This research investigated the preparation of activated carbon derived from Krabok (Irvingia malayana) seed shells to improve the quality of crude glycerol obtained during biodiesel production. The activated carbon was prepared using a dry chemical activation method with NaOH, utilizing an innovative biomass incinerator. The results revealed that the resulting KC/AC-two-step exhibited favorable physicochemical adsorption properties, with a high surface area of 758.72 m2/g and an iodine number of 611.10 mg/g. These values meet the criteria of the industrial product standard for activated carbon No. TIS 900-2004, as specified by the Ministry of Industry in Thailand. Additionally, the adsorption efficiency for methylene blue reached an impressive 99.35 %. This developed activated carbon was then used to improve the quality of crude glycerol obtained from biodiesel production. The experimental results showed that the KC/AC-two-step increased the purity of crude glycerol to 73.61 %. In comparison, commercially available activated carbon (C/AC) resulted in a higher crude glycerol purity of 81.19 %, as analyzed by the GC technique. Additionally, the metal content (Zn, Cu, Fe, Pb, Cd, and Na) in purified glycerol using KC/AC-two-step was below the standards for heavy metals permitted in food and cosmeceuticals by the Food and Drug Administration of Thailand and the European Committee for Food Contact Materials and Articles. As a result, it can be inferred that Krabok seed shells have favorable properties for producing activated carbon suitable as an adsorbent to enhance crude glycerol purity. Furthermore, the improved crude glycerol from this research has potential for various industrial applications.

The present study was undertaken to evaluate the effect of trisaccharides supplementation in glycerol-free tris (GFT) for the cryopreservation of dog spermatozoa. In the first experiment (E1), dog spermatozoa were resuspended with 50, 75, 100 or 125 mM of raffinose, melezitose or maltotriose and cooled at 4 ℃ for 10 min. To determine the effect of different cooling time, the spermatozoa resuspended with 100 mM of raffinose, melezitose or maltotriose were cooled during 10, 20, 30 or 40 min at 4 ℃ (second experiment; E2). The straws were then aligned horizontally for 10 min on the rack and then plunged into LN2. In the third experiment (E3), to determine the effect of different vapor freezing time, the spermatozoa resuspended with 100 mM raffinose were cooled at 4 ℃ for 20 min and frozen in LN2 for 5, 10, 15 or 20 min and then plunged into LN2. In the fourth experiment (E4), to compare different freezing methods [cooling plus vapor freezing (CV), cooling plus step-down freezing (CS) and direct step-down freezing (SD)], the spermatozoa resuspended with 100 mM raffinose were cooled for 20 min and frozen in LN2 vapor for 5 min in case of CV method. In case of CS method, spermatozoa were cooled for 20 min at 4℃ and then frozen by the step-down freezing method. The straws were then aligned horizontally at 18, 15, 5, and 2 cm respectively from the surface of LN2 for 1, 1, 1.4, and 5 min, respectively in an L shaped straw holder and then plunged into LN2. For SD method, the straws were directly aligned horizontally at the same levels as CS from the surface of LN2 for 1, 1, 1.9, and 5 min, respectively and then plunged into LN2. After thawing at 37℃ for 25 sec, the spermatozoa were then incubated for 30 min in the freezing extender (E1) or in the 50 mM sucrose supplemented GFT (E2, E3, and E4) at 24℃. Following post-thaw incubation, sperm progressive motility and viability were assessed in E1, E2, E3, and E4. In addition, acrosome integrity, and gene expression related to apoptosis (BAX, BCL2, and Caspase10) and sperm motility (SMCP) were evaluated in E4. The results demonstrated that, in E1, using 75 mM trisaccharides resulted in significantly (p<0.05) higher sperm motility in all sugar groups. Using 100 mM melezitose significantly (p<0.05) improved the post-thaw viability than the 100 mM raffinose. The viability in 100 mM maltotriose was similar with 100 mM raffinose and melezitose group. In E2, the different cooling time has no significant effect on post-thaw sperm progressive motility in all the sugar types. In addition, the viability was variable among the different groups. In E3, liquid nitrogen vapor freezing for 5 min resulted in improved motility and viability. The sperm progressive motility was significantly (p<0.05) higher in CV and SD group compared to CS group and the sperm viability was significantly (p<0.05) higher in CV group compared to the other groups in E4. However, the acrosomal integrity of spermatozoa in the group CV was significantly (p<0.05) higher than the group CS and SD. In addition, the expression of SMCP gene was significantly (p<0.05) higher in the CV group than the CS group. In contrast, the expression of Caspase10 significantly (p<0.05) lower in the group CV and SD than the group CS. Furthermore, the ratio of gene expression of BAX and BCL2 was significantly (p<0.05) lower in the group CV than the group CS. Therefore, cryopreservation of dog spermatozoa in 100 mM of raffinose supplemented GFT cooled for 20 min and vapor freezing for 5 min provides better progressive sperm motility, viability, and acrosome integrity with higher expression of SMCP gene and lower expression of caspase10 and BAX/BCL2 ratio following post-thaw incubation in 50 mM sucrose supplemented GFT for 30 min at 24℃.

본 연구는 한우 체외 수정란의 동결보존에 hyaluronate(HA), ethylene glycol(EG), glycerol(G), sucrose(S)를 사용한 동결배양액의 효과를 검증하고자 수행되었다. 체외 수정란 생산을 위해 도축장에서 회수된 난소로부터 미성숙 난자를 채취하여 체외성숙, 수정, 발달 배양을 실시하였다. 성숙배양은 0.5 ug/ml FSH, 5 ug/ml LH, 0.125% BSA(v/v)가 첨가된 mTCM199 배양액에서 38.5 ℃, 5 % CO2 의 조건으로 22 h 동안 실시하였으며 체외수정은 mTALP 배양액에서 38.5 ℃, 5 % CO2 의 조건에서 22 h 동안 이루어졌고 발달배양은 mSOFaa 배양액을 사용하여 38.5 ℃, 5 % CO2, 5 % O2 의 조건에서 이루어졌다. 동결에 이용된 수정란은 수정배양 후 7 일차의 배반포를 대상으로 하였다. 실험에 사용된 동결배양액은 대조군으로써 vigro 의 1.8M EG 에 0.1 M S 가 첨가된 제품을 사용하였고 처리군으로써 1.8M EG 배양액, 1.8M EG 에 0.1M S 와 1.7M G 이 첨가된 배양액, 1.8M EG 에 0.1M S, 1.7M G 및 0.0007 g/ml HA 가 첨가된 배양액을 사용하였다. 모든 처리군은 0.01 g/ml Albumax 가 첨가된 CJ buffer 를 동결배양액의 base 로 사용하였다. 동결방법은 0.25 ml 스트로에 장착된 수정란을 CL-8800i(CryoLogic)에 의한 –0.3℃/min 의 완만동결로 –32℃까지 냉각시킨 후 액체질소에 침지하여 실시하였다. 동결과정 중, -6℃에서 스트로의 말단 부분에 식빙을 실시하였다. 동결융해 후 생존율 조사를 위해 동결보존 중인 스트로 32℃의 온수에서 25 초간 융해를 실시하여 발달배양액에서 48 시간 배양하여 총 동결융해 수정란에 대한 재확장율 및 부화율을 통해 생존율을 확인하였다. 실험결과, 동결융해 후 재확장율은 대조군(EG+S)과 EG, EG+S+G, EG+S+G+HA 에서 각 90.1±0.5, 76.0±1.0, ±85.4±2.1, 84.9±0.5 %로 관찰되었으며 부화율은 80.2±1.0, 70.8±4.2, 74.0±1.0, 82.8±1.6 %로 확인되었다. 재확장율은 대조군인 1.8M EG + 0.1M sucrose 배양액에서 90.1±0.5 %로 가장 유의적으로 높게 관찰되었으며 부화율은 대조군과 1.8M EG + 0.1M sucrose + 1.7M glycerol + 0.0007 g/ml HA 처리군에서 각 80.2±1.0 %와 82.8±1.6 %로 가장 유의적으로 높게 관찰되었다(p<0.05). 추가적으로 본 동결방법에 의해 생산된 동결수정란의 수정란이식을 실시하여 수태율을 조사하고 있지만 결과가 부족하여 본 연구결과에서 포함시키지 못하였다. 하지만 수정란 이식과정에서 EG+S+G 처리군의 경우 30 분이 경과된 시점부터 다른 처리군보다 수태율이 감소되는 경향이 뚜렷하게 관찰되었다. 반면, glycerol 과 HA 가 함께 첨가된 다른 동결배양액의 경우에는 이러한 경향이 관찰되지 않았다. 다른 체외수정란의 발달연구에서 HA 의 첨가는 부화율을 증가시킨다는 연구결과가 보고된 바 있는데 이러한 연구결과와 일치된다고 생각된다. 본 연구결과는 농촌진흥청 연구사업(세부과제번호:PJ012695032018)의 지원에 의해 이루어진 것임.

In the present study, we evaluated the effect of glucose-fructose and sucrose supplementation in glycerol-free tris (GFT) on sperm motility, viability, ROS level, apoptosis (BAX and BCL2) and motility (SMCP) related gene expression of dog sperm according to different post-thaw incubation time. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 86 mM glucose and 86 mM fructose (GF-GFT) or 100 mM sucrose (S-GFT). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. The progressive motility, viability, ROS (H2O2) level and mRNA expression of spermatozoa were evaluated according to post-thaw incubation time (0 h, 3 h and 6 h) at 24℃. ROS was assessed using H2DCFDA stain by flow cytometry. The relative abundances of BAX, BCL2 and SMCP were assessed using quantitative real-time polymerase chain reaction (RT-PCR). The motility of spermatozoa cryopreserved in GF-GFT was increased throughout the post-thaw incubation time. The motility of spermatozoa cryopreserved in S-GFT was increased at 3 h of post-thaw incubation. Whereas, the sperm ROS level in GF-GFT group was decreased at 6 h of post-thaw incubation. However, the ROS level in the group S-GFT was gradually increased with the progress of post-thaw incubation period. The post-thaw incubation had no substantial effect on mRNA expression of BAX, BCL2 and SMCP genes of dog spermatozoa in both the GF-GFT and S-GFT groups. These results indicate that GF supplementation in GFT improves the progressive sperm motility during the 6 h of post-thaw incubation with maintaining similar sperm viability and is more efficient in reducing ROS after 3 h of post-thaw incubation. The addition of GF in GFT for the cryopreservation of dog spermatozoa and post-thaw incubation would open an option to achieve more functioning spermatozoa for future assisted reproduction practices.

The objective of the present study was to evaluate the effect of disaccharides supplementation in glycerol-free tris (GFT) on dog sperm cryopreservation with respect to pH adjustment of extender and post-thaw incubation. The spermatozoa collected from five dogs were resuspended (5×107 cell/ml) with GFT containing 100 mM of lactose (L), trehalose (T) or sucrose (S) or pH adjusted (6.85) 100 mM of lactose (LP), trehalose (TP) or sucrose (SP). The sperm (500 μl) were loaded in straws, cooled for 50 min at 4℃, frozen using liquid nitrogen (LN2) vapor for 20 min and plunged in LN2. After thawing at 37℃ for 25 s in a water bath, the spermatozoa were incubated at 24℃ for 30 min. The progressive motility, viability, mitochondrial membrane potential (MMP) and mRNA expression of SMCP gene were then assessed. The MMP was evaluated by combined JC-1 plus PI staining. The relative abundance of SMCP was assessed using quantitative real-time polymerase chain reaction (RT-PCR). Adjustment of pH in GFT extender supplemented with disaccharides did not improve sperrm motility and viability. In general, post-thaw incubation increased the progressive motility of spermatozoa. The sperm motility in the group S was significantly (P<0.05) higher than other groups regardless of post-thaw incubation time. Similarly, the sperm viability in the group S was significantly (P<0.05) higher following post-thaw incubation. The higher sperm motility in the group S was also supported with the significantly (P<0.05) higher live sperm having high MMP. There was no significant difference in mRNA expression of SMCP gene among the experimental groups. These results indicate that cryopreservation of dog sperm in GFT supplemented with S and 30 min post-thaw incubation at 24℃ could provide better freezability of dog spermatozoa with improved motility and higher MMP.

This study aimed to indentify single nucleotide polymorphisms (SNPs) in exon region of the glycerol-3-phosphate dehydrogenase 1 (GPD1) gene and to evaluate their associations with meat yield and quality traits in Hanwoo (Korean cattle). Two SNP markers, g.2766C>T and g.5105A>G were identified in the exons 5 and 8, respectively. Genotyping of the two SNPs was carried out using PCR-RFLP analysis in 309 Hanwoo steers to evaluate their associations with meat yield and quality traits. As a result, g.2766C>T in exon 5 was significantly associated with carcass weight (CW) and marbling score (MS). Animals with the CC genotype of g.2766C>T had heavier CW than those with the CA or AA genotype. Animals with the CC genotype of g.2766C>T also had higher MS than those with the CA or AA genotype. Additive effect was also observed with CW and MS traits. We constructed haplotypes by linkage disequilibrium analysis and analyzed association between haplotypes and meat yield and quality traits. Haplotype of GPD1 gene was associated with CW. As a result, animals with CA haplotype had heavier CW than TG haplotype. These findings suggest that the SNPs of bovine GPD1 gene may be a useful molecular marker for selection of meat yield and quality traits in Hanwoo.

The red-spotted apollo butterfly, Parnassius bremeri, immatures grow during winter and spring. Supercooling point of larvae during January goes much below -20℃. Morphologically, the larvae appear to be adapted to cold temperatures. Dark-colored body surface is useful to absorb solar energy and spiny integument may prevent any external ice formation on the body surface. Biochemically, P. bremeri larvae elevate glycerol as a cryoprotectant. This study reports two genes associated with glycerol biosynthesis in P. bremeri. Larval transcripts were analyzed using RNA-Seq technique. A total of 14 Gb transcripts were read by Illumina HiSeq and assembled to be 127,279 contigs. To specify the the genes associated with glycerol biosynthesis, a biosynthetic pathway to synthesize glycerol from dihydroxyacetone-3-phosphate was predicted with two genes of glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK). Both genes were annotated in the transcriptome of P. bremeri. Pb-GPDH encodes 166 amino acid residues containing NAD+-binding region, catalytic site, and calcium binding region. The predicted amino acid sequence was clustered with other lepidpopteran GPDH genes. Three Pb-GK genes were annotated from the transcriptome. Pb-GK1 encodes a full open reading frame of 514 amino acid residues. A ohylogenetic analysis showed that these three GKs were separately clustered. Interestingly, Pb-GK1 was clustered with other GKs that were known to be associated with rapid cold hardiness.

Vegetable leather 표면 코팅에 사용된 폴리우레탄 수지는 glycerol의 함유를 mole% 비로 달 리하면서 합성하였다. 합성된 폴리우레탄 수지의 기계적 특성은 SEM, FT-IR, UTM 등을 이용하여 측 정하였다. 친환경적인 고분자 수지의 관심이 고조됨에 따라 용제의 사용을 최소화한 vegetable leather 코팅에 사용되는 수분산 수지를 합성하였다. 지방족 3가 알콜인 glycerol의 mole% 비가 증가함에 따라 내마모도, 인장강도가 증가함을 알 수 있었다. 반대로 연신율, 내굴곡 물성은 감소함을 알 수 있었다. Toluene을 이용한 내용제성 물성측정 결과에는 glycerol의 mole% 증가에 따른 물성 증감 효과는 없었 다.

The boar sperm has more lipid droplets and specialty of seminal plasma compared with other species, causing difficulties of freezing sperm and decreases for the utilization of frozen semen into the artificial insemination. However, several studies reported significant results for the recovery of sperm motility and reproductive by addition of cryoprotectants and seminal plasma after thawing. This study was designed to investigate the effects of supplementation of trehalose or glycerol in the LEY (lactose and egg yolk in BTS) solution for the conventional freezing and vitrification process. Two boars aged 16 months were used to collect semen for 2 times in a week. The samples were allotted to 3 freezing solutions (LEY + glycerol 10.5% + OEP 1.5%, LEY + trehalose 1M + OEP 1.5%, and sucrose 1.5M + trehalose 1 M + OEP 1.5%) after centrifugation at 800 g for 10 minutes. Semen was equilibrated in freezing solutions for 10 minutes and injected into plastic straws with 2∼3 air bubbles to minimize freezing damages. Vitrification was performed to locate sperm in 5 cm above LN2 for 5 minutes, and the conventional freezing was conducted with an automatic freezer. Motility and survival rates were measured by CASA (Computer assisted sperm an alyzing system) and FITC (Fluorescein isothiocyanate), respectively after thawing semen at 50℃ for 12 seconds. The results were analyzed by ANOVA with STATVIEW statistical program. The vitrificatioin solution (LEY + 10.5% glycerol + 1.5% OEP) presented higher motility (20.9%) than other solutions while the solution (LEY + 1M trehalose + 1.5% OEP) showed the lowest (motility : 5.2%). However, survival rates of vitrified sperms detected by FITC showed 1~4% live sperms in almost of dead sperms at all vitrification solutions’ groups, but survival rate of freezing solution of LEY + 1M trehalose + 1.5% OEP LEY and LEY + 10.5% glycerol + 1.5% OEP were showed 49%, and 79%, respectively. There were differences (P<0.05) survival rate of conventional freezing in LEY + 10.5% glycerol + 1.5% OEP and LEY + 1M trehalose + 1.5% OEP and the remaining showed no differences. The results suggested that vitrified boar semen was not enough to be utilized for the artificial insemination, but it showed possibility to utilize for ICSI and conventional freezing with glycerol would be useful method for artificial insemination in pig while we choose the outstanding semen against tolerance to freezing damages.

Glycerol is a polyol that is responsible for the cold hardiness of insects. Glycerol kinase gene, which is an important key enzyme for glycerol biosynthesis, was predicted from whole genome sequencing data from the diamondback moth, Plutella xylostella. Four of P. xylostella glycerol kinase genes (PxGKs) were determined as a functional glycerol kinase through in silico study. Pre-exposure of P. xylostella larvae to 4°C for 7 h significantly enhanced survival (rapid cold hardiness: RCH) under a freezing temperature (-10°C) and increased glycerol titers. To determination of functional GK gene, expressions of all GK genes were measured by RT-PCR analysis. All GK genes were expressed in all larval stage and tissues (gut, hemocyte, and fat body). Expressions of all GK genes were suppressed by its specific dsRNA treatment into 4 th instar larva. Each 150 ng of dsRNA PxGK2 treatment significantly decreased glycerol amount in hemolymph by HPLC analysis. Larval treated by dsRNA PxGK2 also significantly lost the RCH under -10°C exposure. These results indicate that glycerol is a crucial RCH agent and its synthesis is regulated by a specific PxGK2 gene among GK gene isoforms in P. xylostella. In addition, the beet armyworm, Spodpotera exigua, encodes RCHassociated SeGK1, which has been functionally identified by RNA interference.

The beet armyworm, Spodoptera exigua, is a freeze-susceptible species and overwinters without diapause in temperate zone. Depression of supercooling point (SCP) and rapid cold hardiness (RCH) allow S. exigua to survive at low temperatures. This study reports a polyol which is responsible for the cold hardiness of S. exigua. Pre-exposure of S. exigua larvae to 4°C for 6 h significantly enhanced survival under a freezing temperature (-10°C). This pre-exposure treatment also significantly depressed larval SCPs. Analysis of polyols indicated that glycerol titers significantly increase with increase of pre-exposure time. Glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) are involved in glycolysis pathway of insect. The S. exigua GK (SeGK1) and G3PDH (SeG3PDH1) genes were predicted from 454 pyrosequencing transcripts from fifth instar larvae of the beet armyworm, S. exigua. The SeGK1 and SeG3PDH1 genes both were expressed in all larval stage by RT-PCR analysis. Expression of SeGK1 and SeG3PDH1 genes were suppressed by its specific dsRNASeGK1 or dsRNASeG3PDH1 injection into hemocoel of 5th instar larva. Each 200 ng of dsRNASeGK1 or dsRNASeG3PDH1 injection also significantly decreased glycerol amount in hemolymph. Larval treated by either dsRNASeGK1 or dsRNASeG3PDH1 significantly lost the RCH under -10°C exposure. These results indicate that glycerol is a crucial RCH agent and its synthesis is regulated by SeGK1 and SeG3PDH1 genes in S. exigua.

Cryopreservation induces sublethal damage to the spermatozoa, which leads to their reduced fertile life. This study was designed to determine effect of glycerol and ethylene glycol as cryoprotectant in extender on improve the freezability of Jeju horse semen. The semen was cryopreserved with glucose-EDTA extender containing each 5% glycerol, 5% ethylene glycol, 8% glycerol or 8% ethylene glycol, respectively. Post-thawed sperm were evaluated motility, viability, Membrane integrity and acrosome integrity. Post-thawed sperm motility were not significantly differences among treatments. However, sperm viability were significantly higher (p<0.05) in 8% glycerol (39.85% ±11.41) than in 5% glycerol treatment (18.08%±1.61). In membrane integrity, swelling sperm ratio was significantly higher (p<0.05) in 8% glycerol (34.12%±11.02) than other treatments. In the percentage of capacitated sperm assessed by CTC staining, F pattern was significantly higher in 8% ethylene glycol than 5% glycerol and 5% ethylene glycol (p<0.05). B pattern ratio was significantly increased in 5% ethylene glycol compared with 8% glcerol and 8% ethylene glycol (p<0.05). Moreover, 8% ethylene glycol treatment was significantly decreased AR pattern ratio compared with other treatments (p<0.05). It is concluded that treatment of 8% glycerol was improved the sperm viability and 8% ethylene glycol was improved the sperm ascrosome integrity after thawing. However, they were not significantly difference between 8% glycerol and 8% ethylene glycol on post-thawed sperm viability. Therefore, 8% ethylene glycol was more effective sperm cryoprotectant than 8% glycerol in Jeju Horse.

Cryopreservation of avian semen is a useful tool to preserve genetic resource for aim of preventing extinction induced by infectious disease like avian influenza. Unlike those of mammals, data from chicken cryopreserved semen has not been showed feasible results. So, various cryoprotectants and diluents have been examined in many methods. In this report, as a major ingredient of avian seminal plasm, glutamine was substituted by alanyl glutamine to enhance physiological stability of chicken semen during freezing. We studied effect of glycerol and Dimethylacetamide(DMA) on motility and progressive motility of spermatozoa using glutamine diluent(EK-G) or alanyl glutamine diluent(EK-A) condition. The semen of Ogye was collected twice a week by the dorso-abdominimal massage method and diluted with same volume of EK-G or EK-A at 25℃ and stored for 10 min at 4℃ in cold chamber. Glycerol or DMA was added to diluted semen to reached 7% of final concentration at 4℃. After 3min of equilibration, the diluted semen was packed into 0.25ml straws and subjected to cryopreservation used freezing equipment. The packed straw were placed on height 5 cm above surface of liquid nitrogen(LN2) and held for 10min. After preserved for 2 weeks, the straw was thawed onto the 4℃ cooling bath. The images of motility and progressive motility spermatozoa were recorded by digital image recorder and analyzed by manual. The results showed 68.5% motility and 34.1% progressive motility in DMA/EKA diluent, 31.45% and 17.6% in glycerol/EKA, 45.4% and 8.6% in DMA/EKG, and 9.7% and 6.4% in glycerol/EKG. With these results, the alanyl glutamine and DMA could be used as a main composition of diluent and cryoprotectant for cryopreservation of chicken semen.

This study was carried out to establish most suitable freezing condition, to evaluate the different glycerol concentration of freezing and thawing rates on motility, viability, membrane integrity and acrosome intecrity of frozen Korean Jeju Black Bull spermatozoa, Semen was collected from a Korean Jeju Black Bull using an artificial vagina and transported to the laboratory. The semen was extended gradually 1:5 then cooled slowly for 2 hrs to 4. The semen was diluted 1:1 with cryoprotectant extenders (3%, 5% and 7% glycerol) and equilibrated for 2 hrs at cold chamber and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 min and above 8 cm for 10 min. And then the frozen straw was plunged into LN. The presented straws were examined the viability and motility after thawed at 37 water bath. The viability and membrane integrity immediately post-thawing were significantly higher in samples frozen in 7% glycerol than 3% and 5% glycerol (p<0.05). After CTC staining to assess acrosome integrity, F pattern was significantly increased, but B pattern was significantly decreased in 7% glycerol (p<0.05). Freezing distance of 5 cm from liquid nitrogen and pre-cooling for 10 min yield better survival and membrane integrity, but not significant difference. However, AR pattern according to CTC staining was significantly decreased in 3 cm for 5 min.

Ganoderma lucidum has long been used as a potent medicinal plant. In addition, recent studies showed that G. lucidum is used to prevent or treat various human diseases such as allergy, hepatopathy, hypertension, cancer and diabetes. In this study showed that ethanol extract from G. lucidum have potential inhibitory effects of glycerol 3-phosphat acyltransferase (GPAT) activity and increase of glucose uptake on skeletal muscle cells. Inhibition of GPAT, which catalyze the first step in de novo TAG synthesis, has been proposed as one of the drug targets for insulin resistance and type-2 diabetes. G. lucidum extract inhibited GPAT activity. Bioassay-guided fractionation and isolation of bio-active substances from ethanol extracts of G. lucidum were carried out by using chromatographic techniques and in vitro GPAT enzyme assay. One of bioactive molecules, ergosterol peroxide, was isolated and identified by physical and spectral properties. In this study showed that ethanol extract from G. lucidum and ergosterol peroxide have potential anti- diabetes effects through the increase of glucose uptake. This was as sociated with increased activity AMP-activated protein kinase(AMPK). AMPK is another regulatory protein in the glucose uptake pathway and energy metabolism. The result was that the increase of glucose uptake by G. lucidum extract might be mediated by AMPK.