NL2032833B1

NL2032833B1 - Preparation method for bombyx batryticatus extraction and application thereof

Info

Publication number: NL2032833B1
Application number: NL2032833A
Authority: NL
Inventors: Li Chunyan; Xie Yuliang; Qi Hengtian; Xi Chenlong; Zhao Fanrong; Yin Yanyan; Sun Wenze; Zhu Tiantian; Yang Yanyan
Original assignee: Univ Xinxiang Medical; The Third Affiliated Hospital Of Xinxiang Medical Univ
Priority date: 2022-07-07
Filing date: 2022-08-23
Publication date: 2023-02-15
Also published as: NL2032833A; CN116585357A

Abstract

The present invention provides a preparation method for a bombyx batryticatus extraction and an application, and belongs to the technical field of medicines. The bombyx batryticatus extraction provided by the present invention is relatively simple in extraction method conditions, and low cost in raw material, and the total yield reaches about 40%. After cell experiments in vitro and animal experiments, it is found that the bombyx batryticatus extraction may inhibit the proliferation and migration of mouse Lewis lung cancer (LLC) in vitro, may signiﬁcantly enhance the anti-tumor effect of cisplatin (DDP) when used in combination with DDP, and may effectively improve the anti-tumor effect of patients.

Description

PREPARATION METHOD FOR BOMBYX BATRYTICATUS EXTRACTION

AND APPLICATION THEREOF

TECHNICAL FIELD

[01] The present invention belongs to the technical field of medicines, and specifically relates to extraction of a bombyx batryticatus extraction (BBE) and an application thereof.

BACKGROUND ART

[02] Alung cancer is a cancer that causes the most deaths, and is a main reason for morbidity and mortality in male cancers, and the morbidity of the lung cancer is second only to a breast cancer in women. The lung cancer is clinically divided into two categories: a small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) according to histological subtypes, immunohistological and genetic tumor characteristics, wherein SCLC accounts for about 15%, and NSCLC accounts for about 85%, wherein lung adenocarcinoma and lung squamous cell carcinoma account for about 90% of NSCLC solid tumors. At present, an adjuvant chemotherapy after a lung cancer surgery is usually recommended for treatment of the lung cancer, wherein cisplatin (DDP) is the earliest and most widely used platinum drug, but due to the lack of selection specificity, it is often accompanied by many toxic and side effects that are positively related to dosages.

[03] Traditional Chinese medicines have a significant auxiliary effect in treatment of lung cancer patients, it may increase the efficacy of conventional treatments (a surgical treatment, a chemical treatment and a radiation treatment), alleviate the drug resistance, reduce adverse reactions and toxic and side effects, reduce the suffering of the patients and improve the life quality. Therefore, substances with anti-tumor activity extracted from the traditional Chinese medicines may be used as an adjuvant treatment for a lung cancer drug chemotherapy, thereby the efficacy of drugs are enhanced, and the toxic and side effects of this type of the drugs are reduced.

[04] Bombyx batryticatus is a special product, it is also known as a white muscardine silkworm, and is a dry and hardened dead worm body that is infected with beauveria bassiana before a silkworm larva spins. Because its body surface is densely covered with white hyphae and conidia, and shaped like a layer of a white membrane, itis named hence. It is proved from existing researches that the bombyx batryticatus is effective against a variety of cancers clinically, and it is indicated that a bombyx batryticatus extractsion has a potential effect in treatment of tumors, but its therapeutic effects on the lung cancer and related mechanisms are currently less researched.

SUMMARY

[05] In view of this, the present invention was designed is to provide a bombyx batryticatus extraction and a preparation method thereof, which may inhibit the proliferation and migration of mouse Lewis non-small-cell lung cancer (LLC) in vitro, enhance the anti-tumor effect of DDP, and effectively improve the quality of life of cancer patients.

[06] The BBE provided by the present invention has a plurality of anti-cancer active components.

[07] The preparation method for the BBE provided by the present invention includes the following steps:

[08] 1) under the action of soaking in 95% alcohol, separating to obtain a bombyx batryticatus initial extraction and preparing into an extract; and

[09] 2) extracting the bombyx batryticatus initial extraction (extract) with a mixture of petroleum ether and distilled water (the volume ratio is 1:1), collecting an aqueous phase and concentrating, to obtain BBE.

[10] The present invention provides an application of the BBE in preparation of a drug for preventing and treating a lung cancer.

[11] The present invention provides an application of the BBE in preparation of a drug for inhibiting proliferation and migration of a lung cancer cell.

[12] The present invention provides an application of a reagent for enhancing a

DDP curative effect in a drug for treating a lung cancer, and the reagent is the BBE.

[13] The present invention provides an application of the BBE in preparation of a drug for preventing and controlling tumor growth and migration of a lung cancer.

[14] The BBE extracted in the present invention is relatively simple in extraction method conditions, and low in raw material cost, and the total yield reaches about 40%.

After cell experiments in vitro and animal experiments, it is found that the BBE can inhibit the proliferation and migration of LLC in vitro, significantly enhance the anti-tumor effect of DDP when used in combination with DDP, and effectively improve the anti-tumor effect of patients.

BRIEF DESCRIPTION OF THE DRAWINGS

[15] FIG. 1 shows an effect of BBE on the proliferation of LLC cell in vitro;

[16] FIG. 2 shows an effect of BBE on the migration of LLC cell in vitro;

[17] FIG. 3 shows an effect of BBE on tumor growth in an LLC tumor-bearing mouse; and

[18] FIG. 4 shows an effect of BBE on tumor proliferation in an LLC tumor-bearing mouse.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[19] The present invention provides a bombyx batryticatus extraction, which contains a plurality of active components.

[20] The present invention provides a preparation method for the BBE, which includes the following steps:

[21] 1) After a bombyx batryticatus is crushed and filtered, it is soaked under the action of 95% ethanol and a supernatant is collected, and after being concentrated, an initial extraction (extract) is obtained; and

[22] 2) after petroleum ether and distilled water are mixed in a ratio of 1:1, the initial extraction is extracted, an aqueous phase is taken, and concentrated, to obtain

BBE.

[23] In the present invention, in the step 1), bombyx batryticatus powder needs to be soaked in 95% alcohol for 36 h, and it needs to be thoroughly stirred every two hours. After standing for stratification, the supernatant is collected, and a lower filter residue was added to 95% alcohol and soaked for 36 h, this operation is repeated, until the color of supernatant no longer changes after the alcohol is added, all extraction is collected together,and filtered by three layers of filter paper for three times to remove impurities.

[24] In the present invention, the ratio of petroleum ether: distilled water in the step 2) 1s 1:1. It is necessary to repeat the extraction for three times to completely remove residues such as the alcohol and water-insoluble oil and fat, and finally the aqueous phase is collected for concentrating.

[25] The present invention provides an application of a reagent for inhibiting proliferation and migration of a mouse LLC in preparation of a drug for preventing and treating a lung cancer, and the reagent is the BBE.

[26] The BBE and the preparation method thereof and the application provided by the present invention are described in detail below in combination with embodiments, but they may not be construed as limiting a scope of protection of the present invention.

[27] Material description

[28] Experimental animal: C57 male mice in about 8 weeks old, purchased from

Beijing SPF Biotechnology Co., Ltd. (license number: SCXK (Beijing) 2019-0010), the rearing and handling of the mice strictly follows animal experimental procedures guided by the National Health Guidelines Institute.

[29] Cell: mouse Lewis non-small-cell lung cancer cells.

[30] Reagent: purchased and obtained by conventional purchase channels.

[31] Instrument: purchased and obtained by common commodity channels.

[32] Embodiment 1

[33] Extraction method for bombys batryticatus ethanol extract

[34] (1) A bombyx batryticatus with a relatively large individual is selected and placed in a baking oven, after being baked at 50°C for 16 h, it is completely crushed into powder by using a crusher, and sieved by a 100-mesh sieve to remove impurities. kg of the bombyx batryticatus powder is taken and soaked in 10 L of 95% alcohol for 36 h (it is thoroughly stirred once every two hours), after standing for stratification, 5 a supernatant is collected, and a lower filter residue is added to 10 L of the 95% alcohol and soaked for 36 h, this operation is repeated, until the supernatant color no longer changes after the alcohol is added, all extract liquid is collected together and filtered by three layers of filter paper for three times to remove impurities , a filtrate is placed on a rotary evaporator for spin-drying concentration, and place on a fume 10 cupboard to drain the residual alcohol to obtaine a bombyx batryticatus initial extraction (extract).

[35] (2) 200 g of the bombyx batryticatus initial extraction (extract) is taken and placed in a 1000 ml separatory funnel, and 500 ml of each of petroleum ether and distilled water is added for extraction in a ratio of 1:1. The extraction is repeated for three times to completely remove residues such as the alcohol and water-insoluble oil and fat, an aqueous phase is collected and added to a rotary evaporator to be rotary-evaporated and concentrated, then residual liquid is drained in a fume cupboard to obtain a bombys batryticatus ethanol extract, hereinafter referred to as BBE, it is dissolved in Dulbecco's modified eagle medium (DMEM), to prepare BBE mother solution (1 g/ml).

[36] The extraction method is relatively simple in conditions, and low cost in raw material, and may quickly obtain the pure extract.

[37] Embodiment 2

[38] The inhibitory effect of BBE on mouse LLC cell (Lewis) in vitro

[39] Mouse LLC cells are purchased from Wuhan Procell Company. This cell strain is semi-adherent and semi-suspended, and epithelial-like. A growth medium is DMEM + 10% fetal bovine serum (FBS) + 1% penicillin/streptomycin (P/S), the passage ratio is 1:2-1:4, and a cryoprotectant condition is 50% basal medium + 40% FBS + 10% dimethylsulfoxide (DMSO). The cell culture is passaged to the fourth passage for a subsequent experiment.

[40] (1) Effect of BBE on LLC cell activity

[41] Experimental grouping and method: LLC cells are inoculated into a 96-well plate at a density of 0.5x10%*well on the day before an experiment, and the BBE mother solution is diluted according to gradient concentrations and added to the cells.

The final experimental grouping is a 0 mg/ml group, a 2.5 mg/ml group, a 5 mg/ml group, a 10 mg/ml group, a 20 mg/ml group, a 40 mg/ml/group and an 80 mg/ml group.

The cell viability is detected by a CCK8 method at 24 h, 36 h, 48 h and 60 h after BBE were added to cells in each group.. All data are expressed as mean mean + SEM. The mean number of two groups is compared by Student's t test with an independent sample, and the multiple comparison of the mean number of a plurality of the groups is analyzed by one-way ANOVA and Newman-Student-Keuls test. The statistical analysis is performed by using SPSS 19.0 software, and P < 0.05 on both sides is considered to be statistically significant.

[42] Result

[43] BBE inhibits the proliferation of the LLC cells in a concentration-dependent manner (see FIG. 1). The half maximal inhibitory concentration (IC50) measured of

BBE is 37.5 mg/ml, so 20 mg/ml (low concentration), 30 mg/ml (medium concentration) and 40 mg/ml (high concentration) are respectively selected as subsequent experimental drug concentrations in a subsequent experiment.

[44] (2) Effect of BBE on LLC cell migration

[45] Experimental grouping and method: a control group (0 mg/ml), a low-dose group (20 mg/ml), a medium-dose group (30 mg/ml) and a high-dose group (40 mg/ml). The LLC cells are inoculated into a 6-well plate at a density of 1x10%well on the day before the experiment, as to guarantee that the cells are fully grown on the next day. Scratches are made with a ruler and a 10 pl pipette tip, a low-serum medium (DMEM + 2% FBS) is replaced to culture continuously, and photographs are taken at 0 h, 24 h and 48 h respectively. The width of the scratches or the area of the scratches is analyzed at each time point by using Image J software.

[46] Result

[47] BBE may significantly inhibit the migration ability of the LLC cells (see FIG. 2). Results show that both the medium and high concentrations of BBE inhibit the migration ability of the LLC cells, but there is no significant difference between the two groups.

[48] Embodiment 3

[49] The anti-tumor effect of BBE combined with DDP on LLC tumor-bearing mouse in vivo [S0] Establishment of LLC tumor-bearing mouse model

[51] C57 mice (n=24, 18-22 g) in about 6-8 weeks old are selected for an experiment, and after 1 week of adaptive raising, the mice are randomly divided into the following 5 groups (n=10) according to the body weight: a model group (Model), a

BBE group (1440 mg/kg/day BBE), a DPP group (DPP, 2 mg/kg/2 day); a low-dose

BBE + DPP group (900 mg/kg/day BBE-low dose, BBEL+DPP); a high-dose BBE +

DPP group (1440 mg/kg/day BBE-high dose, BBEH+DPP). On the first day of the experiment, the five groups of the mice are subcutaneously injected with 200 ul of

LLC cell suspension (diluted to 1x 107/ml with PBS) under the left anterior axilla. After about one week, when subcutaneous transplantation tumors of the most mice are grown to 50 mm’, the treatment of each experimental group with DDP and/or BBE is started, in which DPP is dissolved in PBS for intraperitoneal injection, 1 time/2 days, and BBE is diluted with PBS for intragastric administration, 1 time/day, the successive administration is performed for 31 days (the administration dosage of BBE is determined according to the human body dosage inquired by a pharmacopoeia).

[52] After the administration is started, the body weight of each group of the mice is weighed every 3 days, and the volume of the tumor is measured at the same time.

After 31 days, the mice are anesthesized with 0.1% sodium pentobarbital via intraperitoneally inject. After the mice are anesthetized, blood is collected from an orbital vein, and centrifuged at 3,000 r/min for 10 minutes, and a supernatant is collected and stored in a -80°C refrigerator. After the blood collection, the mice are sacrificed by a cervical dislocation method, and the tumors and spleens are dissected, and weighed. Tumor tissues are rapidly frozen in liquid nitrogen, and stored in the -80°C refrigerator for subsequent gene and protein expression detection. [S3] Result

[54] 1. BBE inhibits the tumor growth in the LLC tumor-bearing mice.

[55] Compared with the model group, the tumor growth of the mice in the BBE group is significantly inhibited, it is indicated that BBE has the anti-tumor effect in vivo. Compared with the DDP group, after the drug combination of BBE+DPP, the inhibitory effect of BBE on the tumor is dose-dependent (FIG. 4), which may enhance the chemotherapeutic effect of DDP.

[56] 2. BBE inhibits the proliferation of the tumor tissue.

[57] A Western blot technology is used to detect protein expression conditions of a proliferation marker protein Ki-67 (Ki67), a Proliferating cell Nuclear Antigen (PCNA), and an apoptosis inhibitor survivin (FIG. 5). Compared with the model group, both BBE and DPP may reduce the protein expressions of Ki67, PCNA and survivin, and the combined use of BBE and DPP may significantly enhance the inhibitory effect of DPP on the expression of the three proteins, and the inhibitory effect of BBE on the protein expression is dose-dependent.

[58] The above are only preliminary implementation modes of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, a plurality of improvements and modifications may also be made, and these improvements and modifications should also be regarded as a scope of protection of the present invention.

Claims

Conclusions

Bombyx batryticatus (BBE) extract characterized by containing a plurality of active anti-cancer components.

The BBE preparation method according to claim 1, characterized by comprising the following steps: 1) separating under the effect of soaking in 95% alcohol to obtain an initial extract of bombyx batryticatus; and 2) extracting the initial extract of bombyx batryticatus with a mixture of petroleum ether and distilled water, collecting an aqueous phase and concentrating to obtain BBE.

The preparation method according to claim 2, characterized in that the volume ratio of the petroleum ether and the distilled water is 1:1.

Use of BBE according to claim 1 in the preparation of a medicament for the prevention and treatment of a non-small cell lung cancer.

Use of BBE according to claim 1 in the preparation of a medicament for inhibiting proliferation and migration of a non-small cell lung cancer cell.

Use of BBE according to claim 1 in enhancing an antitumor effect of cisplatin (DDP).

Use of a reagent for inhibiting in vivo and in vitro proliferation of a mouse Lewis lung cancer cell in treatment of a non-small cell lung cancer, wherein the reagent is the BBE of claim 1.