A method for breaking the cell wall of pteris spore powder, pteris spore powder and use thereof

Abstract

This invention discloses a method for breaking the cell wall of Pteris vittata spore powder, comprising grinding and breaking the cell wall: placing Pteris vittata spore powder in a grinding tube, and mixing it with a material-to-liquid ratio of 1:(80-90) g / mL. ‑1 Distilled water was added in a specific ratio, and the grinding tube containing the *Pteris vittata* spore powder and distilled water was placed in a tissue homogenizer. The homogenizer was turned on to lower the temperature to below 4°C, and then the cells were ground to break the cell walls, resulting in a suspension of broken *Pteris vittata* spore powder. This cell wall breaking method can break the outer shell of *Pteris vittata* spores, achieving a cell wall breaking rate of over 95.15%, while greatly preserving the active ingredients in the broken *Pteris vittata* spore powder. This invention also confirms that *Pteris vittata* spores have the same efficacy as the whole *Pteris vittata* herb extract and are an important component of *Pteris vittata* medicinal materials. This invention also discloses a *Pteris vittata* spore powder and its uses.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for breaking the cell wall of Pteris vittata spore powder, Pteris vittata spore powder and its uses. Background Technology

[0002] *Pteris vittata*, a plant belonging to the Pteridaceae family, has been the subject of research on the chemical composition and pharmacological effects of the whole herb. It mainly contains flavonoids, terpenes, phenolic acids, lignins, alkaloids, aliphatic compounds, and volatile oils. Among these, flavonoids, terpenes (kauranes, sesquiterpenes), and sterols can effectively inhibit tumors. Terpenes have shown good inhibitory effects on *Escherichia coli*, *Salmonella typhimurium*, and *Staphylococcus aureus*. Studies have shown that a 95% extract of *Pteris vittata* can increase the thymus level in mice. The spleen index and spleen index have a promoting effect on the proliferation of mouse spleen cells, indicating that the extract of Pteris vittata can enhance the body's immunity. Researchers also isolated the anti-tumor active ingredient tetracyclic diterpenoid kaurane (ene) compounds from the rhizome of Pteris vittata for the first time. In recent years, most scholars have focused on the study of the whole herb (root, stem and leaves) of Pteris vittata. At present, there are no reports on the medicinal value of Pteris vittata spore powder at home and abroad. Moreover, because a large amount of spore powder is easily lost during the harvesting, washing and drying process of Pteris vittata, the spore powder of Pteris vittata is difficult to collect and is therefore ignored. Summary of the Invention

[0003] To address the aforementioned shortcomings, the present invention aims to provide a method for breaking the cell wall of *Pteris vittata* spore powder. This method can break the outer shell of *Pteris vittata* spores, achieving a cell wall breakage rate of over 95.15%, while significantly preserving the active ingredients in the broken *Pteris vittata* spore powder. The present invention also confirms that *Pteris vittata* spores possess the same efficacy as the whole *Pteris vittata* herb extract, making them an important component of *Pteris vittata* medicinal materials.

[0004] The technical solution adopted by the present invention to achieve the above-mentioned objective is: a method for breaking the cell wall of Pteris vittata spore powder, comprising grinding and breaking the cell wall: taking Pteris vittata spore powder and placing it in a grinding tube, and mixing it at a material-to-liquid ratio of 1:(80-90)g.mL. -1 Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water into a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to below 4°C, and then grind and break the cell walls to obtain a suspension of Pteris vittata spore powder with broken cell walls.

[0005] A method for breaking the cell wall of Pteris vittata spore powder includes the following steps:

[0006] (i) Ultrasonic cell disruption: Place Pteris vittata spore powder in a centrifuge tube, and adjust the ratio of the solid to the liquid to a concentration of 1:(80-90) g / mL. -1After adding distilled water in the specified proportion, immediately place the mixture in an ultrasonic cell disruptor. Insert the amplitude transformer of the ultrasonic cell disruptor into the liquid surface and perform ultrasonic cell disruption according to the following parameters: ultrasonic temperature below 40℃, ultrasonic power 600-800W, ultrasonic on time 20-30 seconds, ultrasonic off time 20-30 seconds, and each ultrasonic treatment lasts 8-10 minutes, which completes one cycle of ultrasonic treatment. After no more than 6 ultrasonic treatment cycles, dry the mixture at 40-50℃ to constant weight. The entire ultrasonic cell disruption process is now complete.

[0007] (ii) Grinding and cell wall disruption: Place the above-mentioned ultrasonically disrupted Pteris vittata spore powder into a grinding tube, and mix at a material-to-liquid ratio of 1:(80-90)g.mL. -1 Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water into a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to below 4°C, and then grind to obtain a suspension of Pteris vittata spore powder with broken cell walls.

[0008] A method for breaking the cell wall of Pteris vittata spore powder includes the following steps:

[0009] Ⅰ Freeze-thaw cell wall breaking: Weigh out the Pteris vittata spore powder and place it in a cryovial. First, freeze it at a temperature of -60 to -80℃ for 20-30 minutes, then thaw it at a high temperature within 4-5 minutes. Repeat the freeze-thaw cycle as described above at least 6 times to complete the freeze-thaw cell wall breaking.

[0010] II. Ultrasonic cell disruption: Place the freeze-thawed and disrupted *Pteris vittata* spore powder into a centrifuge tube, and mix at a material-to-liquid ratio of 1:(80-90) g / mL. -1 After adding distilled water in the specified proportion, immediately place the mixture in an ultrasonic cell disruptor. Insert the amplitude transformer of the ultrasonic cell disruptor into the liquid surface of the centrifuge tube and perform ultrasonic cell disruption according to the following parameters: ultrasonic temperature below 40℃, ultrasonic power 600-800W, ultrasonic on time 20-30 seconds, ultrasonic off time 20-30 seconds, and each ultrasonic treatment lasting 8-10 minutes, which completes one cycle of ultrasonic treatment. After no more than 6 ultrasonic treatment cycles, dry the mixture at 40-50℃ to constant weight. The entire ultrasonic cell disruption process is now complete.

[0011] III. Grinding and Cell Wall Disruption: Place the above-mentioned ultrasonically disrupted Pteris multifida spore powder into a grinding tube, and add it at a material-to-liquid ratio of 1:(80-90) g·mL. -1 Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water into a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to below 4°C, and then grind to obtain a suspension of Pteris vittata spore powder with broken cell walls.

[0012] As a further improvement of the present invention, the grinding conditions in the grinding and cell wall breaking process include: a grinding frequency of 60-70 Hz, a total of 3-5 grinding cycles, a grinding cycle time of 75-90 seconds, and grinding at least 1-2 times under the above conditions.

[0013] As a further improvement of the present invention, a pretreatment is included before cell wall breaking. The pretreatment method is as follows: soaking the Pteris vittata spore powder in distilled water and performing ultrasonic treatment, and after precipitation, taking the lower precipitate and drying it to constant weight, thus completing the pretreatment.

[0014] As a further improvement of the present invention, the ratio of the powder to distilled water is 1:(20-30)mg / mL; the conditions for ultrasonic treatment are: ultrasonic frequency of 70-90W and ultrasonic time of 180-200 minutes.

[0015] The present invention also discloses a method for preparing broken cell wall Pteris vittata spore powder. The broken cell wall Pteris vittata suspension as described above is taken, and after standing and settling, the lower layer of precipitate is taken and placed in an oven and dried to constant weight at a temperature of 40-50℃ to obtain broken cell wall Pteris vittata spore powder.

[0016] The present invention also discloses the uses of Pteris vittata spore powder, which can be used directly as a medicine, or used directly as a medicine after cell wall breaking, or combined with other medicines to prepare a drug composition for treating spleen deficiency syndrome, or for treating diarrhea caused by enteritis, and for improving the body's immunity to prevent diseases.

[0017] The beneficial effects of this invention are as follows: This invention employs multiple methods to break the cell wall of Pteris vittata spore powder, wherein...

[0018] By using a rapid grinding method with multiple cycles at low temperature to break the outer shell of Pteris vittata spores, the cell wall breakage rate can reach 95.15%. The high cell wall breakage rate results in Pteris vittata spore powder with small particle size and uniform particle size distribution, which is easy to absorb. At the same time, it avoids the oxidation of active ingredients in Pteris vittata spores caused by the high temperature generated during grinding, thus greatly preserving the active ingredients in the broken cell wall Pteris vittata spore powder.

[0019] A comprehensive cell wall disruption method combining ultrasonic and grinding was adopted. First, the amplitude rod of the ultrasonic disruptor was directly inserted into the Pteris vittata spore powder liquid for multi-cycle contact ultrasonic disruption. The ultrasonic waves emitted by the ultrasonic disruptor acted on the Pteris vittata spores, causing the outer shell of the spores to break under the action of ultrasonic waves. Then, low-temperature multi-cycle grinding further pulverized the outer shell of the Pteris vittata spores, achieving a cell wall disruption rate of 97.91%, effectively improving the cell wall disruption rate. Compared with the method of simply using grinding, the comprehensive cell wall disruption method combining ultrasonic and grinding resulted in Pteris vittata spore powder with smaller particle size and more uniform particle size distribution, which is conducive to the full release of the effective components in the Pteris vittata spores and easy absorption.

[0020] A comprehensive cell wall breaking method combining freeze-thaw cell wall breaking, ultrasonic cell wall breaking, and grinding cell wall breaking is adopted. After repeated freezing and thawing treatment, the hard outer shell of the Pteris vittata spores becomes brittle. Then, ultrasonic cell wall breaking treatment is carried out multiple times to break the cell wall of the Pteris vittata spores, causing the outer shell of the Pteris vittata spores to break under the action of ultrasound. Finally, low-temperature grinding can better pulverize the outer shell of the Pteris vittata spores, and the cell wall breaking rate can reach 99.11%, which effectively improves the cell wall breaking rate. The Pteris vittata spore powder after cell wall breaking has a smaller particle size and a more uniform distribution, which is conducive to the full release of the effective ingredients in the Pteris vittata spores and easy absorption.

[0021] By treating mice with spleen deficiency syndrome with both Pteris multifida spore powder and whole herb extract, the results showed that both Pteris multifida spore powder and whole herb extract could increase the mice's food intake and weight, improve intestinal inflammation, and alleviate anemia. This indicates that both whole herb extract and broken-cell wall Pteris multifida spore powder can help mice recover intestinal function more quickly and correct gastrointestinal dysfunction caused by spleen deficiency. Among them, Pteris multifida spore powder showed significantly better effects on improving the mice's food intake, weight, and blood indicators than whole herb extract, indicating that the active ingredients of broken-cell wall Pteris multifida spore powder are easily absorbed by mice. Therefore, Pteris multifida spore powder has the same efficacy as whole herb extract and can be used directly as a medicine, making it an important component of Pteris multifida medicinal materials.

[0022] The ferns spore powder of the present invention can be used directly as a medicine, or after cell wall breaking treatment, it can be used directly as a medicine, or it can be combined with other medicines to prepare a drug composition for spleen deficiency syndrome, or for treating diarrhea caused by enteritis, and for improving the body's immunity to prevent diseases.

[0023] The above is an overview of the invention's technical solution. The invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0024] Figure 1 This is a microscopic image of the unbroken cell wall of *Pteris vittata* spore powder according to the present invention;

[0025] Figure 2 for Figure 1 A magnified view of a complete spore at the location indicated by the middle arrow;

[0026] Figure 3 This is a microscopic photograph of the *Pteris vittata* spore powder after cell wall disruption according to Embodiment 1 of the present invention;

[0027] Figure 4 for Figure 3 A magnified view of a complete spore at the location indicated by the middle arrow;

[0028] Figure 5 This is a microscopic photograph of step (i) of embodiment two of the present invention after ultrasonic cell disruption;

[0029] Figure 6 for Figure 5 A magnified view of a complete spore at the location indicated by the middle arrow;

[0030] Figure 7 This is a microscopic photograph of the cell wall after a combination of ultrasonic and grinding cell wall disruption according to Embodiment 2 of the present invention.

[0031] Figure 8 This is a microscopic photograph of the cell wall breaking process after freeze-thaw cycle in step I of embodiment three of the present invention;

[0032] Figure 9 This is a microscopic image of the cell wall disruption process following the combination of freeze-thaw disruption and ultrasonic disruption in Embodiment 3 of the present invention.

[0033] Figure 10 This is a microscopic photograph of the cell wall after a combination of freeze-thaw disruption, ultrasonic disruption, and grinding disruption according to Embodiment 3 of the present invention.

[0034] Figure 11 This is a graph showing the changes in average food intake of mice in each group during different drug administration periods in this invention.

[0035] Figure 12 This is a graph showing the change in average body weight of each mouse in each group before and after drug administration in this invention. Detailed Implementation

[0036] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the specific implementation of the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments.

[0037] The *Pteris vittata* used in this invention embodiment were all harvested from Meiling Scenic Area in Nanchang, Jiangxi Province, and after identification, were preserved in the Animal Pharmacy Laboratory of the College of Animal Science and Technology, Jiangxi Agricultural University. The *Pteris vittata* spore powder used in this invention embodiment was obtained by cleaning the harvested *Pteris vittata*, placing it in a tray to dry naturally, and collecting the spore powder in the tray.

[0038] To evaluate the cell wall breaking effect of the method for breaking the cell wall of Pteris vittata spore powder in the embodiments, it is necessary to examine the Pteris vittata before and after the cell wall breaking treatment under a microscope and calculate the cell wall breaking rate.

[0039] The specific microscopic examination method includes the following steps:

[0040] (1) Preparation of microscopic examination solution: Take the spore powder of Pteris vittata to be examined under a microscope, and prepare a suspension with a concentration of 35 mg / mL using chloral hydrate test solution to obtain the microscopic examination solution of Pteris vittata spore powder.

[0041] (2) Microscopic examination: The above-mentioned Pteris vittata spore powder microscopic examination solution was examined under a microscope at 100x magnification and photographed. At the same time, the microscopic examination photos under 6 fields of view were taken, and the number of complete spores in each field of view was counted. The average value was taken as the number of Pteris vittata spores N.

[0042] (3) Calculate the cell wall breakage rate: Calculate the cell wall breakage rate using the following formula:

[0043] Cell wall breakage rate = (N0-N1) / N0×100%;

[0044] Wherein, N0 represents the number of Pteris vittata spores before cell wall disruption treatment;

[0045] N1 represents the number of *Pteris vittata* spores after cell wall disruption treatment.

[0046] Example 1

[0047] Embodiment 1 of the present invention provides a method for breaking the cell wall of Pteris vittata spore powder, including grinding and breaking the cell wall: taking Pteris vittata spore powder and placing it in a grinding tube, with a material-to-liquid ratio of 1:86 g / mL. -1 Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water into a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to 4°C, and then grind and break the cell walls to obtain a suspension of Pteris vittata spore powder with broken cell walls.

[0048] In Example 1, the grinding conditions in the grinding and cell wall breaking process include: a grinding frequency of 60 Hz, a total of 3 grinding cycles, a grinding cycle time of 75 s, and the grinding is repeated at least 2 times under the above conditions.

[0049] The tissue grinder used in Example 1 is a high-speed low-temperature grinder produced by Wuhan Sewell Biotechnology Co., Ltd. It can be used to grind and pulverize various tissue samples of plants or animals under low-temperature conditions. Traditional grinding and cell wall breaking methods are prone to generating heat and destroying the effective substances in Pteris vittata spore powder. By using a high-speed low-temperature grinder, the temperature during the grinding process can be controlled below 4°C, which effectively solves the disadvantage of temperature rise caused by traditional grinding methods.

[0050] During grinding, place two small grinding beads with a diameter of 3mm and one large grinding bead with a diameter of 4mm into the grinding tube, place it in a high-speed low-temperature grinder, select the plant stem function, and grind according to the above grinding conditions.

[0051] Example 1 involves mixing Pteris multifida spore powder with distilled water at a specific material-to-liquid ratio, and then grinding it multiple times at a low temperature of 4°C using a high-speed low-temperature grinder. Each grinding cycle includes multiple cycles, which effectively crushes the outer shell of the spores in the Pteris multifida spore powder. At the same time, it avoids the oxidation of the active ingredients in the Pteris multifida spores caused by the high temperature generated during grinding, thus greatly preserving the active ingredients in the broken-cell wall Pteris multifida spore powder.

[0052] Since the spores of Pteris vittata are located in sporangia, the sporangia are oblong, slightly narrow at the base, with a whorl-shaped edge of the cap, brownish-yellow in color, and the stalks vary in length, consisting of 4-6 cells, usually arranged in 2 rows, and containing about 10 spores.

[0053] To better break down the spore shells and accurately count the number of intact spores in the *Pteris vittata* spore powder before cell wall breaking, it is necessary to fully release the spores from the sporangia. Therefore, a pretreatment is included before cell wall breaking. The pretreatment method is as follows: soaking *Pteris vittata* spore powder in distilled water and ultrasonically treating it, then collecting the sediment and drying it to constant weight after precipitation, thus completing the pretreatment. Specifically, in the pretreatment, the weight-to-volume ratio of *Pteris vittata* spore powder to distilled water is 1:20 mg / mL. The ultrasonic treatment conditions are: ultrasonic frequency of 90 W and ultrasonic time of 200 minutes.

[0054] During pretreatment, distilled water easily enters the sporangia, causing them to swell. Combined with ultrasonic treatment, this causes the sporangia to crack, releasing the spores. In this way, during the subsequent cell wall breaking process, the water can directly act on the spore shell, which helps to improve the spore cell wall breaking rate.

[0055] The broken cell wall of the *Pteris vittata* spore powder suspension obtained by the method in Example 1 was examined under a microscope and the cell wall breakage rate was calculated.

[0056] Specifically, the suspension of broken-cell wall Pteris vittata spore powder obtained by grinding and breaking the cell wall was allowed to settle and the lower precipitate was taken and placed in an oven to dry to constant weight at a temperature of 50°C to obtain broken-cell wall Pteris vittata spore powder. The broken-cell wall Pteris vittata spore powder was prepared into a microscopic examination solution for microscopic examination and the number of complete Pteris vittata spores was counted. This number of spores is the number of Pteris vittata spores N1 after cell wall breaking treatment. The Pteris vittata spore powder obtained after pretreatment was prepared into a microscopic examination solution for microscopic examination and the number of complete Pteris vittata spores was counted. This number of spores is the number of Pteris vittata spores N0 before cell wall breaking treatment. The cell wall breaking rate of the method in Example 1 was calculated.

[0057] Microscopic images of *Pteris vittata* spore powder obtained after preprocessing are shown below. Figure 1 and Figure 2 As shown in Table 1, the number of complete spores in the *Pteris vittata* spore powder obtained after pretreatment is shown in Table 1.

[0058] Microscopic images of *Pteris vittata* spore powder obtained in Embodiment 1 of this invention are shown below. Figure 3 and Figure 4 As shown in Table 1, the number of intact spores and the corresponding cell wall breakage rate in the *Pteris vittata* spore powder obtained after the cell wall breakage treatment in Example 1 are shown in Table 1.

[0059] Depend on Figure 1 As can be seen, the complete spore is obtusely triangular, about 45 μm in diameter, and the outer wall surface has tubercles.

[0060] Depend on Figure 3 As can be seen, the Pteris vittata spore powder obtained by crushing the outer shell of Pteris vittata spores using the method of Example 1 has small particle size and uniform particle size distribution. At the same time, as can be seen from the results in Table 1, the cell wall breaking rate of Pteris vittata spore powder using the method of Example 1 can reach 95.15%, which is a high cell wall breaking rate.

[0061] Example 2

[0062] The main difference between Example 2 and Example 1 is that Example 2 is a method for breaking the cell wall of Pteris vittata spore powder, which includes the following steps:

[0063] (i) Ultrasonic cell disruption: Place Pteris vittata spore powder in a centrifuge tube, and adjust the ratio of the solid to the liquid to 1:80 g / mL. -1 After adding distilled water in the specified proportion, immediately place it in an ultrasonic cell disruptor, insert the amplitude rod of the ultrasonic cell disruptor into the liquid surface, and perform ultrasonic cell disruption treatment according to the following parameters: ultrasonic temperature below 40℃, ultrasonic power 800W, ultrasonic on time 20 seconds, ultrasonic off time 20 seconds, and each ultrasonic treatment time is 10 minutes, which completes one cycle of ultrasonic treatment. After 6 cycles of ultrasonic treatment, dry at 50℃ to constant weight, and the entire ultrasonic cell disruption treatment is completed.

[0064] (ii) Grinding and Cell Wall Breaking: Place the above-mentioned ultrasonically broken cell wall powder of *Pteris vittata* spores into a grinding tube, and add it at a material-to-liquid ratio of 1:80 g / mL. -1 Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water into a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to 3°C, and then grind to obtain a suspension of Pteris vittata spore powder with broken cell walls.

[0065] In Example 2, ultrasonic cell disruption was performed using a contact-type fully automatic ultrasonic disruptor (manufacturer: Ningxinzhi Biotechnology Co., Ltd., model: SCIENTZ-1200E), which has a power of 400W-1500W.

[0066] In the pretreatment, the weight-to-volume ratio of the Pteris vittata spore powder to distilled water was 1:30 mg / mL; the conditions for the ultrasonic treatment were: ultrasonic frequency of 70 W and ultrasonic time of 180 minutes.

[0067] In Example 2, the spore powder of Pteris vittata was subjected to multiple cycles of ultrasonic cell disruption. Each cycle of ultrasonic cell disruption lasted 10 minutes, and the ultrasonic temperature was controlled below 40°C to avoid the temperature from rising due to continuous ultrasonic cell disruption, which would affect the biological activity of the spore powder.

[0068] Example 2 describes a method for controlling the ultrasonic temperature: centrifuge tubes containing Pteris vittata spore powder are placed in a container filled with ice and then placed together in the soundproof working chamber of a fully automatic contact ultrasonic disruptor for ultrasonic treatment. Ice is added to the container before each cycle to ensure that the temperature during the entire ultrasonic process remains below 40°C.

[0069] In Example 2, the grinding conditions in step (ii) of grinding and breaking the cell wall include: a grinding frequency of 70 Hz, a total of 5 grinding cycles, a grinding cycle time of 90 s, and grinding is repeated once under the above conditions.

[0070] The suspension of broken cell wall Pteris vittata spore powder obtained by grinding and breaking the cell wall was allowed to stand and settle. The lower layer of precipitate was then placed in an oven and dried to constant weight at a temperature of 40℃ to obtain broken cell wall Pteris vittata spore powder.

[0071] The *Pteris vittata* spore powder obtained in each step of Example 2 was examined under a microscope and the cell wall breakage rate was calculated respectively.

[0072] Example 2, step (i) Microscopic images after ultrasonic cell disruption, as shown below. Figure 5 and Figure 6 As shown in Table 1, the number of intact spores and the corresponding cell wall disruption rate in the *Pteris vittata* spore powder obtained after ultrasonic cell wall disruption are shown in Table 1.

[0073] Example 2: Microscopic images after step (ii) of cell wall disruption (i.e., a combined cell wall disruption method integrating ultrasonic disruption and grinding disruption) are shown below. Figure 7 As shown in Table 1, the number of intact spores and the corresponding cell disruption rate in the broken-cell wall powder of *Pteris vittata* obtained by the combined cell disruption method of ultrasonic disruption and grinding disruption are shown in Table 1.

[0074] Depend on Figures 5 to 7 As shown in Table 1, the cell wall breaking rate after ultrasonic cell wall breaking was only 64.56%. Ultrasonic cell wall breaking alone cannot efficiently crush the spores, and the spore fragments are relatively large. However, the comprehensive cell wall breaking method combining ultrasonic cell wall breaking and grinding can effectively crush the spores in the Pteris vittata spore powder, and the cell wall breaking rate can reach 97.91%. This shows that the spore shell of Pteris vittata is relatively hard, and grinding is necessary to improve the cell wall breaking rate.

[0075] Therefore, Example 2 employs a comprehensive cell-wall breaking method combining ultrasonic disruption and grinding disruption. First, the spores of *Pteris vittata* are subjected to low-temperature multi-cycle cell-wall breaking using an ultrasonic disruptor. The ultrasonic waves emitted by the ultrasonic disruptor directly act on the spores, causing the outer shell of the spores to rupture under the action of the ultrasonic waves. Then, through low-temperature multi-cycle grinding, the outer shell of the spores can be better pulverized, effectively improving the cell-wall breaking rate. Compared with Example 1, which simply uses grinding disruption, Example 2 uses a comprehensive cell-wall breaking method combining ultrasonic disruption and grinding disruption. The resulting *Pteris vittata* spore powder has a smaller particle size and a more uniform particle size distribution, which is conducive to the full release of the effective components in the spores and easy absorption.

[0076] Example 3

[0077] The main difference between Example 3 and Example 1 or Example 2 is that the method for breaking the cell wall of Pteris vittata spore powder includes the following steps:

[0078] Ⅰ Freeze-thaw cell wall breaking: Weigh out the spore powder of Pteris vittata and place it in a cryovial. First, freeze it at a low temperature of -80℃ for 20 minutes, and then thaw it at a high temperature within 4 minutes. Repeat the freeze-thaw cycle 6 times to complete the freeze-thaw cell wall breaking.

[0079] II. Ultrasonic cell disruption: Place the freeze-thawed and disrupted *Pteris vittata* spore powder into a centrifuge tube, and mix at a material-to-liquid ratio of 1:90 g / mL. -1 After adding distilled water in the specified proportion, immediately place it in an ultrasonic cell disruptor. Insert the amplitude rod of the ultrasonic cell disruptor into the liquid surface and perform ultrasonic cell disruption according to the following parameters: ultrasonic temperature below 40℃, ultrasonic power 600W, ultrasonic on time 30 seconds, ultrasonic off time 30 seconds, and each ultrasonic treatment time is 8 minutes, which completes one cycle of ultrasonic treatment. After 3 cycles of ultrasonic treatment, dry at 40℃ to constant weight, and the entire ultrasonic cell disruption treatment is completed.

[0080] III. Grinding and Cell Wall Disruption: Place the above-mentioned ultrasonically disrupted Pteris multifida spore powder into a grinding tube, and add it at a material-to-liquid ratio of 1:90 g / mL. -1 Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water in a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to 2℃, and then grind to obtain a suspension of Pteris vittata spore powder with broken cell walls.

[0081] Specifically, in Example 3, the grinding conditions in step III, grinding and cell wall breaking, include: a grinding frequency of 65 Hz, a total of 4 grinding cycles, a grinding cycle time of 80 seconds, and grinding is repeated twice under the above conditions.

[0082] In the pretreatment, the weight-to-volume ratio of the Pteris vittata spore powder to distilled water was 1:25 mg / mL; the conditions for the ultrasonic treatment were: ultrasonic frequency of 80 W and ultrasonic time of 190 minutes.

[0083] In Example 3, a Haier refrigerator was used for freezing during freeze-thaw cell wall breaking. This Haier refrigerator's lowest temperature can be adjusted to -80℃. During thawing, microwave high-power heating was used. Microwave heating is a heating method that relies on the material absorbing microwaves and converting them into heat energy, causing the entire material to heat up simultaneously. This is completely different from other conventional heating methods. Traditional heating methods rely on the principles of heat conduction, convection, and radiation to transfer heat from the outside to the inside of the material. Heat is always transferred from the surface to the inside, resulting in a temperature gradient within the material. Therefore, the material is heated unevenly, leading to localized overheating. Microwave heating technology raises the temperature of the material by generating "internal friction heat" through the high-frequency reciprocating motion of dipole molecules within the heated material. It does not require any heat conduction process, allowing the material to be heated simultaneously from the inside out, with fast and uniform heating speed. Therefore, in this embodiment, microwave high heat is used for thawing, which can thaw frozen Pteris vittata spore powder in a short time. The Pteris vittata spores are heated evenly, which effectively avoids the destruction of active ingredients in the Pteris vittata spores due to prolonged high temperature treatment, or the phenomenon of incomplete thawing due to uneven heating.

[0084] The suspension of broken cell wall Pteris vittata spore powder obtained by grinding and breaking the cell wall was allowed to stand and settle. The lower layer of precipitate was then placed in an oven and dried to constant weight at a temperature of 45℃ to obtain broken cell wall Pteris vittata spore powder.

[0085] The ferns spore powder obtained in each step of Example 3 were prepared into microscopic examination solutions for microscopic examination and the cell wall breakage rate was calculated respectively.

[0086] Microscopic images of the freeze-thaw cell disruption process after step I are shown below. Figure 8 As shown in Table 1, the number of intact spores and the corresponding cell wall breakage rate in the *Pteris vittata* spore powder obtained after freeze-thaw disruption in step I are shown in Table 1.

[0087] Microscopic images after step II, ultrasonic cell disruption (i.e., a pre-disruption method combining freeze-thaw disruption and ultrasonic disruption), are shown below. Figure 9 As shown in Table 1, the number of intact spores and the corresponding cell disruption rate in the *Pteris vittata* spore powder obtained after the cell disruption method combining freeze-thaw disruption and ultrasonic disruption are shown in Table 1.

[0088] Microscopic images after step III (i.e., a combined method of cell disruption including freeze-thaw disruption, ultrasonic disruption, and grinding disruption) are shown below. Figure 10 As shown in Table 1, the number of intact spores and the corresponding cell wall breakage rate in the *Pteris vittata* spore powder were obtained after a comprehensive cell wall breaking method combining freeze-thaw cell wall breaking, ultrasonic cell wall breaking, and grinding cell wall breaking.

[0089] Depend on Figures 8 to 10 It is evident that freeze-thaw disruption and / or ultrasonic disruption are not very effective in breaking down spores, although the overall spore size decreases. Before disruption, the average spore size was 38-45 μm, after freeze-thaw disruption it was 27-42 μm, and after ultrasonic disruption it was 32-41 μm. As shown in Table 1, freeze-thaw disruption alone only achieved a disruption rate of 19.74%, while the combined freeze-thaw and ultrasonic disruption method achieved a disruption rate of 67.25%. However, a comprehensive disruption method combining freeze-thaw disruption, ultrasonic disruption, and grinding effectively pulverizes the spores in *Pteris vittata* spore powder, achieving a disruption rate of 99.11%.

[0090] Therefore, it can be seen that Example 3 adopts a comprehensive cell-wall breaking method combining freeze-thaw cell-wall breaking, ultrasonic cell-wall breaking, and grinding cell-wall breaking. After repeated freezing and thawing treatment, the hard outer shell of the Pteris vittata spores becomes brittle. Then, ultrasonic cell-wall breaking treatment is performed on the Pteris vittata spores, causing the outer shell of the Pteris vittata spores to break under the action of ultrasound. Finally, low-temperature grinding can better pulverize the outer shell of the Pteris vittata spores, effectively improving the cell-wall breaking rate. The Pteris vittata spore powder after cell-wall breaking has a smaller particle size and a more uniform distribution, which is conducive to the full release of the effective ingredients in the Pteris vittata spores and easy absorption.

[0091] Example 4

[0092] The main difference between Example 4 and Example 3 is that Example 4 is a method for breaking the cell wall of Pteris vittata spore powder, which includes the following steps:

[0093] Ⅰ Freeze-thaw cell wall breaking: Weigh out the spore powder of Pteris vittata and place it in a cryovial. First, freeze it at a low temperature of -60℃ for 30 minutes, and then thaw it at a high temperature within 5 minutes. Repeat the freeze-thaw cycle of freezing and thawing 4 times to complete the freeze-thaw cell wall breaking.

[0094] II. Ultrasonic cell disruption: Place the freeze-thawed and disrupted *Pteris vittata* spore powder into a centrifuge tube, and mix at a material-to-liquid ratio of 1:85 g / mL. -1 After adding distilled water in the specified proportion, immediately place it in an ultrasonic cell disruptor, insert the amplitude rod of the ultrasonic cell disruptor into the liquid surface, and perform ultrasonic cell disruption treatment according to the following parameters: ultrasonic temperature below 40℃, ultrasonic power 700W, ultrasonic on time 25 seconds, ultrasonic off time 25 seconds, and each ultrasonic treatment time is 9 minutes, which completes one cycle of ultrasonic treatment. After 4 cycles of ultrasonic treatment, dry at 45℃ to constant weight, and the entire ultrasonic cell disruption treatment is completed.

[0095] III. Grinding and Cell Wall Disruption: Place the above-mentioned ultrasonically disrupted Pteris multifida spore powder into a grinding tube, and add it at a material-to-liquid ratio of 1:83 g / mL. -1Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water into a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to 4°C, and then grind to obtain a suspension of Pteris vittata spore powder with broken cell walls.

[0096] Specifically, in Example 4, the grinding conditions in step III, grinding and cell wall breaking, include: a grinding frequency of 67 Hz, a total of 4 grinding cycles, a grinding cycle time of 85 seconds, and grinding is repeated once under the above conditions.

[0097] The results of the number of intact Pteris vittata spores and the cell wall disruption rate using different cell wall disruption methods in Examples 1 to 4 are shown in Table 1.

[0098] Table 1. Number of intact *Pteris vittata* spores and cell wall disruption rate under different cell wall disruption methods.

[0099]

[0100] Note: ① A combined cell disruption method that combines ultrasonic cell disruption and grinding cell disruption; ② A pre-disruption method that combines freeze-thaw cell disruption and ultrasonic cell disruption; ③ A combined cell disruption method that combines freeze-thaw cell disruption, ultrasonic cell disruption, and grinding cell disruption.

[0101] To confirm that the spore powder of *Pteris vittata* has the same efficacy as the whole plant of *Pteris vittata*, the broken-cell wall spore powder suspension obtained by any of the methods in Examples 1 to 4 was prepared with double-distilled water to a concentration of 83 mg / mL as the spore drug group. At the same time, crude extract drug group and refined extract drug group of *Pteris vittata* were prepared using the whole plant of *Pteris vittata*. The spore drug group, crude extract drug group and refined extract drug group of *Pteris vittata* were respectively used to conduct spleen deficiency treatment experiments on mice.

[0102] The preparation method of the crude extract of Pteris vittata is as follows: Pteris vittata whole herb is crushed to obtain Pteris vittata whole herb powder. The Pteris vittata whole herb powder is mixed with 70% ethanol solution at a weight ratio of 1:20 and soaked for 30 minutes. Then, it is placed in an electric heating mantle and the temperature is controlled at 50℃ for reflux extraction for 1 hour. The extract is filtered out, and 70% ethanol is added again for reflux extraction under the above conditions. The extracts obtained from the two extractions are combined and placed in a centrifuge. The centrifuge is performed at a speed of 7500 rpm for a centrifugation time of 3 minutes. The supernatant is taken and concentrated to 10 mL by rotary evaporation at 40℃ to prepare a crude extract of Pteris vittata containing a crude drug concentration of 1 g / mL. The extract is then stored in a refrigerator at 4℃ for later use.

[0103] The preparation method of the *Pteris vittata* extract is as follows: A portion of the crude *Pteris vittata* extract and petroleum ether are added to a separatory funnel at a volume ratio of 1:1 and shaken thoroughly. The mixture is then allowed to stand for 15 minutes to allow sufficient precipitation. The lower layer of liquid is collected, which is the first extract of *Pteris vittata*. After discarding the upper layer of liquid, the first extract is poured back into the separatory funnel, and petroleum ether is added at a volume ratio of 1:1. The mixture is shaken thoroughly and allowed to stand for 15 minutes to allow sufficient precipitation. The lower layer of liquid is collected, which is the second extract of *Pteris vittata*. The second extract is concentrated to 10 mL by rotary evaporation at 40°C to prepare a *Pteris vittata* extract with a crude drug concentration of 1 g / mL. This extract is then stored in a refrigerator at 4°C for later use.

[0104] Before conducting the spleen deficiency treatment experiment, it is necessary to prepare a decoction of senna leaves and administer it to mice by gavage to establish a spleen deficiency model.

[0105] The preparation method of the senna leaf decoction is as follows: weigh 25g of senna leaves and add them to 5 times their weight of boiling water. Simmer over low heat for 3.5 minutes and then concentrate under reduced pressure to a decoction with a raw drug content of 1g / mL (1mL of decoction contains 1g of raw senna leaves). This is the senna leaf decoction. After cooling, store it in a refrigerator at 4℃ for later use.

[0106] The experimental method for treating spleen deficiency includes the following steps:

[0107] S1 grouping: 30 SPF-grade male Kunming mice were randomly divided into 5 groups of 6 mice each, namely blank control group, model control group, crude drug extraction group, refined drug extraction group, and spore drug group.

[0108] S2 Spleen Deficiency Model Construction (Modeling): Except for the blank control group, all other groups were administered 0.4 mL of senna leaf decoction by gavage, while the blank control group was administered an equal volume of physiological saline by gavage. Gavage was performed once a day at a fixed time for 4 consecutive days, with the mice fasted every other day. The symptoms of the mice were observed. The criteria for determining the spleen deficiency animal model were formulated with reference to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines": The model was considered to be successfully constructed if the mice had two main symptoms (loose stools, weight loss or slow growth, and huddling together); or one main symptom and two secondary symptoms (listlessness, dry fur, arched back, and easy fatigue).

[0109] S3 Spleen Deficiency Treatment: After the spleen deficiency model was successfully established, each mouse in the crude extract group and the refined extract group was administered 2g of crude extract of Pteris vittata by gavage, and the spore drug group was administered 1g of crude extract of Pteris vittata by gavage. The blank control group and the model control group were administered the same volume of physiological saline by gavage. The spleen deficiency treatment was terminated after 5 consecutive days of gavage in each group.

[0110] S4 Sample Collection: After the treatment for spleen deficiency was completed, the eyeballs were removed 20 minutes later and blood was collected into anticoagulant tubes to obtain whole blood from the mice.

[0111] S5 Indicator Testing:

[0112] 5.1 Food intake and body weight: The daily food intake and body weight of mice in each group were recorded. The food intake results for different drug groups are shown in Table 2 and [Table data missing]. Figure 11 The body weight of mice in different drug groups is shown in Table 3. Figure 12 ;

[0113] 5.2 Complete blood count: Fourteen indicators in mouse whole blood were measured using a fully automated blood analyzer: white blood cell count (WBC), lymphocyte count (Lymph), monocyte count (Mon), neutrophil count (Gran), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelet count (PLT), mean platelet volume (MPV), and platelet distribution width (PDW).

[0114] S3 Data Processing Method: The data obtained in the experiment were processed using SPSS Statistics 23, and an independent samples t-test was performed between groups. Numerical values ​​were expressed as follows: This indicates that the 95% confidence interval for body weight and blood cell count is used as the normal reference range.

[0115] Table 2. Average food intake per mouse in each group during different drug administration periods.

[0116]

[0117] Note: - indicates fasting.

[0118] Table 3. Average body weight of each mouse in each group before and after drug administration.

[0119]

[0120] Note: Different lowercase letters in the same column indicate significant differences (P<0.05); identical letters or no label indicate no significant differences (P>0.05).

[0121] From Table 2, and Figure 11It is evident that, regarding food intake: before the experiment, the food intake of mice in each group was not significantly different. After three days of gavage administration of senna leaf decoction, the food intake of mice in all four groups significantly decreased. Among them, the food intake of mice in the spore drug group and the refined drug group was significantly lower than that of other groups, indicating that gavage administration of senna leaf decoction leads to poor appetite in mice. After successfully replicating the spleen deficiency model, on the first day of gavage administration, the overall food intake of the crude drug group, the refined drug group, and the spore drug group was greater than that of the model control group, with the spore drug group having the highest food intake. In addition, the food intake of mice changed with the increase of the number of days of gavage. On the first and second days, the food intake of the spore drug group was the best, and the food intake reached its highest point on the third day. After the fourth day of gavage administration, the food intake of the spore drug group and the crude drug group were comparable, indicating that the effective components of broken cell wall Pteris vittata spore powder are easily absorbed by mice and can effectively improve the food intake of mice. The mice in the refined drug group had a higher food intake than the model control group before day 3, but a lower intake after day 4, indicating that the refined extraction of the whole plant of Pteris vittata leads to the loss of some active ingredients.

[0122] Regarding weight: as shown in Table 3, and Figure 12 The results showed that, after 4 days of gavage administration of senna leaf decoction, the weight of mice in the model control group was significantly lower than that in the blank control group, proving that the spleen deficiency model was successfully established. There was no significant difference in weight between the spore powder group and the spleen deficiency model group after gavage administration of senna leaf decoction. However, after 5 days of continuous gavage administration of broken-cell wall spore powder, the weight of mice in the spore powder group was significantly higher than that in the spleen deficiency model group. This indicates that broken-cell wall spore powder can help mice recover intestinal function more quickly, correct gastrointestinal dysfunction caused by spleen deficiency, increase food intake, and promote growth. Moreover, the efficacy of broken-cell wall spore powder at a dose of 1 g / kg bw is comparable to that of the whole herb extract of Pteris vittata at a dose of 2 g / kg bw.

[0123] Table 4. Effects of different drug groups on blood routine tests in spleen-deficient mice.

[0124]

[0125] Note: Different lowercase letters in the same row's shoulder label indicate significant differences (P<0.05), while identical letters or no label indicate no significant differences (P>0.05).

[0126] In routine blood tests, white blood cell count (WBC), neutrophil count (Gran), and lymphocyte count (Lymph) are commonly used to reflect the body's level of inflammation. RBC and HGB can indicate whether anemia is present.

[0127] As shown in Table 4, the WBC, Lymph, MON, and Gran indices in the model control group were significantly higher than those in the normal control group (P<0.05), indicating that senna leaf administration to the intestines for 4 days caused acute inflammation. The HCT, MCV, and MCH indices in the model control group were significantly lower than those in the blank control group. The RBC and HGT indices in the model control group mice were not significantly different from those in the blank control group, but were significantly lower than those in the blank control group, indicating that the diarrhea caused by senna leaf in the model control group mice led to malabsorption and a certain degree of anemia.

[0128] Compared with the blank control group, only one item in the crude extract group and the spore drug group showed a significant difference from the blank control group, while the rest showed no significant difference. Two items in the refined extract group showed significant differences from the blank control group, and eight items in the model control group showed significant differences. This indicates that gavage administration of *Pteris vittata* whole herb extract and *Pteris vittata* spore powder improved blood indicators in mice. Specifically, the crude extract group showed significantly higher HCT than the spore drug group, while other indicators were not significantly different from the spore drug group, suggesting that the crude extract and spore powder of *Pteris vittata* whole herb had essentially the same effect on improving blood indicators in spleen-deficient mice. Specifically, the WBC, Lymph, MON, and Gran indicators in the crude extract group, refined extract group, and spore drug group were not significantly different from the blank control group (P>0.05); however, the WBC, MON, and Gran indicators in all three drug groups were significantly lower than those in the model control group (P<0.05), further demonstrating that all three drug groups effectively improved intestinal inflammation. The RBC and HGT indices of the three drug groups were not significantly different from those of the model group, but were significantly higher. The HCT, MCV, and MCH indices of the crude extract drug group and the spore drug group were significantly higher than those of the model control group, indicating that both the crude extract drug group and the spore drug group could significantly improve the anemia status of mice.

[0129] In this embodiment of the invention, mice with spleen deficiency were treated with broken cell wall Pteris vittata spore powder and extract of whole Pteris vittata herb, respectively. Both the spore drug group and the crude extract drug group significantly increased the food intake and body weight of the mice, improved intestinal inflammation and anemia in the mice with spleen deficiency, indicating that broken cell wall Pteris vittata spore powder has the same efficacy as the whole Pteris vittata herb extract. In particular, the effect of Pteris vittata spore powder on improving the body weight, food intake and blood indicators of mice was significantly better than that of the whole Pteris vittata herb extract, indicating that broken cell wall Pteris vittata spore powder can be used directly as a medicine and is an important component of Pteris vittata herb.

[0130] In other embodiments, unbroken cell wall Pteris vittata spore powder also has the same effect, but its effect is significantly lower than that of broken cell wall Pteris vittata spore powder.

[0131] In this embodiment of the invention, only broken cell wall Pteris vittata spore powder was used to treat spleen deficiency in mice. However, based on the above experimental results of spleen deficiency treatment and the existing uses of the whole Pteris vittata herb, it is foreseeable that Pteris vittata spore powder can also be combined with other drugs to prepare drug compositions for treating spleen deficiency syndrome, or for treating diarrhea caused by enteritis, and for improving the body's immunity to prevent diseases.

[0132] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, other structures obtained by using the same or similar technical features as the above embodiments of the present invention are all within the protection scope of the present invention.

Claims

1. Use of the spores of broken cell of Pteridium aquilinum in the preparation of a medicament for the treatment of symptoms of spleen deficiency associated with intestinal inflammatory symptoms and anemic states, characterized in that, The broken cell wall spore powder of Pteris multifida is prepared into a medicine for treating symptoms of spleen deficiency accompanied by intestinal inflammation and anemia.

2. Use according to claim 1, characterized in that, The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: Grinding and cell wall breaking: Place the fern spore powder in a grinding tube, and mix at a material-to-liquid ratio of 1:(80-90) g·mL. -1 Add distilled water in the specified proportion, place the grinding tube containing Pteris vittata spore powder and distilled water in a tissue homogenizer, turn on the tissue homogenizer to lower the temperature to below 4°C, and then grind and break the cell wall to obtain a suspension of Pteris vittata spore powder with broken cell wall. The broken cell wall spore powder of Pteris multifida is prepared into a medicine for treating symptoms of spleen deficiency accompanied by intestinal inflammation and anemia.

3. Use according to claim 2, characterized in that, The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: Take the spore powder of Pteridium aquilinum to centrifugal tube, add distilled water according to the ratio of 1: (80-90) g.mL -1 Immediately, put the ultrasonic cell wall breaking instrument into the liquid surface, and carry out ultrasonic cell wall breaking treatment according to the following parameters: ultrasonic temperature is below 40℃, ultrasonic power is 600-800W, ultrasonic opening time is 20-30 seconds, ultrasonic closing time is 20-30 seconds, and ultrasonic treatment time is 8-10 minutes each time, that is, one cycle of ultrasonic treatment is completed, and after not more than 6 cycles of ultrasonic treatment, drying is carried out at 40-50℃ until the weight is constant, and the ultrasonic cell wall breaking treatment is completed.

4. Use of Pteris Spora powder according to claim 3, characterized in that, The broken cell wall spore powder of Pteris multifida is prepared into a medicine for treating symptoms of spleen deficiency accompanied by intestinal inflammation and anemia. The preparation method of the broken cell wall spore powder of Pteris multifida is as follows:

5. Use according to any one of claims 2 to 4, characterized in that, The preparation method of the broken cell wall spore powder of Pteris multifida is as follows:

6. Use according to claim 5, characterized in that, The broken cell wall spore powder of Pteris multifida is prepared into a medicine for treating symptoms of spleen deficiency accompanied by intestinal inflammation and anemia. The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The broken cell wall spore powder of Pteris multifida is prepared into a medicine for treating symptoms of spleen deficiency accompanied by intestinal inflammation and anemia. The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows: The preparation method of the broken cell wall spore powder of Pteris multifida is as follows

Images