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Purification and Biological Activity of Ecdysterone from Korean Achyranthes radix (韓國産 牛膝의 Ecdysterone 抽出과 그 生理活性에 관한 硏究)

  • Kim, Jeong-Il;Lee, Jae-Yong;Kim, Chun-Su;Park, Kwang-E.
    • Journal of Sericultural and Entomological Science
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    • v.25 no.1
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    • pp.1-20
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    • 1983
  • It has been known that the insect molting hormone and its analogues exist also in plant kingdom and their concentration has been found to be about 0.1~2.0% of dry matter, which is equivalent to $10^3{\sim}10^5$ times of those in insects. This study was carried out; 1) to isolate the phytoecdysones from Korean Achyranthes radix and characterize their physico-chemical properties. 2) to investigate the biological activity of this phytoecdysone on Bombyx mori larvae. The resuls were summarized as follows; 1. The extraction method of phytoecdysones was optimized by three consecutive reflux for 1hr using 200g of dried and milled radix per 1l methanol. 2. The purification from the crude extract was made by a series of steps such as precipitation of gum-type polymer with n-Butyl acetate, adsorption on technical grade silica and chromatography with neutral alumina. The conditions of each step were optimized and the resulting crude crystal was about 500mg per kg dry radix. 3. The crude crystal from the cultivated Achyranthes(Achyranthes japonia) contained ecdysterone (20-hydroxyecdysone) and inokosterone in the proportion of one to one. In order to separate these, a series of processes such as acetylation, separation by alumina column chromatography deacetylation by alcoholysis, deionization and crystallization were introduced and optimized 125mg of ecdysterone and 18mg of inokosterone per kg dry radix were thus obtained. 4. The wild Achyranthes (Achyranthes obtusifolia) radix was found to contain the ecdysterone only. A 285mg of ecdysterone was crystallized per kg dry radix. 5. Isolated ecdysterone, inodosterone and acetylated compounds were characterized by IR., UV., NMR spectroscopy, mp, TLC and densitometry. 6. Ligation experiment was undertaken to confirm the biological activity of the purified ecdysterone; the ecdysterone could induce larval-pupal metamorphosis in the ligated abdomen of 4th instar larvae injecting 0.5~1.0${\mu}g$. 7. By ecdysterone feeding experiment using artificial diet, it was elucidated that the critical time of feeding would be the first half of each instar resulting in increased weight of silk layer. 8. The ecdysterone was fed to 5th instar silkworm at the level of 1, 2, 3, 5ppm of dry feed of artificial diet containing 5% mulberry leaves for 72hrs. At 2ppm of the concentration. body weight and silk layer weight were arrived at maximum. But at higher concentrations body weight and silk layer weight decreased than the control group. At 2ppm of the concentration, body weight was increased by 12.5%. 9. Feeding 2ppm of ecdysterone at the later half of 5th instar, the duration of larvae was shortened.

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Defence Mechanism of Java Tilapia, Tilapia mossambica, to Clonorchis sinensis (Tilapia mossambica의 간흡충에 대한 방어기전)

  • RHEE Jae-Ku;PAIK Young-Ki;LEE Ho-Il;YANG Hong-Hyun
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.20 no.4
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    • pp.317-327
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    • 1987
  • As a series of studies on the suitability as a second intermediate host of Clonorchis sinensis, artificial infection experiments were applied to Tilapia mossambica. And then, in order to elucidate the defence mechanism of the fish to Clonorchis, clonorchicidal substance in the epidermal mucus of the fish was isolated by silica gel column and thin layer chromatography and analyzed for its chemical structure by UV, IR and NMR-spectroscopy. The results obtained are summarized as follows: 1. The cercariae which attempted to contact with the fish in the water were observed under stereomicroscope. After contact, the cercariae began to separate their tail from the body after several minutes and then the number increased to $80\%$ more than 10 minutes after the encounter. But very few cercariae could actually invade the epidermis of the fish. 2. The fish were reared with Parafossarulus manchouricus which were shedding numerous cercariae of Clonorchis in the aquarium for 24 hours. Only a few cercariae could invade the epidermis but most of the invaded cercariae died out before forming their cysts. Very few number of the remaining encysted cercariae were also found to be in a state of suspended animation within 42 hours. 3. In the cases of the control fish, Pseudorasbora parva, numerous cercariae of Clonorchis were found to invade the fish through the epidermis under stereomicroscope. Then many metacercariae of Clonorchis were also found in the fish while they were kept in the aquarium. 4. A sample of the epidermal mucus of Tilapia mossambica was extracted with ethly ether 6 times repeatedly. In silica gel column chromatography, using petroleum ether: chloroform/30:70(v/v) as a first solvent and MeOH as a second solvent, the extract was fractionated into the yellow and brownish red solutions in the first solvent and the clonorchicidal brownish yellow solution in the second sovent. 5. The clonorchicidal brownish yellow solution was added to petroleum ether, and the mixture was stored for 5 days at $5^{\circ}C$ and was, then, separated into supernatant fraction and precipitate. Ten mg/ml of the supernatant fraction killed, in vitro, the excysted metacercariae in 45 minutes but the precipitate in 600 minutes. 6. In silica gel column chromatography, using acetone: benzene/10:90(v/v) as a solvent, the more clonorchicidal supernatant fraction was fractionated into the first fraction with Rf. 0.2966 and the second fraction with Rf. 0.072. In vitro, 10mg/ml of the first fraction killed the excysted metacercariae in 28 minutes, the second fraction in 80 minutes and the first fraction was, therefore, determined to be a final clonorchicidal substance. 7. By this purification procedures, the most clonorchicidal substance from the epidermal mucus of Tilapia mossambica was purified 71-folds with $0.2075\%$ yield. Infra red, nuclear magnetic resonance and ultraviolet spectrometric analysis of the purified substance revealed that the substance is linoleic acid. According to the results of the present studies it seemed that this species can not serve as a proper intermediate host of Clonorchis sinensis, and that defence mechanism to the fluke seems to be correlated with linoleic acid in the epidermal mucus of this species.

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Carotenoid Pigments in Echiurid, Urechis unicinctus (개불의 carotenoid 색소성분)

  • KIM Soo Young;HA Bong Seuk
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.32 no.2
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    • pp.223-231
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    • 1999
  • Carotenoid pigments of echiurid, Urechis unicinctus were investigated during March, April and May as a part of comparative biochemical studies of carotenoid pigments for the marine organisms other than pisces. Total carotenoid contents were found to be 1.19 mg/100 g in March, 0.98 mg/100 g in April and 0.84 mg/100 g in May, indicating that total carotenoid content was negatively affected by the temperature of sea water that echiurid resided. The carotenoid isolated in March composed of $16.3\%$ diatoxanthin monoester, $14.8\%$ $\beta$-carotene and $12.6\%$ cynthiaxanthin monoester, $8.4\%$ cynthiaxanthin diester, $8.2\%$ zeaxanthin monoester, $7.3\%$ diatoxanthin diester $4.2\%$ astaxanthin $2.9\%$ diatoxanthin, $2.4\%$ triol, $2.3\%$ cynthiaxanthin, $1.7\%$ isocrytoxanthin, $1.5\%$ zeaxanthin diester, $0.8\%$ zeaxanthin and $0.5\%$ lutein. The carotenoid isolated in April composed of $21.9\%$ diatoxanthin monoester, $17.2\%$ cynthiaxanthin monoester and $16.6\%$ $\beta$-carotene $10.9\%$ zeaxanthin monoester, $5.6\%$ cynthiaxanthin diester, $4.9\%$ diatoxanthin diester, $3.1\%$ astaxanthin, $2.4\%$ triol, $2.3\%$ diatoxanthin, $1.7\%$ isocrytoxanthin, $1.5\%$ lutein, $1.1\%$ zeaxanthin, $1.0\%$ cynthiaxanthin and $1.0\%$ zeaxanthin diester. Similarly, the carotenoid isolated in May composed of $25.3\%$ diatoxanthin monoester, $19.7\%$ cynthiaxanthin monoester, $13.0\%$ $\beta$-carotene, and $12.6\%$ zeaxanthin monoester, $5.8\%$ cynthiaxanthin diester, $5.1\%$ diatoxanthin, $3.0\%$ astaxanthin, $2.4\%$ triol, $2.2\%$ diatoxanthin, $1.3\%$ isocrytoxanthin, $1.2\%$ zeaxanthin, $1.1\%$ zeaxanthin diester, $1.0\%$ lutein and $0.9\%$ cynthiaxanthin. Based on these data, monoester-type carotenoids ($37.1\~57.6\%$) and diester-type carotenoids ($11.5\~17.2\%$) of total carotenoids in echiurid were the major carotenoids. Meanwhile, when the sea water temperature was elevated and the contents of total carotenoid in echiurids were decreased, the contents of zeaxanthin monoester, diatoxanthin monoester and cynthiaxanthin monoester were increased, but the contents of zeaxanthin diester, diatoxanthin diester and cynthiaxanthin diseter were decreased, indicating that changes in ester-type caroteoids were differently affected by the sea water temperature.

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