, and mitigated APAP-induced toxicity in liver development in liver-specific RFP-expressing transgenic zebrafish. HSYC attenuated CCl4-induced mouse liver injury and regulated the levels of HIF-1α, iNOS, TNF-α, α-SMA, and NFκB in liver tissues. HSYA was also protective in a dual thrombotic and liver toxicity zebrafish model. By UHPLC-MS, HSYA accelerated the excretion of APAP. CONCLUSION: HSYA and C are the bioactive constituents of Safflower that are responsible for the herbal drug's traditional use in promoting blood circulation to remove blood stasis. Safflower and its chalcone constituents may protect from damage due to exogenous or disease-induced endogenous toxins by enhancing the excretion velocity of toxins.

In vivo hepatoprotective activity and the underlying mechanism of chebulinic acid from Terminalia chebula fruit.

BACKGROUND: The fruit of Terminalia chebula Retz. is one of the most widely used herbal drug in Traditional medicine prescriptions including those for liver diseases. In the screening of bioactive constituents that have potential hepatoprotective activity, chebulinic acid (CA) which is a major chemical constituent of T. chebula fruit showed potent activity. PURPOSE: This work was conducted to investigate the hepatoprotective activity and mechanisms of CA. METHODS: The hepatoprotective effect of CA was examined on hepatotoxic models of cells, zebrafish larvae and mice caused by tert-butyl hydrogen peroxide (t-BHP), acetaminophen (APAP) and CCl4, respectively. RESULTS: Pretreatment with CA could prevent t-BHP-induced damage in L-02 hepatocytes by blocking the production of ROS, reducing LDH levels and enhancing HO-1 and NQO1 expression via MAPK/Nrf2 signaling pathway. In animal experiments, CA significantly protected mice from CCl4-induced liver injury, as demonstrated by reduced ALT, AST and MDA levels, enhanced SOD activity, improved liver histopathological changes, and the activation of the Nrf2/HO-1 signaling pathway. CA metabolized to chebulic acid isomers with DPPH radical scavenging activity. In a transgenic zebrafish line with liver specific expression of DsRed RFP, CA diminished the hepatotoxicity induced by 10 mM APAP. CONCLUSION: Experiments in cell and two animal models demonstrated consistent results and comprehensively expounded the hepatoprotective effects of CA.

Inhibitory effects and molecular mechanisms of pentagalloyl glucose in combination with 5-FU on aggressive phenotypes of HepG2 cells.

This study examined the inhibition and mechanism of natural product pentagalloyl glucose (PGG) against HepG2 cells and determined the effects of its combination with the clinical chemotherapeutic drug, 5-FU. PGG was found to inhibit the proliferation, migration and invasion of HepG2 cells, induced G1 arrest and apoptosis in both concentration- and time- dependent manners. The combination of PGG and 5-FU had synergistic effects on reversal the aggressive phenotypes of HepG2 cells, increasing the proportion of Bax/Bcl-2, promoting the activation of caspase-9 and caspase-3, and inducing apoptosis. This combination upregulated P27 and cyclin B1, and downregulated cyclin E1, leading to G1 phase arrest. The combination significantly downregulated MDR1 and LRP1, suggesting the potential to reverse the resistance to 5-FU.

Technical note: Quantification of lignans in the urine, milk, and plasma of flaxseed cake-fed dairy sheep.

Mammalian lignans are phytoestrogens with important bioactivities, and their concentrations in livestock milk may influence the health of consumers. This research aimed to establish a method to quantify multiple mammalian lignans in the biofluids of dairy sheep using ultra-HPLC-triple quadropole mass spectrometry with multiple-reaction monitoring. Secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, enterodiol (ED), enterolactone (EL), ED-sulfate (ED-S), and EL-sulfate (EL-S) were purified from the urine of flaxseed cake-fed dairy sheep. The structures of these lignans were identified by a combination of mass and nuclear magnetic resonance spectra. These purified lignans were used as standards to optimize their quantification conditions in urine, milk, and plasma of dairy sheep. On this basis, the lignan metabolites in biofluids were quantified. To improve analysis sensitivity, plasma and milk were pretreated with acetonitrile containing 1% formic acid and passed through a HybridSPE-PL 55261-U column (Supelco, Bellefonte, PA). The limit of quantification of the lignans ranged from 1.43 to 18.3 ng/mL in plasma, and from 1.01 to 18.7 ng/mL in milk. The linearity of the calibration curves ranged from their limit of quantification to at least 217 ng/mL in plasma, and 217 ng/mL in milk. Regression coefficient of the calibration curves were above 0.99 for secoisolariciresinol, 2-[(4-hydroxy-3-methoxyphenyl)methyl]-3-[(3-hydroxyphenyl)methyl]-1,4-butanediol, ED, EL, ED-S, and EL-S, indicating satisfactory relationships between the peak areas and concentrations in the quantification range. The relative concentrations of ED-glucuronide and EL-glucuronide (EL-G) in different biofluids were compared based on their chromatogram peak areas. The sheep plasma contained all forms of mammalian lignans (i.e., ED, EL, ED-S, EL-S, ED-glucuronide, and EL-G.); the urine contained ED, EL, ED-S, and EL-S; and the milk contained ED, EL, ED-S, EL-S, and EL-G. Milk-to-plasma concentration ratios of the mammalian lignans indicated that the free forms were more permeable than the sulfated conjugates. Mammalian lignans found in sheep plasma and milk may provide health benefits to the sheep and sheep-product consumers. The analytical method established in this work could be used to quantify mammalian lignans in livestock products.

Insecticidal activity of triterpenoids and volatile oil from the stems of Tetraena mongolica.

Tetraena mongolica Maxim is a species of Zygophyllaceae endemic to China. Because few insect pests affect its growth and flowering, we speculated that this plant produces defensive chemicals that are insect repellents or antifeedants. The effects of different fractions from crude stem and leaf extracts on Pieris rapae were examined. The results confirmed that the ethyl acetate (EtOAc) fraction from the stems had insecticidal potential. Five compounds were isolated from the EtOAc fraction: a volatile oil [bis(2-ethylhexyl) benzene-1,2-dicarboxylate (1)], three triterpenoids 2E-3ß-(3,4-dihydroxycinnamoyl)-erythrodiol (2), 2Z-3ß-(3,4-dihydroxycinnamoyl)-erythrodiol (3), and 2E-3ß-(3,4-dihydroxyphenyl)-2-propenoate (4)], and one steroid [ß-sitosterol (5)]. Compounds 1-5 exhibited different degrees of insecticidal activity, including antifeedant and growth-inhibition effects. Compounds 1-5 inhibited the activity of carboxylesterase (CarE) and acetylcholinesterase (AChE) to different degrees. Compound 1 had the strongest antifeedant and growth-inhibition effects, and significantly inhibited the activity of CarE and AChE. Our results indicate that compounds 1-4 are the major bioactive insecticidal constituents of Tetraena mongolica. This work should facilitate the development and application of plant-derived botanical pesticides.

Protective effect of triterpenes of Ganoderma lucidum on lipopolysaccharide-induced inflammatory responses and acute liver injury.

Ganoderma lucidum is a popular medicinal mushroom, which has been used as therapeutic for centuries in traditional Chinese medicine. Although G. lucidum showed strong protective effects in prevention or treatment of a variety of inflammatory diseases, the mechanisms underlying the anti-inflammatory properties of triterpenes of G. lucidum remain undefined. In the current study, we demonstrated that ethanol extract and triterpenes of G. lucidum specifically suppressed LPS-mediated inflammatory responses. Notably, ganodermanontriol inhibited the expressions and interactions of TLR4 and MyD88, NF-κB translocation to nucleus and its DNA binding activity, phosphorylation of p38, ErK1/2 and JNK. In vivo, we showed that ganodermanontriol effectively prevented LPS/D-Galactosamine-induced liver injury by reducing TNF-α and IL-6 production, and decrease of ALT/AST release. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for triterpenes that may act through potential inhibition of TLR4-MyD88-mediated NF-κB and MAPK signaling pathways.

Constituents from the Leaves of Tetraena mongolica and Their Protective Activity in HEK 293t Cells Damaged by CdCl2.

Tetraena mongolica Maxim, a relict originating from the Tertiary Period, is an endemic species of Zygophyllaceae in China. Three new monoterpenoids (1-3), two new phenols (4, 5) with unusual O-sulfoglucosyl groups, a new flavonoid (6), and nine known compounds were isolated from the leaves of T. mongolica. The structures of these compounds were determined by interpretation of NMR, MS, and ECD data. Some of the isolated compounds showed protective effects on HEK 293t cells damaged by CdCl2, with IC50 values being 55.7 and 80.3 µM for compounds 7 and 8, respectively, at the time point of 48 h after treatment.

Structures and In Vitro Antihepatic Fibrosis Activities of Prenylated Dihydrostilbenes and Flavonoids from Glycyrrhiza uralensis Leaves.

Glycyrrhiza uralensis is the major plant source of licorice. This study was to identify bioactive compounds from the plant's leaves in order to make better use of its aerial part. An ethanol extract of the leaves was subjected to repeated chromatography to yield 15 compounds. The structures were determined to be three novel dihydrostilbenes, based on their various spectroscopic data-glycypytilbene A (1), glycydipytilbene (2), and glycypytilbene B (3)-and 12 known compounds, α,α'-dihydro-3,5,4'-trihydroxy-4,3'-diisopentenylstilbene (4), α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diisopentenylstilbene (5), 6-prenyleriodictyol (6), 5'-prenyleriodictyol (7), 6-prenylquercetin-3-Me ether (8), 5'-prenylquercetin (9), 6-prenylquercetin (10), 6-prenylnaringenin (11), 3'-prenylnaringenin (12), sigmoidin C (13), 8-[(E)-3-hydroxymethyl-2- butenyl]-eriodictyol (14), and quercetin-3-Me ether (15). Most of these chemical constituents inhibited α-glucosidase activity, with the two prenylated quercetin derivatives (9 to 10) being the greatest active (IC50 < 4.0 µg/mL). Compounds 1, 3 to 4, 6 to 7, 9 to 12 impeded the growth of human hepatic stellate cells, with the prenylated flavonoids (6 to 7, 9 to 12) being more robust than their unprenylated counterparts. PRACTICAL APPLICATIONS: This study found that Glycyrrhiza uralensis leaves contain prenylated dihydrostilbenes and flavonoids with inhibiting effects on α-glucosidase and on the proliferation of human hepatic stellate cells, which should prompt the development of G. uralensis leaves for healthy products with anti-diabetic or liver fibrosis-preventing effects.

Lignans, flavonoids and coumarins from Viola philippica and their α-glucosidase and HCV protease inhibitory activities.

Two lignans including a new one, five flavonoids and five coumarins were isolated from the whole plant of Viola philippica (synonymised as Viola yedoensis Makino). The new compound was structurally determined as (7R,8S,8'S) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside by analysis of its NMR, MS and CD spectroscopic data. The known compounds were characterised by comparing their NMR and MS data with those reported. Among the known compounds, 5-hydroxy-4'-methoxyflavone-7-O- rutinoside, 6,7-di-O-ß-D- glucopyranosylesculetin, and 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-ß-D- glucopyranoside were isolated and identified from this genus for the first time. Of these compounds, 5-hydroxy-4'-methoxyflavone-7-O-rutinoside and (7R,8S,8'S) -3,3'-dimethoxy- 4,4',9-trihydroxy- 7,9'-epoxy-8,8'-lignan 9-O-rutinoside were potently active against α-glucosidase, while the two dimeric coumarins, 5, 5'-bi (6, 7-dihydroxycoumarin) and 6,6',7,7'-tetrahydroxy-5,8'-bicoumarin potently inhibited HCV protease.

Comparison of Antioxidant Constituents of Agriophyllum squarrosum Seed with Conventional Crop Seeds.

Twelve chemical constituents were identified from the Agriophyllum squarrosum seed (ASS). ASS contained large amounts of flavonoids, which were more concentrated in the seed coat. ASS-coat (1 g) contained 335.7 µg flavonoids of rutin equivalent, which was similar to the flavonoid content in soybean (351.2 µg/g), and greater than that in millet, wheat, rice, peanut, and corn. By LC-MS analysis, the major constituents in ASS were 3-O-[α-L-rhamnopyranosyl-(1→6)-ß-D- glucopyranosyl]-7- O-(ß-D-glucopyranosyl)-quercetin (1), rutin (4), quercetin-3-O-ß-D- apiosyl(1→2)-[α-L-rhamnosyl(l→6)]-ß-D-glucoside (2), isorhamnetin-3-O-rutinoside (5), and allantoin (3), compared with isoflavonoids-genistin (16), daidzin (14), and glycitin (18) in soybean. Among constituents in ASS, compounds 1, 2, 4, protocatechuic acid (8), isoquercitrin (11), and luteolin-6-C-glucoside (12) potently scavenged DPPH radicals and intracellular ROS; strongly protected against peroxyl radical-induced DNA scission; and upregulated Nrf2, phosphorylated p38, phosphorylated JNK, and Bcl-2 in HepG2 cells. These results indicate that ASS is rich in antioxidant constituents that can enrich the varieties of food flavonoids, with significant beneficial implications for those who suffer from oxidative stress-related conditions. PRACTICAL APPLICATION: This study found that A. squarrosum seed contains large amounts of antioxidative flavonoids and compared its chemical constituents with those of conventional foods. These results should increase the interest in planting the sand-fixing A. squarrosum on a large scale, thus preventing desertification and providing valuable foods.

Identification and Enrichment of α-Glucosidase-Inhibiting Dihydrostilbene and Flavonoids from Glycyrrhiza uralensis Leaves.

To exploit Glycyrrhiza uralensis resources, we examined the bioactive constituents of G. uralensis leaves. Seven chemical components were isolated by repeat column chromatography, and using spectroscopic methods, their structures were determined to be a novel prenylated dihydrostilbene, α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diprenylstilbene (1); a methylated flavonoid, quercetin-3-Me ether (4); and 5 prenylated flavonoids: 5'-prenylquercetin (3), 8-[(E)-3-hydroxymethyl-2-butenyl]eriodictyol (7), 6-prenyleriodictyol (5), 5'-prenyleriodictyol (6), and 6-prenylquercetin-3-Me ether (2). Compounds 1-7 and their unprenylated counterparts, glycosides, and other related compounds (8-13) were quantitatively analyzed. Using a macroporous resin column, most of these compounds could be enriched in the 40% to 60% ethanol-eluted fractions. Compounds 1-7 showed strong radical scavenging activity toward DPPH, and most of them demonstrated greater inhibitory activity against α-glucosidase than their unprenylated counterparts.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry.

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.