Oxidosqualene cyclases in triterpenoids biosynthesis: a review / 生物工程学报

Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.

Construction of squalene producing cell factories and screening, cloning and expression of key genes / 生物工程学报

Squalene is widely used in pharmaceutical, nutraceutical, cosmetics and other fields because of its strong antioxidative, antibacterial and anti-tumor activities. In order to produce squalene, a gene ispA encoding farnesyl pyrophosphate synthase was overexpressed in a previously engineered Escherichia coli strain capable of efficiently producing terpenoids, resulting in a chassis strain that efficiently synthesizes triterpenoids. Through phylogenetic analysis, screening, cloning and expression of squalene synthase derived from different prokaryotes, engineered E. coli strains capable of efficiently producing squalene were obtained. Among them, squalene produced by strains harboring squalene synthase derived from Thermosynechococcus elongatus and Synechococcus lividus reached (16.5±1.4) mg/g DCW ((167.1±14.3) mg/L broth) and (12.0±1.9) mg/g DCW ((121.8±19.5) mg/L broth), respectively. Compared with the first-generation strains harboring the human-derived squalene synthase, the squalene synthase derived from T. elongatus and S. lividus remarkably increased the squalene production by 3.3 times and 2.4 times, respectively, making progress toward the cost-effective heterologous production of squalene.

Construction and optimization of squalene epoxide synthetic pathway in Escherichia coli / 生物工程学报

Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.

Modulation of Melanin Synthesis by Amaranthus spp. L Seed Extract in Melan-a Cells

Anti-melanogenic effects of amaranth (AT), one of the key source of squalene, were investigated in melanocytes. Amaranth seed powder was extracted with water and melan-a cells were treated with various concentrations of AT. By using HPLC, content of myo-inositol, one of potential active components, was measured in the crude extract of AT.AT reduced the melanin content in melan-a melanocytes and down-regulated melanogenic enzyme activity such as tyrosinase, TRP-1 and TRP-2. By regulating melanogenic enzyme activity, AT may be a potential natural source for whitening agent. Myo-inositol was detected in AT by HPLC and may be one of the active compounds from AT involved in the regulation of anti-melanogenesis. In this study, we demonstrated that AT has anti-melanogenesis properties. This new function of amaranth may be useful in the development of new skin-whitening products and its value as food.

A Case of Foreign Body Granuloma after Squalene Injection by Non-dermatologists / 대한피부과학회지

No abstract available.

Downregulation of lanosterol synthase gene expression by antisense RNA technology in Saccharomyces cerevisiae / 药学学报

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.

Construction of Saccharomyces cerevisiae haploid mutant deficient in lanosterol synthase gene / 药学学报

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.

Fluad(R)-MF59(R)-Adjuvanted Influenza Vaccine in Older Adults

Influenza directly or indirectly contributes to the four leading causes of global mortality, at rates that are highest in older adults. As the proportion of older adults in the Korean population is greater than in most other countries, influenza prevention is a greater public health priority in Korea than elsewhere. Conventional inactivated influenza vaccine (IIV) is less immunogenic and efficacious (-50%) in older than in young adults, but adjuvanting the vaccine with oil-in-water emulsion MF59(R) increases immunogenicity, resulting in comparatively higher levels of hemagglutination inhibition antibodies and greater protection against all influenza, as well as cases requiring hospitalization. A recent observational study demonstrated that the adjuvanted vaccine protected older adults against influenza in a year when nonadjuvanted IIV was ineffective. In another multiyear study, the adjuvanted vaccine was estimated to be 25% more effective in preventing pneumonia and influenza hospitalizations compared to nonadjuvanted vaccine. Although MF59-adjuvanted vaccine is transiently more reactogenic than nonadjuvanted vaccine, there is no evidence that it increases risks for serious adverse events, including those with an autoimmune etiology. Experience thus far indicates a favorable balance of benefit to risk for MF59. This may reflect the adjuvant's mechanism of action in which the squalene oil emulsion increases antibody responses to co-administered antigen without acting more generally as an immunopotentiator.

Researches on influence of squalene synthase gene polymorphism on catalytic efficiency of its encode enzyme in Glycyrrhiza uralensis / 中国中药杂志

<p><b>OBJECTIVE</b>To analyse the polymorphism of squalene synthase gene and reveal the influence of squalene synthase (SQS) gene polymorphism on the catalytic efficiency of its encode enzyme in Glycyrrhiza uralensi.</p><p><b>METHOD</b>The total RNA was extracted. PCR was used to amplify the coding sequences of squalene synthase gene, which were sequenced and analysed. The expression vectors containing different SQS gene sequences, including SQS1C, SQS1F, SQS2A, SQS2B, were constructed and transformed into Escherichia coli BL21. The fusion protein was induced to express by IPTG, then was isolated, purified and used to carry out the enzymatic reaction in vitro. GC-MS was used to analyse the production.</p><p><b>RESULT</b>There were three kinds of gene polymorphism existing in SQS1 gene of G. uralensis, including single nucleotide polymorphism (SNPs), insertion/deletion length polymorphism (InDels) and level of amino acid, the proportion of conservative replace of SQS1 was 53.94%, and there were 2 mutational sites in structural domains. The proportion of conservative replace of SQS2 was 60%, and there was 1 mutational site in structural domains. The production squalene could be detected by GC-MS in all the 4 kinds of enzymatic reactions. The capacity of accumulating squalene of SQS1F was higher than other SQS genes.</p><p><b>CONCLUSION</b>The polymorphism of SQS gene was quite abundant in G. uralensis, which maybe the molecular foundation of the formation of high-quality liquorice.</p>

Study on the hypochlolesterolemic and antioxidative effects of tyramine derivatives from the root bark of Lycium chenese Miller

The aim of the present study was to investigate the hypocholesterolemic effect and potential of tyramine derivatives from Lycii Cortex Radicis (LCR), the root bark of lycium (Lycium chenese Miller) in reducing lipid peroxidation. The activities of enzymes, hepatic 3-hydroxy 3-methylglutaryl (HMG) CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT) and LDL oxidation were measured in vitro and animal experiments were also performed by feeding LCR extracts to rats. The test compounds employed for in vitro study were trans-N-p-coumaroyltyramine (CT) and trans-N-feruloyltyramine (FT), LCR components, N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS) from safflower seeds, ferulic acid (FA) and 10-gingerol. It was observed that FT and FS at the concentration of 1.2 mg/mL inhibited liver microsomal HMG CoA reductase activity by ~40%, but no inhibition of activity was seen in the cases of CT, CS, FA and 10-gingerol. Whereas, ACAT activity was inhibited ~50% by FT and CT, 34-43% by FS and CS and ~80% by 10-gingerol at the concentration of 1 mg/mL. A significant delay in LDL oxidation was induced by CT, FT, and 10-gingerol. For the animal experiment, five groups of Sprague-Dawley male rats were fed high fat diets containing no test material (HF-control), 1 and 2% of LCR ethanol extract (LCR1 and LCR2), and 1% of extracts from safflower seed (Saf) and ginger (Gin). The results indicated that total cholesterol level was significantly lower in Saf, LCR2 and Gin groups, and HDL cholesterol level was lower only in Gin group when compared with HF-control group; while there was no difference in the serum triglyceride levels among the five experimental groups. The level of liver cholesterol was significantly lower in LCR1 and LCR2 groups than HF-control. Serum levels of TBARS were significantly lower only in LCR2 group when compared with HF-control group. From the observed results, we concluded that LCR can be utilized as a hypocholesterolemic ingredient in combination with ginger, especially for functional foods.

A Case of Superficial Acral Fibromyxoma / 대한피부과학회지

Superficial acral fibromyxoma (SAF) is an uncommon soft tissue tumor that has recently been showed to be a separate disease entity. It is most often located in the ungal region of the fingers and toes of middle-aged adults. It is histologically characterized by a slight to moderate cellular proliferation of spindle cells and stellate cells arranged in a random, storiform or fascicular pattern, within a myxoid, myxocollagenous or collagenous stroma with a prominent vasculature. The neoplastic cells show positive staining for CD34, CD99 and EMA, but negative staining for S100, HMB45, cytokeratin, smooth muscle actin (SMA) and desmin. We report here on a typical case of SAF that occurred on the left index finger of a 44-year-old man.

Contenido de aceite, ácidos grasos y escualeno en variedades crudas y procesadas de grano de amaranto / The oil, fatty acid and squalene content of varieties of raw and processed grain amaranth

Seis variedades de Amaranto fueron procesadas para dar una harina cruda, una nixtamalizada, una cocida en agua, otra expandida, una malteada y una laminada después de un tratamiento térmico. Los valores analíticos en estas muestras se compararon con los valores en una muestra cruda. Los granos crudos contenían de 14.5 por ciento a 15.1 por ciento de proteína, 5.9 a 6.7 de grasa, y 2.3 por ciento a 3.2 por ciento de cenizas. Las harinas de las variedades de diferentes procesos dieron un contenido de grasa que varía entre 6.4 por ciento - 7.0 por ciento. Las harinas de procesos en seco contenían mas aceite que las de procesos en húmedo (cocida en agua). El aceite de tres variedades y de 4 procesos fue analizado por su contenido de ácidos grasos, dando en promedio 17.85 por ciento de C16:0, 68.1 por ciento de oleico + linoléico, 3.86 por ciento C18:3, 5.1 por ciento de C20:0 y cantidades menores de C20:1 y C22:0. El contenido de escualeno en el aceite de las harinas de los diferentes procesos fue de 7.0-9.6 g/100 g para la harina cruda, de 8.1-12.6 g/100 g para la de cocción húmeda, 9.0-12.7 g/100 g para la nixtamalizada, 10.1-12.8 g/100 g para la expandida, 9.0-11.2 g/100 g para la malteada y 6.0-9.5 g/100 g para la harina laminada.

Six amaranth grain varieties were processed to yield a nixtamalized flour, one cooked in water, one expanded, a malted one and a laminate samples after a thermic treatment. The chemical values of the raw samples contained from 14.5 percent to 15.1 percent protein, 5.9 to 6.7 percent ether extract and from 2.3 percent to 3.2 percent ash on a dry weight basis. The flours from the different processes yield products with a fat content which varied from 6.4 percent to 7.0 percent for the 6 varieties. The flours coming from dry heat processing contained higher oil levels than those flours coming from wet processes. The oil from only 3 varieties and from 4 processes were analyzed from its fatty acid composition. The oil contained on the average 17.85 percent of C16:0, 68.1 percent of stearic, olic and linoleic acids, 3.86 percent of C18:3, 5.1 percent of C20:0 and small amounts of C20:1 and C22:0. The squalene content in the oil of the processed flours varied from 7.0 to 9.6 g/100 g for the raw flour, 8.1 - 12.6 g/100 g for the flour from wet cooking in water, 9.0 -12.7g/100 g for the flour from the nixtamalization process, 10.1-12.8g/100 g for the expanded grain flour, 9.0 to 11.2 g/100 g for the malted flour and 6.0 -9.5 g/100 g for the laminated grain flour. The squalene averages per process showed statistical significant differences.

Influence of squalene feeding on plasma leptin, testosterone & blood pressure in rats.

BACKGROUND & OBJECTIVES: Obesity, hyperlipidaemia and hypercholesterolaemia are known risk factors in the pathogenesis of hypertension. Squalene has been shown to reduce serum cholesterol and triglycerides in dogs although its therapeutic use in high BP and obese patients has not been established. This study evaluates the effect of feeding high doses of squalene on plasma leptin, glucose, testosterone, blood pressure (BP) and body fat in rats. METHODS: Wistar rats (male, 22 days old) were randomly divided into two groups receiving either regular control diet or a squalene-containing diet. After feeding squalene for 4 wk, 10 rats each from the squalene and control groups were sacrificed and blood samples were collected for measurement of leptin, cholesterol, triglycerides and testosterone. Blood pressure was monitored weekly. RESULTS: Following squalene feeding, BP and body weight gain were lower in the squalene group. BP was significantly lower from 47 days of age in squalene fed group compared to controls. The levels of plasma leptin, glucose, cholesterol and triglycerides were significantly lower in squalene fed rats than those from the control group at 51 and 75 days of age. However, testicular weights (only 75 days) and testosterone levels were significantly higher in rats from the squalene group than those from the control group at days 51 and 75. INTERPRETATION & CONCLUSION: Our results suggested that squalene may counteract the increase in body fat, BP and levels of plasma leptin, glucose, cholesterol and triglycerides. These effects of squalene may be further explored as a likely new approach for clinical management of high BP and obesity.

A Case of Lipoid Pneumonia Induced by Aspiration of Shark Liver Oil / 소아알레르기및호흡기학회지

Lipoid pneumonia, a rare disease entity, results from accumulation of lipids in the alveoli. It results from the aspiration of vegetable, animal oil or mineral oil. Squalene is a derivative of shark liver oil that is taken as a traditional remedy in some Asian counties and is used widely also in cosmetics. Although many cases with predisposing factors have been reported, lipoid pneumonia can occur in normal individuals without predisposing factors. A 2-year-old boy was admitted to the hospital with a history of fever and cough for during 2 weeks. On physical examination, his lung sound was decreased in the right lung field. He was found to have infiltrate in the right upper lobe, middle and lower lung field on a chest radiograph. His parents fed him with squalene. High resolution computed tomography scan of the chest showed 'Crazy-paving appearance' in the right upper, middle and lower lobes. Lipoid pneumonia is suggested when there is a history of oil- or lipid- based product use or consumption. The characteristic finding of lipid materials, 'Crazy-paving appearance' in chest Computed Tomography (CT) or chest Magnetic Resonance Image (MRI), may be useful in the diagnosis of lipoid pneumonia. We report the case of lipoid pneumonia of a 2-year-old healthy child with a history of squalene.

Bilateral Pulmonary Infiltrate with Milky BAL Fluid / 결핵및호흡기질환

Exogenous lipoid pneumonia is an uncommon disease that's caused by aspirating lipid formulations. Squalene, obtained from shark liver oil, is one of the causative agent and this is commonly used by some Koreans as a health promoting medication. We report here on a case of exogenous lipoid pneumonia that developed after ingestion of squalene capsules. The case showed milky BAL fluid and multiple pulmonary consolidations.

A Case of Exogenous Lipoid Pneumonia after Ingestion of Squalene in Patients with Achalasia / 결핵및호흡기질환

Exogenous lipoid pneumonia (ELP) is a chronic inflammatory reaction of the lungs resulting from the aspiration of vegetable, animal or mineral oils. Squalene, is a derivative of shark liver oil that is taken as a traditional remedy in some Asian countries, and is used widely also in cosmetics. Similar to the symptoms in most cases of oil aspiration, the symptoms of squalene-induced lipoid pneumonia are either absent or nonspecific. Hence, the disease is generally detected incidentally. Although many cases with predisposing factors have been reported, ELP with achalasia is quite rare. We report a 47-year old woman with achalasia who developed ELP after ingesting squalene. The patient was treated successfully by supportive care and surgical treatment of the achalasia.

The Effects of Physiogel(R) Cream on the Allergic Contact Dermatitis / 대한피부과학회지

BACKGROUND: A allergic contact dermatitis has been increasing in the world. The first choice of treatment for the allergic contact dermatitis is topical steroids, but application of topical steroids may lead to many side effects. Physiogel(R) cream, made of ceramide, triglyceride, squalene and others is a moisturizer without any fragnance or preservatives which has effects of normalizing the damaged skin barrier and soothing troubled skin. OBJECTIVES: We carried out this study to evaluate the effects of the Physiogel(R) cream in the treamemt of the allergic contact dermatitis. METHODS: Among the patients clinically suspected to have allergic contact dermatitis due to fragnance or perservatives, patients already diagnosed with allergic contact dermatitis were included. On the skin lesions of the right side of individual patients the Physiogel(R) cream was applied twice a day. No topical agents were applied to the lesions of the left side. Two weeks later the clinical improvement of skin lesions was compared. RESULTS: In fourteen (61%) out of 23 patients the Physiogel(R) cream-applied lesions showed significant clinical improvement compared with the control lesions. In cases of atopic dermatitis or seborrheic dermatitis, the ratio of patients in whom the Physiogel(R) cream was efficacious, was 80% or 70% respectively. The skin lesions of all patients of 2nd and 3rd decades were significantly improved by the Physiogel(R) cream. In patients whose skin lesions were not responsive to the Physiogel(R) cream, patch test results showed the highest positive response rate against nickel (20%) and formaldehyde (20%). CONCLUSION: Physiogel(R) cream could be used as well as topical steroids in the treatment of allergic contact dermatitis reducing the risk of side effects due to long-term use of topical steroids.

Sclerosing Lipogranuloma by Subcutaneous Injection of Squalene into Penis / 대한비뇨기과학회지

A 21 year-old man visited our clinic complaining of a penile skin defect and discharge after self-injection of Squalene into his penis to facilitate an erection and increase his potency. The compromised skin and subcutaneous tissue were completely removed, and a split-thickness skin graft performed using the skin from the right side of the groins. This method was a simple, one stage operation, with a shortened hospital stay. Although a few studies on the oral administration of Squalene have been performed, a Squalene injection for penile augmentation has seldom been reported. Herein, a case in which a patient injected Squalene oil into his penis is reported and the subsequent treatment result described.

Severe Exogenous Lipoid Pneumonia Following Ingestion of Large Dose Squalene: Successful Treatment with Steroid / 결핵및호흡기질환

Exogenous lipoid pneumonia (ELP) is an uncommon condition resulting from aspiration or inhalation of vegetable, animal or mineral oil. It results in foreign body type inflammatory reaction of the lung and can show various clinical presentations from asymptomatic incidental finding to severe pneumonia leading to acute respiratory failure. Although many cases have been reported, severe ELP requiring steroid or whole lung lavage for treatment is rare. We report a case of 51-year old man with esophageal cancer who developed severe ELP following ingestion of large dose shark oil (Squalene) and successfully treated with prednisolone.

Chemical constituents from the mangrove plant Ceriops tagal / 药学学报

<p><b>AIM</b>To investigate the secondary metabolites of the mangrove plant Ceriops tagal.</p><p><b>METHODS</b>Column chromatography techniques including HPLC were used for the separation and purification, and extensive spectral analysis including various 2D NMR spectra were employed for structure elucidation.</p><p><b>RESULTS</b>Nine compounds, namely, tagalsins A (1), ent-5alpha-dolabr-4 (18) -ene-15S,16-diol (2), squalene (3), betulinic acid (4), lup-20 (29) -en-3-on-28-oic acid (5), betulin (6), lup-20 (29) -en-3-on-28-ol (7), beta-sitosterol (8), n-hexacosanylferulate (9) were obtained. Of which 1 and 2 belong to dolabrane diterpene.</p><p><b>CONCLUSION</b>Compound 1 is a new compound, and 2 to 9 are isolated from this species for the first time.</p>