Application of naftifine hydrochloride in in-vitro promotion of development of cordyceps sinensis germ spores to form hyphae

Abstract

The invention discloses an application of naftifine hydrochloride in in-vitro promotion of cordyceps sinensis germ spores to develop and form hyphae. Aiming at the problems of low stiffness rate and the like of the cordyceps sinensis bacteria, the naftifine hydrochloride is added into the spores of the cordyceps sinensis bacteria, so that the conversion rate of the spores (64.57 + / -2.36%) is increased and is obviously higher than the conversion rate of mycelia with only spores (28.48 + / -1.16%), and the conversion rate of the mycelia can be obviously increased by adding the naftifine hydrochloride into the spores of the cordyceps sinensis bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of artificial cultivation of Cordyceps sinensis, and specifically relates to the application of a novel allylamine-type naftifine hydrochloride in promoting the development of blastospores of Cordyceps sinensis into hyphae in vitro. Background Art

[0002] Cordyceps sinensis is a traditional precious biological resource unique to the Qinghai-Tibet Plateau, and is a complex of insect and fungus formed by the infection of the larvae of Hepialus armoricanus Oberthur by Ophiocordyceps sinensis. Cordyceps sinensis has good curative effects in aspects such as immune regulation, anti-tumor, anti-fatigue, and treatment of lung and kidney diseases, and has received extensive attention from the industrial circle. With the in-depth exploration of its active ingredients and pharmacological effects, Cordyceps sinensis has become an important raw material for high-end foods, health products, and drugs. The market demand for this resource has increased sharply. Over-excavation combined with the impact of global warming on the yield of wild Cordyceps sinensis has driven its price to soar. Cordyceps sinensis has been listed as a second-class endangered plant. Fortunately, after decades of research and development, significant breakthroughs have been made in the overall technology of artificial cultivation of Cordyceps sinensis, including the artificial cultivation of the fruiting bodies of Cordyceps sinensis, the large-scale artificial breeding of the host insect Hepialus armoricanus Oberthur at low altitudes, and the use of Cordyceps sinensis to infect the larvae of Hepialus armoricanus Oberthur to obtain Cordyceps sinensis comparable to wild ones. This has greatly promoted the effective protection of the plateau ecological environment and precious wild resources, as well as the high-quality development of the bioeconomy. However, in the process of artificial cultivation of Cordyceps sinensis, the core technology to be overcome is to improve the efficiency of the conversion of blastospores of Cordyceps sinensis into hyphae.

[0003] Dimorphism of fungi refers to the ability of some fungi to transform their mycelial morphology between yeast form and hyphal form under the influence of environmental factors. Cordyceps sinensis is a typical dimorphic fungus (blastospores transform into prehyphae and hyphae). At present, it is known that N-acetylglucosamine, proline, farnesol, tyrosol, methyl farnesoate, ecdysteroid, mannitol, etc. can promote the transformation of blastospores of Cordyceps sinensis into hyphae to varying degrees in vitro.

[0004] Novel allylamine-type agents, especially naftifine hydrochloride, mainly refer to a class of drugs with antifungal activity, which mainly achieve the antifungal effect by interfering with the biosynthesis process of fungal cells, especially the formation of fungal cell membranes. These agents are widely used in the treatment of various skin fungal infections. However, the effect of naftifine hydrochloride in promoting the development of blastospores of Cordyceps sinensis into hyphae in vitro at an appropriate concentration has not been reported. Summary of the Invention

[0005] Based on the above problems, the purpose of the present invention is to provide the application of naftifine hydrochloride in promoting the development of blastospores of Cordyceps sinensis into hyphae in vitro, so as to solve the problem of low rigidity rate in the artificial cultivation of Cordyceps sinensis.

[0006] The present invention discovers that adding naftifine hydrochloride to the blastospores of Cordyceps sinensis can promote the transformation of blastospores into hyphae.

[0007] Therefore, the present invention provides the use of naftifine hydrochloride in the preparation of a preparation for promoting the transformation of blastospores of Cordyceps sinensis into hyphae.

[0008] Preferably, the inoculation dose of the blastospores of Cordyceps sinensis is 11.6 x 10 6 per milliliter.

[0009] Preferably, the concentration of naftifine hydrochloride in the culture medium is 6.48 μg per milliliter.

[0010] Preferably, the preparation method per liter of the culture medium is as follows: boil the juice of 200 g of peeled potatoes, add 20 g of maltose, 10 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 0.02 g of vitamin B1, and after mixing, make up the volume to 1 L with distilled water.

[0011] The present invention also provides a preparation for promoting the transformation of blastospores of Cordyceps sinensis into hyphae, which contains naftifine hydrochloride as an active ingredient.

[0012] The said preparation may contain available excipients.

[0013] In view of the problems such as the low rigidification rate of Cordyceps sinensis, the present invention adds naftifine hydrochloride to the blastospores of Cordyceps sinensis, and improves the transformation rate of blastospores (64.57 ± 2.36%), which is significantly higher than the transformation rate of hyphae of only blastospores (28.48 ± 1.16%), indicating that adding naftifine hydrochloride to the blastospores of Cordyceps sinensis can significantly improve the transformation rate of hyphae. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is that naftifine hydrochloride promotes the transformation of blastospores of Cordyceps sinensis into pre-hyphae. D8, the group adding blastospores (final concentration of 1.16×10 7 per mL) and DMSO (final concentration of 0.1%); N8, the group adding blastospores (final concentration of 1.16×10 7 per mL) and naftifine hydrochloride (final concentration of 0.2 mM). The data are the transformation rates of blastospores into pre-hyphae on the eighth day, expressed as the mean ± standard error of the transformation rate; t = 11.697, df = 23, P < 0.001.

[0015] Figure 2 It is a photograph of the pre-hyphae (Calcofluor White staining, 400×, day 8, N8) after the transformation of blastospores of Cordyceps sinensis promoted by naftifine hydrochloride in vitro. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following examples are further illustrations of the present invention rather than limitations thereof.

[0017] Example 1:

[0018] The Ophiocordyceps sinensis strain was isolated from wild Ophiocordyceps sinensis in Kangding, Sichuan (No.: KD1223) by tissue isolation method [Cao, L., Ye, Y., & Han, R. (2015). Fruiting body production of the medicinal Chinese caterpillar mushroom, Ophiocordyceps sinensis (Ascomycetes), in artificial medium. International Journal of Medicinal Mushrooms, 17(11), 1107–1112, which is also held and preserved by the applicant and provided to the public for 20 years since the applicant]. After molecular identification, it was stored at -80 °C. Liquid medium for Ophiocordyceps sinensis (PMG): The juice of 200 g of peeled potatoes was boiled, 20 g of maltose, 10 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 0.02 g of vitamin B1, and 5 g of ground fresh Galleria mellonella larvae were mixed and made up to 1 L with distilled water, then dispensed into 250 mL Erlenmeyer flasks, 150 mL per flask, and autoclaved at 121 °C for 30 min and used after cooling. On a super clean bench, the solid-cultured Ophiocordyceps sinensis blocks (about 0.5 cm 3 ) were inoculated into the above liquid medium and cultured on a shaker at 120 rpm and 9 - 13 °C for 45 days to collect blastospores. Method for collecting blastospores: The liquid culture was filtered through three layers of sterile lens paper, and the filtrate was collected into a 50 mL sterilized centrifuge tube and centrifuged at 8000 rpm and 10 °C for 15 min. The supernatant was discarded, resuspended with sterile phosphate buffer (PBS; pH = 6.2), centrifuged again to discard the supernatant, and the collected blastospores were diluted with sterile PBS to 1.16×10 8 cells / mL for subsequent experiments. Meanwhile, PM medium was added to a 96-well plate.

[0019] PM medium: The juice of 200 g of peeled potatoes was boiled, 20 g of maltose, 10 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 0.02 g of vitamin B1 were mixed and made up to 1 L with distilled water, autoclaved at 121 °C for 30 min and used after cooling.

[0020] Using 100% DMSO as the solvent, naftifine hydrochloride (product of Macklin) was prepared into a stock solution with a concentration of 200 mM. Then, 10 μL of the stock solution was taken and diluted 100-fold with sterile ultrapure water to prepare a 2 mM solution, which was filtered through a 0.22 μm bacterial filter (Millipore) before use.

[0021] D8: The group with blastospores (final concentration of 1.16 spores 7 per mL) and DMSO (final concentration of 0.1% by mass fraction) added to the PM liquid medium; N8: The group with blastospores (final concentration of 1.16 spores 7 per mL) and naftifine hydrochloride (final concentration of 0.2 mM) added. 80 μL of the PM liquid medium, 10 μL of blastospores (1.16×10 8 per mL), and 10 μL of the 2 mM naftifine hydrochloride solution were added to each well of a 96-well plate. The total volume per well was 100 μL. Then, using a micro oscillator, it was shaken for 5 min, the 96-well plate was sealed with a sealing film, placed in a sterile plastic box, and cultured in a room at 12 - 14°C; 4 wells were set as replicates for each treatment. After 8 days, a hemocytometer was used to accurately count the number of blastospores and prehyphae. The whole experiment was repeated 3 times.

[0022] The results showed that after 8 days of microscopic examination, the hyphal conversion rate of the group with blastospores (final concentration of 1.16×10 7 per mL) and DMSO (final concentration of 0.1%) (D8) was 28.48 ± 1.16%, and the hyphal conversion rate of the group with blastospores (final concentration of 1.16×10 7 per mL) and naftifine hydrochloride (final concentration of 0.2 mM) (N8) was 64.57 ± 2.36% ( Figure 1 ), which was significantly higher than the hyphal conversion rate of the D8 group, proving that naftifine hydrochloride promoted the conversion of blastospores of Cordyceps sinensis into hyphae ( Figure 2 ).

Claims

1. Use of naftifine hydrochloride in promoting the development of blastospores of Cordyceps sinensis into hyphae in vitro.

2. Use of naftifine hydrochloride in the preparation of a preparation for promoting the transformation of blastospores of Cordyceps sinensis into hyphae.

3. The application according to claim 1 or 2, characterized in that The inoculation dose of the blastospores of the Ophiocordyceps sinensis is 11.6 x 10 6 per milliliter.

4. The application according to claim 1 or 2, characterized in that For the naftifine hydrochloride described, its concentration in the blastospore culture medium is 6.48 μg per milliliter.

5. The application according to claim 4, wherein For the culture medium described, its preparation method per liter is: boil the juice of 200 g of peeled potatoes, 20 g of maltose, 10 g of peptone, 3 g of potassium dihydrogen phosphate, 1.5 g of magnesium sulfate, 0.02 g of vitamin B1, and after mixing, make up the volume to 1 L with distilled water.

6. A preparation for promoting the transformation of blastospores of Ophiocordyceps sinensis into hyphae, characterized in that, Contains naftifine hydrochloride as an active ingredient.

7. The preparation according to claim 6, characterized in that, The preparation described contains pharmaceutically acceptable excipients.

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