Microbial Metabolite; Production of norsolorinic acid (NA，a precursor of aflatoxin)

The purified inhibitory substance was identified as cyclo(l-Leu-l-Pro) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml(-1), as determined by the tip culture method. High concentrations (more than 6.0 mg ml(-1)) of cyclo(l-Leu-l-Pro) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(D-leucyl-D-prolyl) and cyclo(L-valyl-L-prolyl), whereas cyclo(D-prolyl-L-leucyl) and cyclo(L-prolyl-D-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(L-leucyl-L-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production.[1]

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Characterization of the inhibition activities.[1]
We investigated the inhibitory activity of commercially obtained cyclo(l-Leu-l-Pro) for NA accumulation by the A. parasiticus NFRI-95 mutant using the microtiter agar plate assay (Table 1). This substance at a concentration greater than 3.5 mg ml−1 was found to completely inhibit accumulation of NA by A. parasiticus NFRI-95. It also partially inhibited the accumulation of NA at a concentration of 1.0 mg ml−1, suggesting that this concentration may be close to the 50% inhibitory concentration. Cyclo(d-Leu-d-Pro), a stereoisomer of cyclo(l-Leu-l-Pro), showed activity similar to that of cyclo(l-Leu-l-Pro) (Fig. 1D). l-Leucine, d-leucine, l-proline, or d-proline or combinations of these amino acids did not show any inhibitory activity, indicating that the inhibition was specific to the cyclodipeptide. Four other cyclodipeptides containing one of the amino acids of cyclo(l-Leu-l-Pro) were also examined (Table 1). Cyclo(l-Leu-l-Gly) did not show any activity until the concentration was 3.5 mg ml−1, and it weakly inhibited NA production by the fungus at a concentration of 6.0 mg ml−1. The other two cyclodipeptides, cyclo(l-Gly-l-Pro) and cyclo(d-Ala-l-Pro), showed no inhibition. In contrast, cyclo(l-Pro-l-Val) inhibited the accumulation of NA even at a concentration of 0.3 mg ml−1, which was lower than the concentration at which the cyclo(Leu-Pro) showed inhibition. These results suggest that a cyclodipeptide structure consisting of one hydrophobic amino acid and a proline may contribute to the inhibitory activity.

We compared the effects of four isomers, cyclo(l-Leu-l-Pro), cyclo(d-Leu-d-Pro), cyclo(l-Leu-d-Pro), and cyclo(d-Leu-l-Pro), in more detail (Fig. 1D and Table 1). cyclo(l-Leu-l-Pro) completely inhibited accumulation of NA by A. parasiticus NFRI-95 at a concentration of 3.5 mg ml−1 and also inhibited spore formation. Cyclo(d-Leu-d-Pro) also showed complete inhibition of NA accumulation at the same concentration, whereas the effect on spore formation appeared to be weaker because the fungus could produce spores even at a concentration of 6 mg ml−1. In contrast, the other two isomers, cyclo(l-Leu-d-Pro) and cyclo(d-Leu-l-Pro), had much lower activities. Cyclo(l-Leu-d-Pro) could completely inhibit NA accumulation at a concentration of 12.0 mg ml−1, whereas cyclo(d-Leu-l-Pro) could not completely inhibit NA accumulation even at the highest concentrations (Table 1 and Fig. 1D). Interestingly, these isomers affected the colors of the spores, and the higher concentrations changed the spore color from light green to dark green..[1]

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Effects of cyclo(l-Leu-l-Pro) on aflatoxin production.[1]
A. parasiticus SYS-4 (= NRRL2999) was incubated with various concentrations of either cyclo(l-Leu-l-Pro) or cyclo(d-Leu-d-Pro) in tip cultures. As determined by this method, either inhibitor at a concentration of 3.5 mg ml−1 completely inhibited aflatoxin production (Fig. 4A, lanes 2 and 3), whereas the fungal growth was only slightly affected at this concentration. In contrast, with both substances at a concentration of 6 mg ml−1, mycelial weight was significantly decreased, indicating that a high concentration of these substances can also inhibit fungal growth. Addition of these substances did not change the pH of the medium. TLC also showed that there was no accumulation of any intermediates related to the aflatoxin production pathway in the mycelia (Fig. 4A, lanes 5 and 6).

With both substances aflatoxin production decreased at concentrations ranging from 0 to 1.0 mg ml−1 (Fig. 4B) in tip cultures. The 50% inhibitory concentrations of cyclo(l-Leu-l-Pro) and cyclo(d-Leu-d-Pro) were almost same, 0.20 and 0.13 mg ml−1, respectively. In contrast, fungal growth was not affected at these concentrations, indicating that these cyclodipeptides specifically inhibited aflatoxin production. However, higher concentrations (more than 6 mg ml−1) of the substances inhibited fungal growth (Fig. 4B). Inhibition of the transcription of aflatoxin-related genes.[1]
The effects of cyclo(l-Leu-l-Pro) on transcription were analyzed by RT-PCR. A. parasiticus SYS-4 was cultured in YES medium with or without cyclo(l-Leu-l-Pro) at a concentration of 3.5 mg ml−1 by the tip culture method. Expression of the aflatoxin-related genes aflR, hexB, dmtA, and pks was detected in the absence of the inhibitor (Fig. 5A). However, expression of these genes was completely inhibited by cyclo(l-Leu-l-Pro) at a concentration of 3.5 mg ml−1, whereas fungal growth was only slightly affected at this concentration. Complete inhibition of aflatoxin production by the inhibitor at a concentration of 3.5 mg ml−1 was also confirmed by TLC of the culture filtrate (Fig. 5B). Expression of the cmd gene, a constitutively expressed calmodulin gene that is not related to aflatoxin production, was not inhibited in the presence of cyclo(l-Leu-l-Pro) (Fig. 5A).

We also investigated effect of cyclo(l-Leu-l-Pro) on enzyme activity. When sterigmatocystin was added to the culture medium in the feeding experiments, 56.4 ± 5.7 μg of total aflatoxins was produced in 200 μl of the medium. In contrast, addition of 2.0 mg of cyclo(l-Leu-l-Pro) ml−1 caused a drastic decrease in aflatoxin production to 7.1% of the amount in the absence of the inhibitor (4.0 ± 6.4 μg of total aflatoxins in the culture medium). Inhibition of the production of aflatoxin from sterigmatocystin may have been due to the inhibition of expression of the enzyme genes by cyclo(l-Leu-l-Pro).

Endophytic Streptomyces strains are potential sources for novel bioactive molecules. In this study, the diketopiperazine cyclo(l-Leu-l-Pro) or gancidin W (GW) was isolated from the endophytic actinobacterial genus Streptomyces, SUK10, obtained from the bark of Shorea ovalis tree, and it was tested in vivo against Plasmodium berghei PZZ1/100. cyclo(l-Leu-l-Pro) or gancidin W/GW exhibited an inhibition rate of nearly 80% at 6.25 and 3.125 μg kg-1 body weight on day four using the 4-day suppression test method on male ICR strain mice. Comparing cyclo(l-Leu-l-Pro) or gancidin W/GW at both concentrations with quinine hydrochloride and normal saline as positive and negative controls, respectively, 50% of the mice treated with 3.125 μg kg-1 body weight managed to survive for more than 11 months after infection, which almost reached the life span of normal mice. Biochemical tests of selected enzymes and proteins in blood samples of mice treated with cyclo(l-Leu-l-Pro) or gancidin W/GW were also within normal levels; in addition, no abnormalities or injuries were found on internal vital organs. These findings indicated that this isolated bioactive compound from Streptomyces SUK10 exhibits very low toxicity and is a good candidate for potential use as an antimalarial agent in an animal model.[2]

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GW/cyclo(l-Leu-l-Pro) or gancidin W was subjected to antimalarial screening across a narrow range of five different concentrations in order to determine which dose gave the best antimalarial activity. The value for parasitemia density directly reflected the value of percentage inhibition, where the higher the percentage inhibition – the more effective the treatment was. The inhibition rate at 3.125 μg kg−1 bw dose was found to exhibit the greatest value. Reaching almost 80% inhibition rate, there was a significant difference (P<0.05, n=6) between the 3.125 μg kg−1 bw group and the groups given the other four concentrations. Inhibition rate higher than 65%, which was considered a benchmark for in vivo antimalarial activity, was also reached at 6.25 μg kg−1 bw dose, whereby this value was far better than the other three dose concentrations (Table 3).[2]

The survival time of mice treated with 3.125 μg kg−1 bw dose of cyclo(l-Leu-l-Pro) or gancidin W/GW was steadily almost twofold longer than that of mice given 12.5 μg kg−1 bw and was proportionately higher than that for mice given a dose of 6.25 μg kg−1 bw (Figure 3). As previously demonstrated, both in vivo and in vitro analyses predicted that the survival times of the treated mice would be longer at a higher parasite inhibition rate. The longest period of mouse survival (235.53±2.20 days) was achieved by treatment with cyclo(l-Leu-l-Pro) or gancidin W/GW at a dose concentration of 3.125 μg kg−1 bw, which was significantly higher than that with other dose concentrations (P<0.05, n=6). In conjunction with this, surprisingly, the remaining 50% (n=3) of the group treated with cyclo(l-Leu-l-Pro) or gancidin W/GW at this concentration individually managed to survive until 291.13±0.5 days postinfection. Based on previous documentations, this period of survival time was considered to almost reach the life span of normal male mice at approximately 12–18 months. Until 411 postinfection days, all PC mice survived and this observation was in parallel with results of previous in vivo antimalarial studies, while the NC mice survived to 7–9 days postinfection, as also recorded in this study.[2]

RT-PCR.[1]
A. parasiticus SYS-4 (= NRRL2999) was cultured in YES broth or YES broth supplemented with 3.5 mg of cyclo(l-Leu-l-Pro) ml−1. After the mycelia were cultured for 63 h, they were disrupted with TRI REAGENT by using FastPrep FP100A (Q-BIO gene; Bio 101). Total RNA was prepared according to the manufacturer's instructions and then treated with RNase-free DNase. Reverse transcription (RT)-PCR was carried out by using the resulting total RNA and an RT-PCR kit. The primers used were aflR-BamHI-F (CGCGGATCCATGGTTGACCATATCTCCCC) and aflR-HindIII-R (CCCCAAGCTTCATTCTCGATGCAGGTAATC) for aflR (accession no. AF441437), HexB-F1 (CTGCGGGTGGAGCTGCA) and HexB-R1 (CAAGCTCCAAGGGCGGC) for the hexanoate synthase gene (accession no. AF391094), pKSL1-F1 (CCAGGACAGCCCTATTCTAG) and pKSL1-R1 (GGAGTCCAGTGGTATTCAGC) for the polyketide synthetase gene, pksL1 (accession no. L42766), MT-1wholeF1 (ACAAATACCCCTGGCTCAGG) and MT-1wholeR1 (ACCTGTTCCATCAAATCGTC) for the O-methyltransferase I gene, dmtA (accession no. AB022906), and CMDF1 (GGTGATGGCCAGATCACCAC) and CMDR1 (CCGATGGAGGTCATGACGTG) for the calmodulin gene (accession no. AY017584).

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Feeding experiment.[1]
By using the tip culture method, A. parasiticus NIAH-26 was cultured in YES medium supplemented with 40 μM sterigmatocystin in the presence or absence of 2 mg of cyclo(l-Leu-l-Pro) ml−1 at 28°C for 4 days. Aflatoxin formation was measured by extraction of the medium with chloroform, followed by HPLC analysis, as described above.

Assays for the inhibitory substance (e.g. cyclo(l-Leu-l-Pro)). [1]
(i) Visual agar plate assay. [1]
For selection of a bacterium that inhibits aflatoxin production, the visual agar plate assay (14) was used with the NA-accumulating mutant NFRI-95, with minor modifications (Fig. 1A). The spores of the fungus were inoculated in a line at the center of a plate containing GY agar, and an aliquot of a liquid culture of the bacterium was then inoculated in a line 1.5 cm from the centerline. After 3 to 7 days of incubation at 28°C, the effects of the bacterium on either NA accumulation in the mycelium or the growth of the fungus were observed from the underside of the plate. A decrease in the red pigment (NA) in the mycelium of the fungus indicated inhibition of aflatoxin production by the bacterium.

(ii) Microtiter agar plate assay. [1]
Based on the visual agar plate assay, we devised a small-scale assay system, in which a 96-well, flat-bottom tissue culture plate was used as a culture vessel (Fig. 1B and C). Autoclaved GY agar (100 μl) was poured into each well and then solidified. An aliquot (10 to 20 μl) of each fraction from each purification step or the methanol solution containing the inhibitory substance(s) was applied to the agar medium and then incubated for more than 30 min without a lid to let the solution diffuse into the medium and the methanol to evaporate. A spore suspension (1 μl) of A. parasiticus NFRI-95 was inoculated onto the center of the medium. Because the number of spores did not affect the results, we did not adjust it for this method. A small mass of clay or Parafilm was placed at each corner of the plate to produce a thin gap between the lid and the plate, and the gap was sealed with surgical tape (21N, No.12; 12 mm by 9 m) so that the lid would not open. After incubation at 28°C for 2 or 3 days, the color of the mycelium was observed from the underside of the plate.

(iii) Tip culture method. [1]
A spore suspension (5 μl) of A. parasiticus SYS-4 was inoculated into 250 μl of YES medium supplemented with various concentrations of the inhibitory substance by using a Pipetman tip as a culture vessel After incubation at 28°C for 4 days, the mycelium and culture medium were separated by centrifugation. For detection of aflatoxin, 5 μl of the medium was spotted onto a thin-layer chromatography (TLC) silica gel plate (Silica Gel 60), which was then developed with a solution containing chloroform, ethyl acetate, and 90% formic acid (6:3:1, vol/vol/vol). Aflatoxins were inspected under long-wavelength UV light (365 nm) and were photographed under UV light (365 nm) with a Fluor-S MultiImager. To measure the amounts of the aflatoxins, the culture filtrate was extracted with chloroform, and an aliquot of the resulting chloroform extract was injected into a Shimadzu high-performance liquid chromatography (HPLC) apparatus (model LC-10A) equipped with a silica gel column (0.46 by 15 cm; Shim-pack CLC-SI) and a fluorescence monitor (excitation wavelength, 365 nm; emission wavelength, 425 nm; Shimadzu model RF-535) at a flow rate of 1 ml min−1 at room temperature. The solvent system consisted of toluene, ethyl acetate, formic acid, and methanol (178:15:4:3, vol/vol/vol/vol). The retention times of aflatoxins B1, B2, G1, and G2 were compared with those of standard metabolite samples (aflatoxin B-aflatoxin G mixture; Sigma Chemical Co.). To detect precursors of the aflatoxins, precursors in the mycelial mat were extracted with acetone. After the extract was concentrated to dryness, the debris was dissolved in benzene-acetonitrile (98:2, vol/vol) and then analyzed by TLC.

In vivo antimalarial screening of bioactive compound [2]
ICR strain male mice (n=120, 25–30 g, 6–8 weeks old) were used in all animal experiments and were divided into 20 groups (n=6). All groups were housed in stainless steel cages under 12:12 hours with and without light conditions at 28°C with daily ad libitum feed. To initiate the infection, 0.1 mL of 1.0×106 P. berghei PZZ1/100 parasitized red blood cell (RBC) solution was intraperitoneously administered into the host. To determine the best concentration, 50, 25, 12.5, 6.25 and 3.125 μg kg−1 bw of cyclo(l-Leu-l-Pro) or gancidin W/GW solutions were prepared by dissolving the obtained fractions of cyclo(l-Leu-l-Pro) or gancidin W/GW in 1.0 mL dimethyl sulfoxide (DMSO) as the stock compound solution before they were serially diluted with sterile distilled water to achieve the targeted concentrations. The 4-day suppression test was chosen to implement the antimalarial screening, whereby day zero of the treatment was the day when the mice were treated with 0.1 mL of cyclo(l-Leu-l-Pro) or gancidin W/GW at all concentrations, immediately within 2 hours postinfection. In parallel, 0.1 mL of 10 mg kg−1 bw of dH2O-diluted quinine hydrochloride (QH) and 0.9% normal saline were respectively used as positive control (PC) and negative control (NC) solutions.
After 20 days postinfection, the mice were observed daily for their survival period (days). Treatment regime with inhibition rate of >65% was considered as having antimalarial activity, and the mice group with the longest survival time was considered as receiving the best treatment. In vivo toxicity assessment of the compound [2]
Mice with the same characteristics as during antimalarial screening (ICR strain, male, 25–30 g, 6–8 weeks old, n=36) were used for in vivo toxicity assessment. All animal experiments for toxicity assessment were conducted under the same Universiti Kebangsaan Malaysia Animal Ethics Committee (UKMAEC) approval code for antimalarial screening. The mice were divided into 12 groups (n=6), and all groups were subjected to the same conditions (28°C room temperature, stainless steel cage, 12:12 hours with and without light and daily ad libitum feed). At the best-detected concentration during antimalarial screening, toxicity tests were carried out on blood samples of mice treated with cyclo(l-Leu-l-Pro) or gancidin W/GW according to two types of toxicity regime: acute exposure (daily treatment for 7 days) and subacute exposure (daily treatment for 28 days). Each toxicity regime was divided into two groups of treatment: without infection and immediately within 2 hours after infection on day zero. For labeling purposes, all the mice groups were respectively labeled as TA for acute exposure without infection, TB (acute exposure immediately within 2 hours after infection), TC (subacute exposure without infection) and TD (subacute exposure immediately within 2 hours after infection). Data from the two control regimens, namely, normal mice without any infection and treatment (CN) and single-dose infected mice (CI), were also obtained for comparison. The animals were sacrificed under diethyl ether anesthesia, 0.8–1.0 mL of the blood was collected from each mouse by cardiac puncture on day eight and day 29 postexposure and tested for serum total protein (STP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels.
From the same diethyl ether-anesthetized mice used for biochemical toxicity tests, three vital organs, namely, liver, kidney and spleen, were individually collected for organ toxicity and histology studies. The infiltration procedure was done automatically by a tissue processor. The tissues were next embedded in hot paraffin wax using an embedding machine. These tissues were then cut (0.4–0.6 μm thick) by a microtome. Histologic tissue preparation was done using hematoxylin-and-eosin (H–E) staining method. The H–E-stained slides for organ histology were observed using a computerized light microscopic camera at 100× magnification by placing a drop of immersion oil on the surface of the slide or without the immersion oil at 40× magnification; this method was vital to give a clear picture of the internal structure of the targeted tissues, as well as for assessing and identifying abnormalities, toxicity and injuries in the tissues.

[1]. Cyclo(L-leucyl-L-prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus. Appl Environ Microbiol. 2004 Dec;70(12):7466-73.

[2]. Gancidin W, a potential low-toxicity antimalarial agent isolated from an endophytic Streptomyces SUK10. Drug Des Devel Ther. 2017 Feb 8;11:351-363

Cyclo(L-Leu-L-Pro) is a homodetic cyclic peptide composed from leucyl and prolyl residues. It has a role as a marine metabolite and a bacterial metabolite. It is a dipeptide, a homodetic cyclic peptide and a pyrrolopyrazine. ChEBI (3S,8aS)-3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione has been reported in Epichloe typhina, Peroneutypa scoparia, and other organisms with data available.

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Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(l-Leu-l-Pro) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml(-1), as determined by the tip culture method. High concentrations (more than 6.0 mg ml(-1)) of cyclo(L-leucyl-L-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(D-leucyl-D-prolyl) and cyclo(L-valyl-L-prolyl), whereas cyclo(D-prolyl-L-leucyl) and cyclo(L-prolyl-D-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(l-Leu-l-Pro) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production. [1]

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In purifying the inhibitor, we found that the culture medium of the A1 bacterium contained at least three inhibitory substances other than cyclo(l-Leu-l-Pro). The amounts of these substances were not large enough for further characterization. Although the combination of these unknown substances and cyclo(l-Leu-l-Pro) might result in synergetic inhibitory activity, detailed relationships among the substances remain to be studied.
The biosynthetic mechanism and the secretion mechanism of the cyclodipeptide of A. xylosoxidans NFRI-A1 are still unknown. It has been reported that cyclodipeptides might be degradation products of proteins following spontaneous cyclization. However, it has been found that many cyclodipeptides play various biological roles. These substances are now regarded as important metabolic substances rather than as protein artifacts.
For purification of the inhibitor from the bacterial culture, a simple, sensitive, and small-scale detection system is of primary importance. Although the visual agar plate assay provided sensitive and clear results, the experimental scale seemed to be too large even if we used a small plate (diameter, 6 cm). Instead, we devised a small-scale assay system using a microtiter plate. The microtiter plate is quite useful. Magnusson et al. have independently reported use of a similar microtiter plate well assay to monitor antifungal activity of the metabolites produced by Lactobacillus coryniformis subsp. coryniformis. We have already isolated other inhibitory substances from soil bacteria by using the microtiter agar plate assay. We hope that some of our results will soon be useful in preventing aflatoxin contamination.[1]

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Administration of antimalarial drugs totally depends on the regime combination, stage of infection, the side effects experienced by the infected host and the type of plasmodial species. Quinine has been proven to kill all Plasmodium cells, regardless of the species. However, it is only effective in the early stages of malarial infection, during which generally, the host will experience asymptomatic signs. All the untreated control mice in this study died between 7 and 9 days postinfection, as described in previous in vivo antimalarial studies, regardless of the type of animal model used. Although cyclo(l-Leu-l-Pro) or gancidin W/GW has been previously reported from other Streptomyces species, its antimalarial properties have not yet been revealed. In fact, our results indicate that cyclo(l-Leu-l-Pro) or gancidin W/GW is one of the metabolites responsible for the in vivo antimalarial activity of the ethyl acetate crude extract from SUK10. The outcomes of the presented study indicate that Streptomyces SUK10 living endophytically in S. ovalis tree is a good source of a potential antimalarial agent with relatively very low toxicity. Apart from the fact that the progression of the erythrocytic cycle in the Plasmodium-infected host is still poorly understood, there is also a tendency to obtain more significant values for certain antimalarial parameters in particular cycles or stages of the Plasmodium life cycle, which have not been considered in this study.[2]

C11H18N2O2

210.272822856903

210.136

C, 62.83; H, 8.63; N, 13.32; O, 15.22

2873-36-1

Cyclo(Pro-Leu);5654-86-4; 2873-36-1; 32510-93-3

7074739

White to off-white solid powder

1.1±0.1 g/cm3

427.6±34.0 °C at 760 mmHg

163-165ºC

212.4±25.7 °C

0.0±1.0 mmHg at 25°C

1.530

1.1

1

2

2

15

288

2

O=C1[C@H](CC(C)C)NC([C@@H]2CCCN21)=O

SZJNCZMRZAUNQT-IUCAKERBSA-N

InChI=1S/C11H18N2O2/c1-7(2)6-8-11(15)13-5-3-4-9(13)10(14)12-8/h7-9H,3-6H2,1-2H3,(H,12,14)/t8-,9-/m0/s1

(3S,8aS)-3-(2-methylpropyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione

2873-36-1; Gancidin W; Maculosin 6; Cyclo(-Leu-Pro); cyclo(L-leu-L-pro); (3S,8aS)-3-Isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione; Gancidin W; cyclo(Leu-Pro); Cyclo(L-prolyl-L-leucyl); Cyclo-L-leu-L-pro;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 250 mg/mL (1188.95 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)