A trans-septum extract, its preparation method and use

Abstract

The application discloses a preparation method of Hengjingxi extract, which comprises the following steps: crushing Hengjingxi medicinal materials, adding purified water to the obtained medicinal powder to perform micro-boiling extraction, filtering, adding purified water to medicinal residues to continue micro-boiling extraction, filtering, and combining the two extraction solutions and adding cool boiled water to quantitatively obtain the Hengjingxi extract. The preparation method has low cost and is easy to operate, and can be applied to industrial continuous production. The obtained Hengjingxi extract has the effect of improving lipid metabolism disorder and can be used for preparing medicines for treating hyperlipidemia and fatty liver.

Description

Technical Field

[0001] This invention belongs to the field of drug extraction and application technology, specifically relating to a transverse meridian extract, its preparation method, and its uses. Background Technology

[0002] The root and leaves of *Hedyotis diffusa*, also known as thin-leaved *Hedyotis diffusa*, comb wood, basket king, thin-leaved red thick shell, bruise general, bone-crushing lotus, skin yellow (Guangzhou Military Region's "Handbook of Commonly Used Chinese Herbal Medicines"), are often used medicinally. It has a slightly bitter taste and is neutral in nature. It has the functions of dispelling wind and dampness, strengthening muscles and bones, promoting blood circulation and relieving pain. It has broad medicinal prospects and is mainly distributed in Guangdong and Guangxi.

[0003] Pharmacodynamic studies on *Hymenochae lanceolata* are scarce. Only one document (Wei Jianquan et al. Experimental study on anti-inflammatory and analgesic effects and acute toxicity of *Hymenochae lanceolata* [J]. Shizhen Guoyi Guoyao, 2012, 23(03): 639-641.) discloses that *Hymenochae lanceolata* has anti-inflammatory and analgesic effects, and uses modern pharmacological methods to conduct experimental studies on the anti-inflammatory and analgesic effects and acute toxicity of its extracts. Given the preciousness of *Hymenochae lanceolata*, how to further develop its medicinal functions and achieve comprehensive utilization of this medicinal resource is a technical problem that needs to be solved. Currently, there are no reports on other medicinal uses of *Hymenochae lanceolata*. Summary of the Invention

[0004] The present invention aims to provide a method for preparing a transverse meridian extract and the use of the extract in the preparation of drugs for treating lipid metabolism disorders.

[0005] The technical solution of the present invention: a method for preparing a transverse tataricus extract, characterized by comprising the following steps: taking transverse tataricus medicinal material and crushing it, adding purified water to the obtained powder for extraction by gentle boiling, filtering, adding purified water to the residue for further extraction by gentle boiling, filtering, combining the two extracts, adding cooled boiled water to a measured amount, and thus obtaining the transverse tataricus extract.

[0006] Preferably, the *Hypericum perforatum* herb is pulverized, and the resulting powder is extracted with 5 times the amount of purified water. The mixture is kept at a gentle boil for 30 minutes, then filtered. The residue is then extracted with 2-3 times the amount of purified water, and the mixture is kept at a gentle boil for 20 minutes, then filtered. The two extracts are combined, and cooled boiled water is added to bring the volume up to 5 times the original amount of powder to obtain the *Hypericum perforatum* extract.

[0007] Preferably, the medicinal material of *Hymenoplastyum* is the rhizome, branches, leaves, or seeds of *Hymenoplastyum*.

[0008] The beneficial technical effects of this invention are: (1) The present invention provides a method for preparing transverse diaphragm extract. The method is simple, easy to operate, has low equipment requirements, low cost, and is suitable for industrial continuous production. (2) The transverse meridian extract provided by the present invention has the effect of improving lipid metabolism disorder and can be used to prepare drugs for treating hyperlipidemia and fatty liver. Attached Figure Description

[0009] Figure 1 This is a typical image showing the triglyceride staining intensity of the zebrafish tail vein after transverse cross-section sample processing (Note: the dashed box represents the zebrafish tail vein of the analysis site).

[0010] Figure 2 This is a typical image of cholesterol fluorescence intensity in the zebrafish tail vein after transverse cross-section sample processing (Note: the dashed box represents the zebrafish tail vein at the analysis site).

[0011] Figure 3 This is a schematic diagram of the analysis sites for the thickness of blood vessel walls in the eyes of zebrafish after transverse diaphragm sample processing (Note: the dashed box indicates the analysis site).

[0012] Figure 4 This is a typical image of the blood vessels in the eye of a zebrafish after transverse transverse sample processing.

[0013] Figure 5 This is a typical image of the staining intensity of zebrafish liver after transverse cross-section sample processing (Note: the dashed box indicates the analysis area). Detailed Implementation

[0014] The following examples provide further illustration, but the present invention is not limited to these examples.

[0015] Example 1 Take the rhizome of *Hylocereus undatus* and crush it. Weigh 10 g of the obtained powder and add it to 50 mL of purified water for extraction. Keep it at a gentle boil for 30 min, filter, add 20 mL of purified water to the residue and continue extraction. Keep it at a gentle boil for 20 min, filter, combine the two extracts, add cooled boiled water to a final volume of 50 mL, and you will get *Hylocereus undatus* extract with a content of 0.2 g / mL (each milliliter of extract is equivalent to 0.2 g of crude drug, the same below).

[0016] Example 2 Take the branches and leaves of *Echinopsis thunbergii* and crush them. Weigh 10 g of the obtained powder and add 50 mL of purified water for extraction. Keep it at a gentle boil for 30 min, filter, add 30 mL of purified water to the residue and continue extraction. Keep it at a gentle boil for 20 min, filter, combine the two extracts, add cooled boiled water to a final volume of 50 mL, and you will get *Echinopsis thunbergii* extract with a content of 0.2 g / mL.

[0017] Example 3 Take the seeds of *Hymenochloa crus-galli* and crush them. Weigh 10 g of the powder and add 50 mL of purified water for extraction. Keep it at a gentle boil for 30 min, then filter. Add 25 mL of purified water to the residue and continue extraction. Keep it at a gentle boil for 20 min, then filter. Combine the two extracts and add cooled boiled water to a final volume of 50 mL to obtain *Hymenochloa crus-galli* extract with a content of 0.2 g / mL.

[0018] Experimental Example The following experimental example, which establishes a zebrafish model of hyperlipidemia and fatty liver, further illustrates the efficacy of transverse meridian extract in improving lipid metabolism disorders, but the present invention is not limited to these experimental examples.

[0019] 1. Laboratory animals Zebrafish were raised in aquarium water at 28℃ (water quality: 200 mg of instant sea salt added per 1 L of reverse osmosis water, conductivity 450~550 µS / cm; pH 6.5~8.5; hardness 50~100 mg / L CaCO3), bred and provided by the aquarium breeding center of Hangzhou Huante Biotechnology Co., Ltd.

[0020] The Albino strain of translucent zebrafish, with a melanin allele mutation, was bred through natural pair mating. Zebrafish aged 5 days post-fertilization (5 dpf) were used for maximum detectable concentration (MTC) determination and evaluation of its efficacy in lowering blood lipids and improving fatty liver. Transgenic vascular green fluorescent zebrafish were also bred through natural pair mating, with zebrafish aged 5 days post-fertilization (5 dpf) used to evaluate its efficacy in improving blood vessel wall thickness.

[0021] 2. Experimental Consumables and Reagents Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma); Methylcellulose (batch number B2006074, Shanghai Aladdin Biochemical Technology Co., Ltd.); CholEsteryl BODIPY™ 542 / 563 C11 (cholesterol probe, batch number 2291600, Invitrogen); Oil Red O (batch number SHBM5455, Sigma); 1,2-Propanediol (batch number 20211117, Sinopharm Chemical Reagent Co., Ltd.); Pure egg yolk powder (batch number 20200809, Zhejiang Agribusiness Biotechnology Co., Ltd.); D-(+)-glucose (batch number 20201105, Sinopharm Chemical Reagent Co., Ltd.).

[0022] 3. Sample preparation information The transverse suture extracts obtained in Examples 1, 2, and 3 were used as mother liquors, and were aliquoted and stored at -20°C. During the experiment, sample solutions of high, medium, and low concentrations were prepared using purified water.

[0023] Positive control: Atorvastatin calcium tablets (hereinafter referred to as atorvastatin calcium), white tablets, batch number 202105219C, Lepu Medical. Prepared into a stock solution of 11.6 mg / mL with DMSO, aliquoted and stored at -20℃, and diluted before use.

[0024] 4. Detection Method (1) MTC measurement Zebrafish of the Albino strain (5 dpf melanin allele mutant, translucent) were randomly selected and placed in beakers, 30 fish per beaker. Three concentrations (high, medium, and low) of *Imperata cylindrica* rootstock, branches, leaves, and seeds were dissolved in water. A normal control group and a model control group were also included. Each beaker contained 25 mL of liquid. Except for the normal control group, all other experimental groups were fed a high-fat diet (pure egg yolk powder + glucose) to establish a zebrafish hyperlipidemia model. The samples and high-fat diet were co-treated for 15 h (7.5 h per day). During the sample treatment period, the number of dead zebrafish in each experimental group was counted daily and removed promptly. After treatment at 28℃ for 48 h, the MTC of *Imperata cylindrica* samples in the hyperlipidemia model zebrafish was measured.

[0025] (2) Evaluation of the efficacy of lowering triglycerides The zebrafish hyperlipidemia model was established as described in (1). The concentration of atorvastatin calcium in the positive control was increased to 11.6 µg / mL. The samples were treated with a high-fat diet for 15 h (7.5 h per day). After treatment at 28℃ for 48 h, Oil Red O was used for whole-body fat staining. After staining, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The staining intensity of the zebrafish tail vessels was analyzed, and the statistical analysis results of this index were used to evaluate the triglyceride-lowering efficacy of the transverse truncation sample.

[0026] (3) Evaluation of cholesterol-lowering efficacy The zebrafish hyperlipidemia model was established as described in (1). The concentration of atorvastatin calcium in the positive control was increased to 11.6 µg / mL. The samples were treated with a high-fat diet for 15 h (7.5 h per day). After treatment at 28℃ for 32 h, a cholesterol probe was injected. Treatment continued for 16 h. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. The cholesterol fluorescence intensity of the zebrafish tail blood vessels was analyzed, and the cholesterol-lowering efficacy of the transverse cerebral sample was evaluated based on the statistical analysis results of this index.

[0027] (4) Evaluation of efficacy in improving blood flow in hyperlipidemia The zebrafish hyperlipidemia model was established as described in (1). The concentration of atorvastatin calcium in the positive control was increased to 11.6 µg / mL. The samples were treated with a high-fat diet for 15 h (7.5 h per day). After treatment at 28℃ for 48 h, 10 zebrafish were randomly selected from each experimental group and placed under a heart rate blood flow analysis system to record zebrafish blood flow videos. The blood flow velocity of the zebrafish was analyzed and statistically analyzed. The efficacy of the transverse diaphragm sample in improving blood flow in hyperlipidemia was evaluated based on the statistical analysis results of this index.

[0028] (5) Evaluation of efficacy in improving blood vessel wall thickness 30 transgenic green fluorescent zebrafish (5 dpf) were randomly selected and placed in beakers. Three concentrations (high, medium, and low) of *Vitex negundo* rhizomes, branches, leaves, and seeds were administered via water-soluble solutions. Atorvastatin calcium was used as a positive control at 11.6 µg / mL. Normal and model control groups were also included. Each beaker contained 25 mL of liquid. Except for the normal control group, all experimental groups were given a high-fat diet via water-soluble solutions to establish a zebrafish hyperlipidemia model. The samples and high-fat diet were co-treated for 15 h (7.5 h per day). After 48 h of treatment at 28℃, three zebrafish from each experimental group were randomly selected and photographed under a laser confocal microscope. The efficacy of *Vitex negundo* samples in improving blood vessel wall thickness was qualitatively evaluated by assessing the thickness of the blood vessel walls in the zebrafish eyes.

[0029] (6) Evaluation of efficacy in improving fatty liver 30 zebrafish of the Albino strain (5 dpf melanin allele mutant) were randomly selected and placed in beakers. Three concentrations of sample solutions (high, medium, and low) were administered to the rhizomes, branches, leaves, and seeds of *Imperata cylindrica*. The positive control group received atorvastatin calcium at 11.6 µg / mL. A normal control group and a model control group were also included. Each beaker contained 25 mL of liquid. Except for the normal control group, all experimental groups were fed a high-fat diet to establish a zebrafish fatty liver model. After treatment at 28℃ for 48 h, whole-body fat staining was performed using Oil Red O. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The staining intensity of the zebrafish liver was analyzed, and the statistical analysis results of this index were used to evaluate the efficacy of *Imperata cylindrica* samples in improving fatty liver.

[0030] 5. Statistical methods Statistical results are expressed as mean ± standard deviation. The result indicates that, according to statistical analysis performed using SPSS 26.0 software, P < 0.05 indicates that the difference is statistically significant.

[0031] 6. Experimental Results (1) MTC Under the conditions of this experiment, the MTC of lipid-lowering efficacy of the rhizome and branches of *Ixora chinensis* was 125 µg / mL, and the MTC of lipid-lowering efficacy of the seeds was 2000 µg / mL, as detailed in Table 1.

[0032] (2) Evaluation of the efficacy of lowering triglycerides Under the experimental conditions, compared with the normal control group, the staining intensity of triglycerides in the tail vein of the model control group was significantly increased, with a statistically significant difference (P < 0.001), indicating successful model establishment. Compared with the model control group, the positive control drug atorvastatin calcium significantly reduced the staining intensity of triglycerides in the tail vein, with a statistically significant difference (P < 0.001). The staining intensity of triglycerides in the tail vein of the rhizome group at a concentration of 125 µg / mL was significantly lower than that of the model control group, with a statistically significant difference (P < 0.001). The staining intensity of triglycerides in the tail vein of the branches and leaves group at concentrations of 31.2, 62.5, and 125 µg / mL was lower than that of the model control group, with statistically significant differences (P < 0.05 & P < 0.01 & P < 0.01). The staining intensity of triglycerides in the tail vein of the seeds group at concentrations of 1000 and 2000 µg / mL was lower than that of the model control group, with statistically significant differences (all P < 0.05). See Table 2 for details. Figure 1 .

[0033] (3) Evaluation of cholesterol-lowering efficacy Under the experimental conditions, compared with the normal control group, the cholesterol fluorescence intensity of the tail vein in the model control group was significantly increased, with a statistically significant difference (P < 0.001), indicating successful model establishment. Compared with the model control group, the positive control drug atorvastatin calcium significantly reduced the cholesterol fluorescence intensity of the tail vein, with a statistically significant difference (P < 0.01). The cholesterol fluorescence intensity of the tail vein in the 125 µg / mL concentration group of *Imperata cylindrica* rhizome was significantly lower than that in the model control group, with a statistically significant difference (P < 0.001). The cholesterol fluorescence intensity of the tail vein in the 125 µg / mL concentration group of *Imperata cylindrica* branches and leaves was lower than that in the model control group, with a statistically significant difference (P < 0.05). The cholesterol fluorescence intensity of the tail vein in the 1000 and 2000 µg / mL concentration groups of *Imperata cylindrica* seeds was significantly lower than that in the model control group, with statistically significant differences (P < 0.01 & P < 0.001). See Table 3 for details. Figure 2 .

[0034] (4) Evaluation of efficacy in improving blood flow in hyperlipidemia Under the experimental conditions, compared with the normal control group, the blood flow velocity in the model control group was significantly reduced, with a statistically significant difference (P < 0.001), indicating that the model was successfully established. Compared with the model control group, the positive control drug atorvastatin calcium significantly increased blood flow velocity, with a statistically significant difference (P < 0.01). The blood flow velocity in the 62.5 and 125 µg / mL concentration groups of *Phyllostachys edulis* rhizomes was significantly increased compared with the model control group, with statistically significant differences (all P < 0.001). The blood flow velocity in the 31.2, 62.5, and 125 µg / mL concentration groups of *Phyllostachys edulis* branches and leaves was increased compared with the model control group, with statistically significant differences (P < 0.05 & P < 0.05 & P < 0.001). The blood flow velocity in the 500, 1000, and 2000 µg / mL concentration groups of *Phyllostachys edulis* seeds was significantly increased compared with the model control group, with statistically significant differences (all P < 0.001). See Table 4 for details.

[0035] (5) Evaluation of efficacy in improving blood vessel wall thickness observe Figure 3 and Figure 4 It can be seen that, under the conditions of this experiment, the rhizomes, branches and leaves, and seeds of *Phyllostachys edulis* all have the effect of improving the thickness of blood vessel walls.

[0036] (6) Evaluation of efficacy in improving fatty liver From Table 5 and Figure 5 It can be seen that, under the conditions of this experiment, compared with the normal control group, the liver staining intensity of the model control group was significantly increased, with a statistically significant difference (P < 0.001), indicating that the model was successfully established. Compared with the model control group, the positive control drug atorvastatin calcium significantly reduced the liver staining intensity, with a statistically significant difference (P < 0.001). The liver staining intensity of the 125 µg / mL concentration group of *Ipomoea quamoclit* rhizome was significantly lower than that of the model control group, with a statistically significant difference (P < 0.01). The liver staining intensity of the 62.5 µg / mL and 125 µg / mL concentration groups of *Ipomoea quamoclit* branches and leaves was significantly lower than that of the model control group, with statistically significant differences (all P < 0.001). The liver staining intensity of the 1000 µg / mL and 2000 µg / mL concentration groups of *Ipomoea quamoclit* seeds was significantly lower than that of the model control group, with statistically significant differences (all P < 0.01).

[0037] 7. Experimental Conclusions Under the experimental conditions, the rhizome, branches, leaves, and seeds of *Imperata cylindrica* all exhibited lipid-lowering effects, specifically by lowering triglyceride and cholesterol levels and improving abnormal blood flow velocity and vessel wall thickness caused by hyperlipidemia. Furthermore, all three components of *Imperata cylindrica* showed effects in improving fatty liver, with lower liver staining intensity indicating better efficacy. The experimental results suggest that the branches and leaves of *Imperata cylindrica* showed the best efficacy in improving fatty liver, followed by the rhizome. In conclusion, the *Imperata cylindrica* extract obtained in this invention can be used to prepare drugs for treating hyperlipidemia and fatty liver.

[0038] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of a transverse meridian extract in the preparation of a drug for treating hyperlipidemia and fatty liver, characterized in that, The preparation method includes the following steps: take the *Hypericum perforatum* herb and crush it. Add 5 times the amount of purified water to the powder and extract it. Keep it at a gentle boil for 30 minutes. Filter it. Add 2-3 times the amount of purified water to the residue and continue to extract it. Keep it at a gentle boil for 20 minutes. Filter it. Combine the two extracts and add cooled boiled water to a quantitative amount equal to 5 times the original amount of powder to obtain the *Hypericum perforatum* extract.

2. The application according to claim 1, characterized in that, The medicinal material mentioned is the rhizome, branches, leaves, or seeds of the herb *Hymenopterys pubescens*.

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