• Title/Summary/Keyword: In-vivo Study

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EVALUATION OF PERIODONTAL LIGAMENT CELL VIABILITY IN RAT TEETH ACCORDING TO VARIOUS EXTRA-ORAL DRY STORAGE TIMES USING MTT ASSAY (구강 외 노출시간에 따른 흰쥐 치아 치주인대세포 활성도의 MTT 검색법을 이용한 평가)

  • Jeon, In-Soo;Kim, Eui-Seong;Kim, Jin;Lee, Seung-Jong
    • Restorative Dentistry and Endodontics
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    • v.31 no.5
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    • pp.398-408
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    • 2006
  • The purpose of this study was to verify the usefulness of MTT analysis as a tool of measurement of the periodontal ligament cell viability from the extracted rat molar. A total of 80 Sprague-Dawley white female rat of 4 week-old with a body weight of 100 grams were used. The maxillary left and right, first and second molars were extracted under Ketamine anesthesia. Twenty-four teeth of each group (divided as five groups depending upon the time-lapse after extraction such as Immediate, 10, 20, 40 and 60 minutes) were immersed in $200{\mu}l$ of MTT solution (0.5 mg/ml) and processed for optical density measurements. Another 10 teeth of each group were treated as same as above and sectioned at $10{\mu}m$ for microscopic examination. All measurements values were divided by the value of hematoxylin-eosin staining which represented the volume of each corresponding samples. Immediate and 10 minute groups showed highest MTT values followed by 20, 40, and 60 minutes consecutively. Statistical significance (p<0.05) existed between all groups except in immediate versus 10 minute groups and 40 versus 60 minutes. Histological findings also showed similar findings with MTT results in crystal shape and crystal numbers between the experimental groups. These data indicate that in vivo MTT analysis nay be of value for evaluation of the periodontal ligament cell viability without time- consuming cell culturing processes.

Evaluation of Sprouted Barley as a Nutritive Feed Additive for Protaetia brevitarsis and Its Antibacterial Action against Serratia marcescens (흰점박이꽃무지 사료첨가제로서 새싹보리의 곤충병원성 세균에 대한 항균 효과에 관한 연구)

  • Song, Myung Ha;Kim, Nang-Hee;Park, Kwan-Ho;Kim, Eunsun;Kim, Yongsoon
    • Journal of Life Science
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    • v.31 no.5
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    • pp.475-480
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    • 2021
  • Interest in edible insects such as Protaetia brevitarsis has increased rapidly, and several insect producers use these insects in industrialized mass production. However, mass rearing of insects can cause insect diseases. Sprouted barley is a valuable source of nutrients and has antioxidant, antimicrobial, anti-inflammatory, and anti-cancer effects. This study was conducted to investigate the effect of sprouted barley as a feed additive for producing healthy P. brevitarsis larvae. P. brevitarsis larvae were fed feeds with or without sprouted barley, and their body weight and larval period wewe checked weekly. To confirm the antibacterial effects of sprouted barley, in vitro bioassays were performed by counting Serratia marcescens colonies, and in vivo bioassays were performed by determining the survival rate and body weights of the S. marcescens-infected larvae. Larvae fed different feeds were analyzed for their nutrient compositions (i.e., such as proximate composition, minerals, amino acids, and heavy metals). Larvae fed 5% and 10% sprouted barley had maximum weight increases of 19.2% and 23.1%, respectively. Both treatment groups had significantly shorter larval periods than those of the control group. Sprouted barley markedly inhibited the growth of entomopathogenic S. marcescens. Furthermore, larvae fed sprouted barley exhibited higher Cu, Zn, and K levels. Seventeen amino acids were present in larvae fed sprouted barley, of which, tyrosine and glutamic acid were predominant. No heavy metals were detected in any of the investigated groups. Therefore, sprouted barley may be a suitable feed additive for producing high-quality P. brevitarsis larvae.

Development of Polyclonal Antibodies to Abdominal and Subcutaneous Adipocyte for Producing Fat-reduced High Quality Pork (저지방 고품질 돈육 생산을 위한 돼지 복강 및 피하지방 항체 개발)

  • Choi, Chang-Weon;Baek, Kyung-Hoon;Choi, Chang-Bon;Oh, Young-Kyoon;Hong, Seong-Koo
    • Food Science of Animal Resources
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    • v.30 no.1
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    • pp.87-94
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    • 2010
  • The aim of the present study was to develop polyclonal antibodies to regional inedible adipocytes of pigs and investigate the effect of these antibodies on adipocytes in vitro. As antigens, abdominal and subcutaneous adipocyte PMPs from pigs were injected into sheep 3 times per 3 wk intervals for passive immunization, and non-immunized serum, antisera against abodominal (AAb) or subcutaneous adipocyte PMPs (SAb) were collected before and after the injections. Titers of the antisera obtained from sheep and their cross-reactivities with the heart, kidney, liver, lung, muscle, and spleen of pig were determined by ELISA. Isolation and culture of abdominal and subcutaneous adipocytes from pigs were performed to analyze LDH concentration. At a 1:1,000 dilution, little antibody reactivity was observed for non-immunized serum whereas both AAb and SAb had relatively strong reactivity up to a dilution of 1:16,000. These findings may indicate that strong antibodies against adipocyte PMPs can be developed using an immunological approach. Extremely low reactivity of AAb and SAb was detected with the PMPs of the organs. Both antisera most strongly reacted with each adipocyte PMPs and showed statistically (p<0.05) higher cross-reactivities compared with the non-immunized serum. In conclusion, these results may indicate that the present polyclonal antibodies against regional inedible adipocyte PMPs are well developed and are safe against cross-reactivities with the organs of pigs. Further studies on the in vivo nutritional safety and fat reduction of these antibodies in pigs will be required fat-reduced high quality pork production.

Anti-Inflammatory Effect of Sargassum patens C. Agardh Ethanol Extract in LPS-induced RAW264.7 Cells and Mouse Ear Edema (LPS로 유도된 RAW 264.7 cell과 마우스 귀 부종 모델을 통한 쌍발이 모자반 에탄올 추출물의 항염증 효과)

  • Kim, Min-Ji;Kim, Min-Ju;Kim, Koth-Bong-Woo-Ri;Park, Sun-Hee;Choi, Hyeun-Deok;Park, So-Yeong;Kim, Ji-Hyun;Jang, Mi-Ran;Im, Moo-Hyeog;Ahn, Dong-Hyun
    • Microbiology and Biotechnology Letters
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    • v.45 no.2
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    • pp.110-117
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    • 2017
  • The anti-inflammatory effect of Sargassum patens C. Agardh ethanol extract (SPEE) was examined based on the lipopolysaccharide (LPS)-induced inflammatory response in this study. SPEE treatment was not cytotoxic to macrophages compared to the control. The production of NO was suppressed by SPEE by approximately 28% at $100{\mu}g/ml$, and levels of interleukin-6, tumor necrosis $factor-{\alpha}$, and $interleukin-1{\beta}$ decreased in a dose-dependent manner. In addition, the expression of inducible nitric oxide synthase, cyclooxygenase-2, and nuclear $factor-{\kappa}B$ was suppressed by SPEE treatment. In vivo, croton oil-induced mouse ear edema was attenuated by SPEE and the infiltration of mast cells into the tissue decreased. Based on these results, SPEE inhibits the release of LPS-induced pro-inflammatory cytokines and mediators, suggesting that SPEE is a potential agent for anti-inflammatory therapies.

The Effect of Histamine on Polymorphonuclear Leukocyte-induced Pneumocyte Injury in Vitro (다형핵구에 의한 폐포세포 손상에 Histamine이 미치는 영향)

  • Kim, Young-Kyoon;Kwon, Soon-Seog;Kim, Kwan-Hyung;Han, Ki-Don;Moon, Hwa-Sik;Sang, Jeong-Sup;Park, Sung-Hak
    • Tuberculosis and Respiratory Diseases
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    • v.39 no.3
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    • pp.228-235
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    • 1992
  • Background: Although polymorphonuclear leukocytes (PMN) are important in protecting the airways and alveolar surfaces, there is evidence that they can also injure the lung while exercising their defensive role. However it has been unclear whether PMN-induced pneumocyte injury is mediated by their direct cytotoxic effect on target cells or by PMN-derived cytotoxic mediators. On the other hand histamine was known not only to act as an important chemical mediator participated in the pathogenesis of some atotic and allegic disorders, but also to have an inhibitory effect on normal PMN functions. Method: To study the mechanism by which PMN induce pneumocyte injury, we cocultured PMN from four healthy nonsmokers or their PMN-derived supernatants (PMN-SPN) with monolayers of $^{51}Cr$-labeled human A549 pneumocytes and compared PMN-and PMN-SPN-mediated pneumocyte injuries measured by $^{51}Cr$ release assay. We also compared the effects of histamine on each pneumocyte injury. Results: 1) PMN-SPN showed more injurious effect on A549 pneumocytes than that of PMN itself regardless histamine pretreatment of PMN. 2) Pneumocyte injury by PMN with histamine pretreatment was increased or decreased compared with that by PMN without histamine pretreatment, according to histamine concentrations, and PMN stimulating agents and their concentrations. 3) Pneumocyte injury by PMN-SPN with histamine pretreatment tended to be decreased compared with that by PMN-SPN without histamine pretreatment. Conclusion: Our results suggest that PMN-SPN may play more important role in mediating pneumocyte injury than PMN itself and that histamine may partially play a protective role on PMN-induced pneumocyte injury. Alternatively we conclude that the effects of histamine on PMN-induced pneumocyte injury may be affected by microenvironment in vivo.

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Quality Characteristics and Anti-Diabetic Effect of Yacon Vinegar (야콘 식초의 품질특성 및 항당뇨 효과)

  • Lee, Mi-Kyung;Choi, Sa-Ra;Lee, Jin;Choi, Yun-Hong;Lee, Ju-Hye;Park, Kyung-Uk;Kwon, Seung-Hyek;Seo, Kwon-Il
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.41 no.1
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    • pp.79-86
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    • 2012
  • This study was performed to investigate the physiochemical properties and anti-diabetic effect of yacon vinegar by two-step fermentation. Yacon was matured at room temperature for 20 days. The sugar content of yacon juice prepared from mature yacon was approximately $14^{\circ}Brix$. In the first stage, yacon wine was produced from the juice at $28^{\circ}C$ for 6 days. In the second stage, acetic acid fermentation was conducted at $30^{\circ}C$ and 200 rpm for 6 days to produce yacon vinegar with 4.75% acidity. The major free sugars of yacon vinegar were glucose and fructose at 2,072.12 mg% and 463.95 mg%, respectively. The acetic acid content was the highest of the major organic acids at 3,881.44 mg%. The total free amino acid content was 62.88 mg% with the main free amino acids being proline, ${\gamma}$-amino-n-butyric acid and ornithine. The major minerals of yacon vinegar were Ca, K and Mg. The in vivo anti-diabetic activity of yacon vinegar was investigated in high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice. Diabetic mice were administered orally with 10% yacon juice and two yacon vinegars (5% and 10%) at a dose of 7 mL/kg body weight once per day for 4 weeks. Five% yacon vinegar improved the fasting blood glucose levels and glucose tolerance test significantly compared to the diabetic control group (p<0.05). Yacon vinegar increased the pancreatic C-peptide concentration in a dose-dependent manner. These results show that 5% yacon vinegar has a more potent effect on ameliorating hyperglycemia than 10% yacon juice.

Hypoglycemic Effects of Fruits and Vegetables in Hyperglycemic Rats for Prevention of Type-2 Diabetes (고혈압쥐의 과일과 야채의 섭취에 따른 저혈당 효과)

  • Survay, Nazneen Shaik;Ko, Eun-Young;Upadhyay, Chandrama Prakash;Jang, Mi;Park, Se-Won;Lee, Dong-Ha;Jung, Yi-Sook;Yoon, Do-Young;Hong, Sae-Jin
    • Horticultural Science & Technology
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    • v.28 no.5
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    • pp.850-856
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    • 2010
  • An in vivo oral glucose tolerance test (OGTT) was performed on hyperglycemic male Sprague-Dawley rats to assess the effect of fruits and vegetables ($1g{\cdot}kg^{-1}$ body weight) on blood glucose levels (${\Delta}BGLs$) at different time intervals of 0, 5, 15, 30, 60, 90 and 120 min. The areas under glucose curve (${\Delta}AUCs$) were calculated at 120 min of OGTT by trapezoid method. Total phenolic content (TPC) and anti-oxidant activity (AOA) of fruits and vegetables were assayed in vitro by Folin Ciocalteu and DPPH (2, 2-diphenyl-1-picrylhydrazyl) methods, respectively. At the end of the experiment the correlations among the parameters TPC, AOA and ${\Delta}AUC$ was estimated by Pearson's correlations. Among fruit crops, tangerine, plum, grape and pear and among vegetables, blue leaf mustard, cabbage, chicory, broccoli and others exhibited significant hypoglycemic effects by reducing ${\Delta}BGLs$ with significant ${\Delta}AUC$. The effective ${\Delta}AUC$ ranged from $5548.2{\pm}462.1$ to $3823.3{\pm}282.0mg-min{\cdot}dL^{-1}$. The TPC and AOA ranged from $0.063{\pm}0.00$ to $0.913{\pm}0.14mg{\cdot}g^{-1}$ GAE and $01.05{\pm}0.08$ to $75.46{\pm}0.06%$, respectively. Overall, six fruits and fifteen vegetables exhibited higher TPC and one fruit and four vegetables exhibited higher AOA. There was a better correlation among TPC, AOA and ${\Delta}AUC$ of fruits and TPC & AOA of vegetables. We report that hypoglycemically significant fruits and vegetables investigated in this study have pharmacological importance which reduced ${\Delta}BGLs$ through insulin like activity and AOA in prevention of type-2 diabetes.

Screening of Useful Plants with Anti-inflammatory and Antioxidant Activity (항염증 및 항산화 활성 보유 유용 식물 탐색)

  • Lee, Seung-Eun;Choi, Jehun;Lee, Jeong-Hoon;Noh, Hyung-Jun;Kim, Geum-Sook;Kim, Jinkyung;Chung, Hae-Young;Kim, Seung-Yu
    • Korean Journal of Plant Resources
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    • v.26 no.4
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    • pp.441-449
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    • 2013
  • This study was conducted to select some useful plants as functional material candidates. A total of 38 plants were preliminarily screened for the anti-inflammatory and antioxidant activities. The preliminarily selected 8 plants were further investigated to verify the in vitro inhibitory effect on inflammation and oxidative stress. Boehmeria platanifolia (root), Carpinus coreana (branch), and Eupatorium japonicum (leaf) inhibited the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Eupatorium japonicum (leaf) suppressed the expression of cyclooxygenase-2 (COX-2), whereas Boehmeria platanifolia (root) and Prunus yedoensis (branch) inhibited the transcription of nuclear factor-kappa B (NF-${\kappa}B$). Treatment with the extracts ($2.5{\sim}20{\mu}g/ml$) of Abutilon theophrasti (leaf, flower/seed) and Hemistepta lyrata (stem) did not show toxicity on RAW 264.7 cell proliferation, but treatment with $2.5{\mu}g/ml$ of Boehmeria platanifolia (root) exhibited cell toxicity. Carpinus coreana (branch) and Prunus yedoensis (branch) showed potent scavenging activities on peroxynitrite. Akebia quinata (flower), Carpinus coreana (branch), and Prunus yedoensis (branch) effectively inhibited reactive oxygen species (ROS). Abutilon theophrasti (leaf), Boehmeria platanifolia (root), Carpinus coreana (branch), and Eupatorium japonicum (leaf) exhibited strong inhibitory capacity with regard to nitric oxide (NO) production. The results suggested that Abutilon theophrasti (leaf) has in vitro anti-inflammatory and antioxidant activities, and that is a useful functional material candidate.

Purification and Characterization of Sulfated Polysaccharide Isolated from Hot Water Extract of Pachymeniopsis elliptica (Pachymeniopsis elliptica의 열수 추출물로부터 분리한 함황 다당류의 정제 및 특성)

  • Lee, Sun-Hee;Jun, Woo-Jin;Yu, Kwang-Won;Chun, Hyug;Shin, Dong-Hoon;Hong, Bum-Shik;Cho, Hong-Yon;Yang, Han-Chul
    • Korean Journal of Food Science and Technology
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    • v.32 no.5
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    • pp.1191-1197
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    • 2000
  • In the preliminary study, we investigated the anti-complementary activities of 62 extracts from Korean edible seaweeds. Of those, Pachymeniopsis elliptica showed the highest anti-complementary activity. Therefore, it was purified as follows; i) PE-1 by ethanol precipitation, ii) PE-1-C by ultrafiltration, iii) PE-1-CIV by DEAE-Toyopearl 650C, and iv) PE-1-CIV-ii by Sepharose CL-6B. The purified compound, PE-1-CIV-ii, was the complexed homogeneous polysaccharide (molecular mass: 780 kDa) with 82.9% of anti-complementary activity. Also, it contained a significant amount of sulfate group (30.5%), which indicated it as a sulfated algal polysaccharide. Its structural monosaccharides were galactose (44.3%), 3,6-anhydrogalactose (34.0%), glucose (8.2%), fucose (5.4%), xylose (5.2%) and rhamnose (2.9%). After the treatment of periodate on a sample, a significant decrease in anti-complementary activity was found, which was a characteristic of bioactive polysaccharides. And-tumor activity of PE-1-A, B and C was tested in the sarcoma-180 solid tumor model. The PE-1-C with the largest molecular mass (more than 300 kDa) showed 81% of inhibition on the solid tumors, suggesting that the anti-complementary activity was, at least in part, related to anti-tumor activity. Based upon these results, the purified polysacchardes could be an immunopotentiator in vivo.

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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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