Inhibitory Activity of Compounds Isolated from Ligustrum robustum (Roxb.) against HepG2 Liver Cancer Cells: Isocubein and 4-(2-Acetoxyethyl)phenol as Potential Candidates.

Many herbal species in the genus Ligustrum have been shown to contain compounds with anti-cancer biological activity. This study aimed to isolate some compounds from the leaves of Ligustrum robustum (Roxb.) Blume (L. robustum) and evaluate their effects against liver cancer cells. As a result, seven previously reported compounds (1-7) were isolated, including four lignans (1-4) and three phenolic derivatives (5-7). The structures of these compounds were determined using spectroscopic methods and comparison with reported data. All isolates were assessed for their inhibitory effects on HepG2 liver cancer cells. Screening results revealed that two compounds, isocubein (3) and 4-(2-acetoxyethyl)phenol (7), exhibited strong inhibitory activity against cell proliferation, with IC50 values of 3.1±0.9 and 4.5±14 µM, respectively. Further analyses demonstrated that both compounds could suppress the formation and development of 3D tumorspheres in terms of quantity and size. Additionally, isocubein (3) and 4-(2-acetoxyethyl)phenol (7) exhibited the ability to inhibit the migration of HepG2 cells. This study represents the first report on the inhibitory activity against HepG2 liver cancer cells of extracts and isolated compounds from L. robustum, providing valuable information for future research aiming to develop products for liver cancer treatment.

A New HPLC-UV Method Using Hydrolyzation with Sodium Hydroxide for Quantitation of Trans-p-Hydroxycinnamic Acid and Total Trans-p-Hydroxycinnamic Acid Esters in the Leaves of Ligustrum robustum.

Trans-p-hydroxycinnamic acid and its esters in the leaves of Ligustrum robustum might be a new resource to prevent diabetes and its complications. In the present study, a new HPLC-UV method using hydrolyzation with sodium hydroxide for quantitation of trans-p-hydroxycinnamic acid and total trans-p-hydroxycinnamic acid esters in the leaves of L. robustum was developed, since it was difficult and troublesome to analyze no less than 34 trans-p-hydroxycinnamic acid esters by usual HPLC. The extract of L. robustum was hydrolyzed with sodium hydroxide at 80 °C for 2 h, and then, hydrochloride was added. HPLC analysis was performed in reverse phase mode using a C-18 column, eluting with methanol-0.1% acetic acid aqueous solution (40:60, v/v) in isocratic mode at a flow rate of 1.0 mL·min-1 and detecting at 310 nm. The linear range for trans-p-hydroxycinnamic acid was 11.0-352.0 µg·mL-1 (r2 = 1.000). The limit of detection and limit of quantification were 2.00 and 6.07 µg·mL-1, respectively. The relative standard deviations of intra-day and inter-day variabilities for trans-p-hydroxycinnamic acid were less than 2%. The percentage recovery of trans-p-hydroxycinnamic acid was 103.3% ± 1.1%. It is the first HPLC method using hydrolyzation for quantification of many carboxylic esters. Finally, the method was used successfully to determine trans-p-hydroxycinnamic acid and total trans-p-hydroxycinnamic acid esters in various extracts of the leaves of L. robustum. The 60-70% ethanol extracts of L. robustum showed the highest contents of free trans-p-hydroxycinnamic acid (3.96-3.99 mg·g-1), and the 50-80% ethanol extracts of L. robustum displayed the highest contents of total trans-p-hydroxycinnamic acid esters (202.6-210.6 mg·g-1). The method can be applied also to the quality control of the products of L. robustum.

Monoterpenoid Glycosides from the Leaves of Ligustrum robustum and Their Bioactivities (II).

Ligustrum robustum has been not only used as a heat-clearing and detoxicating functional tea (Ku-Ding-Cha) but also consumed as a hypotensive, anti-diabetic, and weight-reducing folk medicine. From the leaves of L. robustum, ten new monoterpenoid glycosides named ligurobustosides T10 (1a), T11 (1b), T12 (2a), T13 (2b), T14 (3a), T15 (3b), F1 (4b), T16 (5a), T17 (5b), and E1 (6b), together with five known ones (4a, 6a, 7, 8a, 8b), were separated and identified using the spectroscopic method and chemical method in this research. The results of biological tests exhibited that the fatty acid synthase (FAS) inhibitory action of compound 5 (IC50: 4.38 ± 0.11 µM) was as strong as orlistat (IC50: 4.46 ± 0.13 µM), a positive control; the α-glucosidase inhibitory actions of compounds 1-4 and 7-8, and the α-amylase inhibitory actions of compounds 1-8 were medium; the ABTS radical scavenging capacities of compounds 1-3 and 5-8 (IC50: 6.27 ± 0.23 ~ 8.59 ± 0.09 µM) were stronger than l-(+)-ascorbic acid (IC50: 10.06 ± 0.19 µM) served as a positive control. This research offered a theoretical foundation for the leaves of L. robustum to prevent diabetes and its complications.

Chemical Constituents from the Leaves of Ligustrum robustum and Their Bioactivities.

The leaves of Ligustrum robustum have been consumed as Ku-Ding-Cha for clearing heat and removing toxins, and they have been used as a folk medicine for curing hypertension, diabetes, and obesity in China. The phytochemical research on the leaves of L. robustum led to the isolation and identification of two new hexenol glycosides, two new butenol glycosides, and five new sugar esters, named ligurobustosides X (1a), X1 (1b), Y (2a), and Y1 (2b) and ligurobustates A (3a), B (3b), C (4b), D (5a), and E (5b), along with seven known compounds (4a and 6-10). Compounds 1-10 were tested for their inhibitory effects on fatty acid synthase (FAS), α-glucosidase, and α-amylase, as well as their antioxidant activities. Compound 2 showed strong FAS inhibitory activity (IC50 4.10 ± 0.12 µM) close to that of the positive control orlistat (IC50 4.46 ± 0.13 µM); compounds 7 and 9 revealed moderate α-glucosidase inhibitory activities; compounds 1-10 showed moderate α-amylase inhibitory activities; and compounds 1 and 10 displayed stronger 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ammonium salt (ABTS) radical scavenging effects (IC50 3.41 ± 0.08~5.65 ± 0.19 µM) than the positive control l-(+)-ascorbic acid (IC50 10.06 ± 0.19 µM). This study provides a theoretical foundation for the leaves of L. robustum as a functional tea to prevent diabetes and its complications.

Phenylethanoid and Phenylmethanoid Glycosides from the Leaves of Ligustrum robustum and Their Bioactivities.

The phytochemical study on the leaves of Ligustrum robustum, which have been used as Ku-Ding-Cha, led to the isolation and identification of three new phenylethanoid glycosides and three new phenylmethanoid glycosides, named ligurobustosides R1 (1b), R2-3 (2), R4 (3), S1 (4b), S2 (5), and S3 (6), and five reported phenylethanoid glycosides (7-11). In the bioactivity test, (Z)-osmanthuside B6 (11) displayed strong fatty acid synthase (FAS) inhibitory activity (IC50: 4.55 ± 0.35 µM) as the positive control orlistat (IC50: 4.46 ± 0.13 µM), while ligurobustosides R4 (3) and S2 (5), ligupurpuroside B (7), cis-ligupurpuroside B (8), ligurobustoside N (9), osmanthuside D (10), and (Z)-osmanthuside B6 (11) showed stronger ABTS radical scavenging activity (IC50: 2.68 ± 0.05~4.86 ± 0.06 µM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 µM). This research provided a theoretical basis for the leaves of L. robustum as a tea with function in treating obesity and diabetes.

Monoterpenoid Glycosides from the Leaves of Ligustrum robustum and Their Bioactivities.

The leaves of Ligustrum robustum have been applied as Ku-Ding-Cha, a functional tea to clear heat, remove toxins, and treat obesity and diabetes, in Southwest China. The phytochemical research on the leaves of L. robustum led to the isolation and identification of eight new monoterpenoid glycosides (1-8) and three known monoterpenoid glycosides (9-11). Compounds 1-11 were tested for the inhibitory activities on fatty acid synthase (FAS), α-glucosidase, α-amylase, and the antioxidant effects. Compound 2 showed stronger FAS inhibitory activity (IC50: 2.36 ± 0.10 µM) than the positive control orlistat (IC50: 4.46 ± 0.13 µM), while compounds 1, 2, 5 and 11 displayed more potent ABTS radical scavenging activity (IC50: 6.91 ± 0.10~9.41 ± 0.22 µM) than the positive control L-(+)-ascorbic acid (IC50: 10.06 ± 0.19 µM). This study provided a theoretical basis for the leaves of L. robustum as a functional tea to treat obesity.

Involvement of the Hydroperoxy Group in the Irreversible Inhibition of Leukocyte-Type 12-Lipoxygenase by Monoterpene Glycosides Contained in the Qing Shan Lu Shui Tea.

We have previously found two novel monoterpene glycosides, liguroside A and liguroside B, with an inhibitory effect on the catalytic activity of the enzyme leukocyte-type 12-lipoxygenase in the Qing Shan Lu Shui tea. Here, two new monoterpene glycosides, liguroside C and liguroside D which inhibit this enzyme, were isolated from the same tea. The spectral and chemical evidence characterized the structures of these compounds as (5E)-7-hydroperoxy-3,7-dimethyl-1,5-octadienyl-3-O-(α-l-rhamnopyranosyl)-(1''→3')-(4'''-O-trans-p-coumaroyl)-ß-d-glucopyranoside and (2E)-6-hydroxy-3,7-dimethyl-2,7-octadienyl-3-O-(α-l-rhamnopyranosyl)-(1''→3')-(4'''-O-trans-p-coumaroyl)-ß-d-glucopyranoside, respectively. These ligurosides, which irreversibly inhibited leukocyte-type 12-lipoxygenase, have a hydroperoxy group in the monoterpene moiety. Additionally, monoterpene glycosides had the same backbone structure but did not have a hydroperoxy group, such as kudingoside A and lipedoside B-III, contained in the tea did not inhibit the enzyme. When a hydroperoxy group in liguroside A was reduced by using triphenylphosphine, the resultant compound, kudingoside B, showed a lower inhibitory effect on the enzyme. These results strongly suggest the involvement of the hydroperoxy group in the irreversible inhibition of the catalytic activity of leukocyte-type 12-lipoxygenase by the monoterpene glycosides contained in the Qing Shan Lu Shui tea.

Hypolipidemic activity and mechanisms of the total phenylpropanoid glycosides from Ligustrum robustum (Roxb.) Blume by AMPK-SREBP-1c pathway in hamsters fed a high-fat diet.

The aim of this study was to evaluate the hypolipidemic effect and mechanisms of total phenylpropanoid glycosides extracted from Ligustrum robustum (Roxb.) Blume (LRTPG) in hamsters fed a high-fat diet and to discover bioactive components in HepG2 cell model induced by oleic acid. LRTPG of high (1.2 g/kg), medium (0.6 g/kg), and low (0.3 g/kg) doses was administrated daily for 21 consecutive days in hamsters. We found that in hamsters fed a high-fat diet, LRTPG effectively reduced the concentrations of plasma triglycerides (TG), free fatty acid, total cholesterol, low-density lipoprotein cholesterol, and hepatic TG and total cholesterol. And the compounds acteoside, ligupurpuroside A, ligupurpuroside C, and ligupurpuroside D significantly inhibited lipid accumulation in HepG2 cell at the concentration of 50 µmol/L. Mechanism research demonstrated that LRTPG increased the levels of phospho-AMP-activated protein kinase and phospho-sterol regulatory element binding protein-1c in liver, further to suppress the downstream lipogenic genes as stearoyl-CoA desaturase 1, glycerol-3-phosphate acyltransferase, 1-acylglycerol-3-phosphate O-acyltransferase 2, and diacylglycerol acyltransferase 2. In addition, LRTPG increased the hydrolysis of circulating TG by up-regulating lipoprotein lipase activities. These results indicate that LRTPG prevents hyperlipidemia via activation of hepatic AMP-activated protein kinase-sterol regulatory element binding protein-1c pathway.

Ultrasound-assisted complex enzyme extraction, structural characterization, and biological activity of polysaccharides from Ligustrum robustum.

Ligustrum robustum is one of the traditional teas in China with a long history of drinking and medicinal use. Through Response surface optimization, the yield of polysaccharides extracted by ultrasonic-assisted complex enzyme (UAE-EN) method was increased to 14.10 ±â€¯0.56 %. Neutral homogeneous polysaccharide (LRNP) and acidic homogeneous polysaccharide (LRAP-1, LRAP-2, LRAP-3) from L. robustum were purified. The molecular weights of them were 5894, 4256, 4621 and 3915 Da. LRNP was composed of glucose (Glc), galactose (Gal), arabinose (Ara) with molar percentage of 24.97, 42.38 and 30.80. Structure analysis revealed that the backbone of LRNP consisted of 1,5-linked α-Araf, 1,4-linked ß-Galp, 1,6-linked ß-Galp, and 1,4-linked ß-Glcp with the branches of 1,2-linked α-Araf, 1,3-linked α-Araf, 1,3-linked ß-Glcp and 1,6-linked ß-Galp residues, some terminal residues of α-Araf, ß-Glcp and α-Galp were also included. In vitro experiments showed that the four polysaccharides possessed excellent antioxidant, antitumor and hypoglycemic activities. LRNP possessed the protective effect against oxidative stress. The studies provide a basis for further exploitation of L. robustum.

Alterations in the phytochemical composition and antioxidant activity of Ligustrum robustum according to continuous wet- and dry-heat treatment.

Small-leaved Kuding tea (SLKDT) obtained from Ligustrum robustum is a traditional tea substitute in southern China and has a range of physiological effects. However, the changes in its phytochemical composition after various heat treatments are not reported yet. Thus, the phytochemical composition and antioxidant activities of fresh leaves of SLKDT (LrF1) and SLKDT after high-temperature wet-heat treatment (LrF2) and wet- and dry-heat treatments (LrF3) were assessed using liquid chromatography-mass spectrometry, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities and lipid peroxidation inhibition activity of LrF1 and LrF3 were investigated. The results indicated that the phytochemical composition of LrF1, LrF2, and LrF3 was significantly different. Overall, 258 and 83 differential constituents, respectively, were obtained in LrF1 versus LrF2 and LrF2 versus LrF3. The differential constituents mainly included amino acids and their derivatives, nucleosides, flavonoids, terpenoids, simple phenylpropanoids, and coumarins. After heat treatment, SLKDT exhibited obvious changes in sensory characteristics and physiological properties, which may be related to the changes in the levels of amino acids, linalool, beta-geraniol, myricetin, naringin, fraxetin, and isoacteoside. Moreover, the antioxidant activities significantly changed after heat treatment of SLKDT. Overall, our study demonstrated that heat treatment can alter the phytochemical composition of SLKDT, thus affecting its sensory properties and physiological properties. PRACTICAL APPLICATION: This study preliminarily assessed the changes in the composition of small-leaved Kuding tea (SLKDT) after various heat treatments and revealed that the composition of SLKDT tea can be adjusted by various heat and temperature treatments.

Potential of phenolic compounds in Ligustrum robustum (Rxob.) Blume as antioxidant and lipase inhibitors: Multi-spectroscopic methods and molecular docking.

Ligustrum robustum (Rxob.) Blume is traditionally served as a functional tea in China. In this work, the antioxidant activities of L. robustum (Rxob.) Blume extract (LRE) were evaluated and its inhibitory effect and mechanism on pancreatic lipase were further investigated. With the high contents of phenols (139.70 ± 1.41 mg gallic acid equivalent/g extract) and flavonoids (326.46 ± 7.36 mg rutin equivalent/g extract), LRE showed significant antioxidant activities (p < 0.05) for scavenging free radicals and hydrogen peroxide, inhibiting lipid peroxidation and providing strong reducing power. Meanwhile, LRE displayed remarkable inhibitory activity on pancreatic lipase with a low half-effective inhibitory concentration (IC50 ) of 2.469 ± 0.005 mg/ml which was further determined as non-competitive inhibition. The spectroscopic results showed that LRE inhibited the activity of pancreatic lipase by modifying the tertiary and secondary structures of lipase. Moreover, four phenolic compounds (acteoside, lipedoside A, oleuropein and ligurobustoside C) were identified from LRE by the high performance liquid chromatography-quadrupole- time of flight-mass spectrometry. In addition, according to molecular docking analysis, the four phenols could interact with pancreatic lipase by hydrogen bonds, so as to change the spatial structure of pancreatic lipase and inhibit its catalytic activity. The present results suggest that LRE not only exhibits strong antioxidant capacity but possesses effectively inhibitory activity on pancreatic lipase, which might have the potential to be developed as functional food and nutraceuticals for the prevention of metabolic diseases. PRACTICAL APPLICATION: Ligustrum robustum (Rxob.) Blume extract has been confirmed to possess antioxidant activity and lipase inhibitory activity, which indicates that the L. robustum extract has the potential to prevent oxidative stress and regulate fat metabolism. This work suggests that L. robustum extract can be served as a novel resource to prepare nutraceuticals and functional food in food industries.

Component analysis using UPLC-Q-Exactive Orbitrap-HRMS and quality control of Kudingcha (Ligustrum robustum (Roxb.) Blume).

Although Kudingcha (Ligustrum robustum (Roxb.) Blume) has been widely used as both traditional medicine and food, systematic studies on their basic active components and quality control are lacking. In this study, a rapid method of identifying the general chemical components of Ligustrum robustum (Roxb.) Blume was established for the first time using UPLC-Q-Exactive Orbitrap-HRMS, and its major basic components were specified as phenylpropanoid, monoterpene, and flavonoid glycosides. The characteristic cleavage pathways of the phenylpropanoid, monoterpene, and flavonoid glycosides were further investigated and elaborated, which could assist in identifying the structures of similar components of other Chinese herbal medicines. A breakthrough was achieved in establishing a chemical fingerprinting profile of Ligustrum robustum (Roxb.) Blume from its original growing areas in China, and chemometric measures were applied to investigate the causes for the variations in its quality stability. The results indicated significant differences in the characteristic compositions of phenylpropanoid and monoterpene glycosides between mature and young leaves of Ligustrum robustum (Roxb.) Blume; however, no significant variation was observed owing to different production areas. Graded harvesting criteria should be established, and harvest period should be specified according to the target active components while considering agricultural metrics, such as leaf shape index, leaf length, and leaf width, to ensure the consistency in quality of active components during their production. From the perspective of overall quality control, an unprecedented quantitative analysis of multi-components by single marker was set up to analyze the signature components of phenylpropanoid glycosides (acteoside, isoacteoside, ligurobustoside N, and ligupurpuroside B) to increase the analytical efficiency and reduce research costs. This study created a scientific basis for the standardized operation, elucidation of the pharmacological materials, and quality control of food and supplements production with Ligustrum robustum (Roxb.) Blume as a raw material.

Effects of Ligustrum robustum (Rxob.) Blume extract on the quality of peanut and palm oils during storage and frying process.

The potential uses of Ligustrum robustum (Rxob.) Blume extract as a natural antioxidant to protect the quality of different oils during storage and frying process were studied. The results showed that L. robustum extract has been shown to retard the decline in the quality of both oils based on the tests of acid value, peroxide value, p-anisidine value, color, volatile flavor, and fatty acid compositions, and the protective effect of L. robustum extract on the quality of peanut oil was better than that of palm oil. By the component analysis, L. robustum extract was found to have a total phenols content of 140.75 ± 1.52 mg/g, and ligurobustoside C was identified as the main phenolic compound. The thermogravimetric and differential scanning calorimetry results showed that L. robustum extract enhanced the oxidative stability of peanut and palm oils. In addition, Fourier transform infrared results indicated that L. robustum extract had protective effects on the C=C bond and ester bond of oil molecule. Moreover, by using electron spin resonance technique, L. robustum extract showed the ability to inhibit and scavenge alkyl-free radicals in both oils. The present results suggested that L. robustum extract may protect the quality of oils during the storage and frying process by inhibiting the oxidation of unsaturated fatty acids and might be a potential natural antioxidant in the food industry. PRACTICAL APPLICATIONS: The excellent antioxidant ability of Ligustrum robustum (Rxob.) Blume extract on the oxidation of different oils and its low price indicated that it could be used as a new low-cost natural antioxidant in oil processing.

Ligustrum robustum (Roxb.) blume extract modulates gut microbiota and prevents metabolic syndrome in high-fat diet-fed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Chinese folk medicine, Ligustrum robustum (Roxb.) Blume has been widely used as a healthy tea beverage for improvement in obesity and lipidemic metabolic disorders. AIM OF THE STUDY: We aimed to investigate the effect of L. robustum extract (LRE) on metabolic syndrome in high-fat diet (HFD)-fed mice and to explore the underlying role of gut microbiota during the treatment. MATERIALS AND METHODS: The ground dried leaves of L. robustum (Roxb.) Blume were extracted with ethanol and then purified by a resin column. The composition of L. robustum extract (LRE) was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). C57BL/6 J mice fed with HFD were treated with LRE for 16 weeks. RT-qPCR and morphological staining were utilized to reveal the impact of LRE on hepatic glucolipid metabolism and gut integrity. The next-generation sequencing of 16 S rDNA was applied for analyzing the gut microbial community of fecal samples. RESULTS: LRE, mainly composed of ligupurpuroside A and aceteoside, alleviated insulin resistance, improved hepatic metabolism, enhanced intestinal integrity, and suppressed inflammatory responses in HFD-fed mice. Moreover, LRE treatment reshaped the gut microbiota structure by increasing the levels of genera Streptococcus, Lactobacillus, and Mucispirillum and decreasing the populations of Alistipes and Lachnospiraceae NK4A136 group in HFD-fed mice. The alteration of gut microbiota was associated with several metabolic pathways of gut bacteria. Spearman's correlation analysis further confirmed the links between the changed intestinal bacteria and multiple disease indices. CONCLUSIONS: LRE prevented gut microbiota dysbiosis and metabolic disorder in HFD-fed mice, which helps to promote the application in LRE-mediated prevention from metabolic syndrome as a gut microbial regulator.

Ligustrum robustum Alleviates Atherosclerosis by Decreasing Serum TMAO, Modulating Gut Microbiota, and Decreasing Bile Acid and Cholesterol Absorption in Mice.

SCOPE: Atherosclerosis (AS) is closely related to gut microbiota. Previous studies demonstrates that Ligustrum robustum (LR), a flavonoid-rich tea like plant, can mitigate several AS-related risk factors and modulate gut microbiota in animal models and human subjects. But its anti-AS effect and mechanisms remain unclear. Therefore, in this study, impacts of LR on AS development are investigated and the potential underlying mechanisms in C57BL/6J and Apoe-/- mice are explored. METHODS AND RESULTS: Female C57BL/6J and Apoe-/- mice are fed a chow diet or high-choline diet, supplemented with vehicle (water) or LR water extract (700 mg kg-1 ) by gavage for 17 weeks. It is found that LR attenuates diet-induced AS by reducing serum trimethylamine and trimethylamine-N-oxide (TMAO) levels likely by modulating gut microbiota. Moreover, LR increases the abundance of the genus Bifidobacterium, which generates bile salt hydrolase, and thus presumably enhances bile acid (BA) deconjugation and increases fecal BA excretion. Meanwhile, LR increases fecal cholesterol excretion, decreases the levels of serum and hepatic cholesterol, but did not affect short-chain fatty acids in feces. CONCLUSION: LR attenuates AS development presumably by decreasing serum TMAO levels and increasing fecal BA excretion likely via gut microbial modulation. These effects are accompanied by increases in fecal cholesterol excretion and decreases in serum and hepatic cholesterol.

Preparation of a functional yogurt with Ligustrum robustum (Rxob.) Blume and its action mechanism.

A functional yogurt was prepared with Ligustrum robustum (Rxob.) Blume extract (LRE), while its antioxidant and hypoglycemic activities were evaluated and its action mechanism was further explored. With the cofermentation of LRE, the yogurt showed the fine quality characteristics, including pH, titratable acidity, texture, syneresis susceptibility, color, microbiological content, and chemical composition. Meanwhile, the yogurt exhibited the remarkable antioxidant capability to enhance the activities of antioxidant enzymes and reduce the malondialdehyde level in animal serums. In addition, the yogurt showed the obvious hypoglycemic activity to inhibit the decrease of glucose tolerance and the increase of postprandial hyperglycemia of diabetes mice. Furthermore, using the analysis of molecular docking, the main compounds of LRE could combine tightly with α-amylase and α-glucosidase by hydrogen bond and hydrophobic interaction, so as to change their spatial structure and inhibit their biocatalytic activity in glucose metabolism. All present results suggested that LRE showed the potential value to be used as supplement to enhance the quality and functions of yogurt in food industry. PRACTICAL APPLICATION: The hypoglycemic and antioxidant activities of a functional yogurt cofermented with LRE were found and its relative action mechanism was also explored. This work provide the experimental and theoretical basis for the application of this yogurt as nutraceuticals to protect human health in food industry.

Preventive effect of small-leaved Kuding tea (Ligustrum robustum) on high-diet-induced obesity in C57BL/6J mice.

Small-leaved Kuding tea (SLKDT; Ligustrum robustum) is a traditional Chinese tea. We systematically investigated the effect of SLKDT extract on obesity. SLKDT-controlled weight gain in mice fed a high-fat diet. Tissue specimen results showed that the SLKDT extract alleviated liver damage and fat accumulation. Meanwhile, SLKDT extract improved dyslipidemia by decreasing total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels and increasing high-density lipoprotein cholesterol levels. Furthermore, SLKDT extract reduced alanine aminotransferase, alkaline phosphatase, and aspartate transaminase levels in the serum and liver tissues; decreased inflammatory cytokines, including interleukin (IL)-1ß, tumor necrosis factor-α, interferon-γ, and IL-6; and increased the anti-inflammatory cytokines, IL-4 and IL-10. The quantitative PCR results showed that SLKDT extract upregulated the mRNA expressions of peroxisome proliferator-activated receptor (PPAR)-α, lipoprotein lipase, carnitine palmitoyltransferase 1, and cholesterol 7 alpha hydroxylase and downregulated PPAR-γ and CCAAT/enhancer-binding protein-alpha mRNA expressions in the obese mouse livers to reduce adipocyte differentiation and fat accumulation, promote fat oxidation, and improve dyslipidemia, thereby inhibiting the immune response and alleviating liver injury. SLKDT shows a potential for preventing obesity and regulating obesity-related syndrome, so it is possible to be further developed as a novel treatment for fighting obesity.

In Vitro and In Vivo Evaluation of Antitumor Activity of Ligustrum robustum, A Chinese Herbal Tea.

OBJECTIVE: To examine the effect of the aqueous extract of Ligustrum robustum on tumor growth in vitro and in vivo and explore the possible molecular mechanisms. METHODS: In in vitro study, cell viabilities of human cervical carcinoma cells (HeLa), human breast cancer cells (MCF-7), human prostate cancer cells (PC-3), human hepatoma cells (7721) and human colon carcinoma cells (SW480) were evaluated with cell counting kit-8. For L. robustum-treated Hela cells, early or late apoptosis were evaluated by annexin V/PI staining. Mitochondrial membrane potential was measured by staining cells with JC-1. Apoptosis was monitored by nuclear morphology based on chromatin condensation and fragmentation by 4',6-diamidino-2-phenylinole (DAPI) staining. Caspase-3 and -8 activity levels were measured by a colorimetric assay. In vivo, to evaluate the possible mechanism of L. robustum-mediated antitumor effect, nude mouse xenograft study was also conducted. RESULTS: In in vitro study, L. robustum was found to be toxic to HeLa, MCF-7, PC-3, 7721, SW480, with an half maximal inhibitory concentration value of 2-5 mg/mL (P<0.05). Moreover, externalization of phosphatidylserine, loss of mitochondrial membrane potential, DNA fragmentation and activation of caspase-3 and -8 were detected in L. robustum-treated Hela cells. Using a nude mouse model bearing Hela xenografts, we found that L. robustum reduced tumor volume and tumor weight (P<0.05), but had no effect on body weight and histological damage of important organs. Intraperitoneal injection of L. robustum caused a significant reduction in serum aspartate transaminase and alanine transaminase levels (P<0.05). Furthermore, cleaved caspase-3-positive and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells were observed in L. robustum-treated tumor tissues. CONCLUSIONS: L. robustum inhibits tumor cell growth both in vitro and in vivo by inducing apoptosis in a caspase-dependent way without apparent hepatic toxicity and histological damage, which may offer partial scientific support for the ethnopharmacological claims of L. robustum as a herbal tea for its antitumor activity.

Acteoside From Ligustrum robustum (Roxb.) Blume Ameliorates Lipid Metabolism and Synthesis in a HepG2 Cell Model of Lipid Accumulation.

We aimed to ascertain the mechanism underlying the effects of acteoside (ACT) from Ligustrum robustum (Roxb.) Blume (Oleaceae) on lipid metabolism and synthesis. ACT, a water-soluble phenylpropanoid glycoside, is the most abundant and major active component of L. robustum; the leaves of L. robustum, known as kudingcha (bitter tea), have long been used in China as an herbal tea for weight loss. Recently, based on previous studies, our team reached a preliminary conclusion that phenylpropanoid glycosides from L. robustum most likely contribute substantially to reducing lipid levels, but the mechanism remains unclear. Here, we conducted an in silico screen of currently known phenylethanoid glycosides from L. robustum and attempted to explore the hypolipidemic mechanism of ACT, the representative component of phenylethanoid glycosides in L. robustum, using RNA-seq technology, quantitative real-time PCR (qPCR) and Western blotting. First, the screening results for six compounds were docked with 15 human protein targets, and 3 of 15 protein targets were related to cardiovascular diseases. Based on previous experimental data and docking results, we selected ACT, which exerted positive effects, for further study. We generated a lipid accumulation model using HepG2 cells treated with a high concentration of oleic acid and then extracted RNA from cells treated for 24 h with 50 µmol/L ACT. Subsequently, we performed a transcriptomic analysis of the RNA-seq results, which revealed a large number of differentially expressed genes. Finally, we randomly selected some genes and proteins for further validation using qPCR and Western blotting; the results agreed with the RNA-seq data and confirmed their reliability. In conclusion, our experiments proved that ACT from L. robustum alters lipid metabolism and synthesis by regulating the expression of multiple genes, including Scarb1, Scarb2, Srebf1, Dhcr7, Acat2, Hmgcr, Fdft1, and Lss, which are involved several pathways, such as the glycolytic, AMPK, and fatty acid degradation pathways.

Exploring mechanism of hypolipidemic effect of total Ligustrum robustum (Roxb. ) Blume on hyperlipidemic golden hamsters based on intestinal flora / 中国药理学通报

Aim To evaluate the hypolipidemic effect of the total phenylpropanoid glycosides extracted from Ligustrum robustum (Roxb.) Blume (LRTPG) on hyperlipidemic golden hamsters and explore its regulatory effect on intestinal flora. Methods Sixty hamsters were randomly divided into a control group, a model group, a positive drug group, LRTPG-L group, LRTPG-M group, and LRTPG-H group. After the successful induction of the model by high-fat diet, the animals were continuously administered for four weeks, and their blood lipids and liver lipids were detected. The formed feces from the colorectal region of the hamsters in the control group, model group and LRTPG-H group were collected for 16S rDNA sequencing. Results LRTPG reduced serum TG, TC, LDL-C and liver TG, TC concentrations significantly in hyperlipidemic hamsters. The results of the intestinal microbiota sequencing showed that compared to the control group, LRTPG significantly decreased the relative abundance of the phylum Firmicutes and increased the relative abundance of the phylum Bacteroidetes and Verrucomicrobia (P < 0.01) at the phylum level. At the family level, LRTPG significantly increased the relative abundance of Christensenellaceae, Peptococcaceae, and Verrucomicrobiaceae (P < 0.05 or P < 0.01). At the genus level, LRTPG significantly increased the relative abundance of Oscillospira, Oscillibacter, Flavonifractor and Akkermansiaceae (P < 0.05 or P < 0.01). These changes in the flora were beneficial to the hypolipidemic effect of LRTPG. Conclusion LRTPG may exert its hypolipidemic effect by improving the intestinal flora disorder caused by a high-fat diet in golden hamsters.