Uses of Pseudobulbus Cremastrae in the Treatment of Osteoarthritis

Abstract

This invention discloses the use of *Cremastra appendiculata* in the treatment of osteoarthritis, belonging to the field of natural active pharmaceutical ingredients. This invention provides the use of *Cremastra appendiculata* alone or its extract in the preparation of a drug for treating osteoarthritis. This invention found that *Cremastra appendiculata* alone or its extract has a certain effect on improving gait in animal models of arthritis pain, significantly increasing the mechanical pain threshold of the model animals, and significantly reducing the weight-bearing difference between the two hind limbs, showing that *Cremastra appendiculata* alone or its extract has a therapeutic effect on osteoarthritis, especially on bone and joint pain and damage caused by osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of natural active pharmaceutical technology, and in particular to the use of Cremastra appendiculata in the treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a degenerative joint disease that severely impacts patients' quality of life, leading to joint pain, deformity, and functional impairment. It also increases the risk of deep vein thrombosis in the lower extremities, hip fractures, and all-cause mortality. It is reported that there are currently over 300 million OA patients worldwide, and the overall prevalence of primary OA in people over 40 years of age in my country has reached as high as 46.3%. Moreover, with the increasing aging of my country's population, the prevalence of OA is showing a gradual upward trend. Knee osteoarthritis (KOA) is the most common site of OA. Pain is a major contributing factor to disability in KOA and severely restricts patients' quality of life, including functional status and mental health. Many patients with chronic pain also experience anxiety and depression. The pathogenesis and mechanisms of OA are not yet fully understood, and there are currently no specific drugs to reverse joint degeneration. Therefore, alleviating joint pain and improving joint function have become the main goals of various OA treatment methods.

[0003] In recent years, traditional Chinese medicine (TCM) has made some progress in the treatment of osteoarthritis (OA). Many clinical studies have shown that TCM can improve symptoms such as limited knee joint movement or pain, reduce adverse reactions, and thus improve patients' quality of life. However, no TCM preparations with proven efficacy have yet been successfully introduced into clinical trials or marketed, providing patients with more treatment options. Summary of the Invention

[0004] To address the aforementioned lack of traditional Chinese medicine preparations that can improve osteoarthritis, this invention provides the use of *Cremastra appendiculata* in the treatment of osteoarthritis. Using *Cremastra appendiculata* alone or its extract as the active component, it exhibits therapeutic effects on osteoarthritis.

[0005] On the one hand, the present invention discloses the use of a single herb, *Cremastra appendiculata* or its extract, in the preparation of a medicament for treating osteoarthritis.

[0006] *Cremastra appendiculata*, also known as Shan Ci Gu, is the dried pseudobulb of one of the orchid species *Cremastra appendiculata* (D. Don) Makino, *Pleione bulbocodioides* (Franch.) Rolfe, or *Pleione yunnanensis* Rolfe, as recorded in the 2020 edition of the Chinese Pharmacopoeia. The former is commonly known as "Mao Ci Gu," while the latter two are commonly known as "Bing Qiu Zi." It is believed to have the effects of "clearing heat and detoxifying, resolving phlegm and dissipating nodules," and is mainly used to treat carbuncles, boils, scrofula, snake and insect bites, and abdominal masses. However, there are no reports on its use in osteoarthritis.

[0007] Through research and experimental studies, the inventors have discovered that single-herb Cremastra appendiculata or its extracts, especially its alcohol extract, have a certain effect on improving the gait of animal models of arthritis pain. It can significantly increase the mechanical pain threshold of the model animals and significantly reduce the weight-bearing difference between the two hind limbs of the model animals, showing that single-herb Cremastra appendiculata or its extracts have a therapeutic effect on osteoarthritis, especially on the pain and damage to the joints caused by osteoarthritis.

[0008] In some protocols, the osteoarthritis referred to is knee osteoarthritis (KOA).

[0009] In some embodiments, the symptoms of osteoarthritis are pain and / or damage to the bones and joints.

[0010] In some embodiments, the *Cremastra appendiculata* is selected from at least one of the following orchid species: *Cremastra appendiculata* (D. Don) Makino, *Pleione bulbocodioides* (Franch.) Rolfe, and *Pleione yunnanensis* Rolfe.

[0011] In some embodiments, the *Pleione bulbocodioides* (Franch.) Rolfe is referred to as *Pleione bulbocodioides* (Franch.) Rolfe.

[0012] On the other hand, the present invention also discloses a *Cremastra appendiculata* extract, which is an alcohol solvent extract of a single *Cremastra appendiculata*.

[0013] In some embodiments, the alcohol solvent is selected from methanol and / or ethanol, and the volume percentage concentration of alcohol in the alcohol solvent is 30%-90%, preferably 50%-65%, more preferably 58%-62%, and most preferably 60%.

[0014] In some embodiments, the alcohol solvent is ethanol.

[0015] In some embodiments, the alcohol solvent is an aqueous ethanol solution with a volume percentage concentration of 50%-65%, preferably 58%-62%, and more preferably 60%.

[0016] In some formulations, the preparation method of the *Cremastra appendiculata* extract is as follows: take *Cremastra appendiculata*, add an alcohol solvent for extraction, obtain the extract, and recover the solvent to obtain the extract.

[0017] On the other hand, the present invention also discloses a method for preparing the above-mentioned *Cremastra appendiculata* extract, comprising the following steps: taking *Cremastra appendiculata*, adding an alcohol solvent for extraction, obtaining an extract, and recovering the solvent to obtain the extract.

[0018] In some methods, extraction is performed using ultrasonic extraction or solvent reflux, and the extraction time can be 15 min to 120 min, for example, 20 min to 40 min, 50 min to 60 min, or 100 min.

[0019] In some methods, extraction is performed using ultrasonic extraction, with an ultrasonic power of 100-800W and a frequency of 20-60kHz.

[0020] In some methods, ultrasonic extraction is used for extraction at a temperature of 5-50°C, preferably 10-40°C, and more preferably 20-30°C.

[0021] In some methods, extraction is performed using solvent reflux, where the reflux temperature is the reflux temperature of the selected alcohol solvent.

[0022] Understandably, the reflux temperature mentioned above refers to maintaining the selected solvent at a boiling point, but the temperature should not be too high, lest it damage the extraction system, such as causing over-boiling or destroying the active ingredients of the medicinal materials. The temperature can be controlled within 5-10°C above the boiling point of the solvent used. For example, if ethanol is used as the alcohol extraction solvent, the reflux temperature can be controlled within the range of 90-110°C.

[0023] In some embodiments, the *Cremastra appendiculata* is a powder of the dried pseudobulbs of *Cremastra appendiculata*, *Cremastra yunnanensis*, or *Cremastra yunnanensis* belonging to the Orchidaceae family.

[0024] In some embodiments, the ratio of the *Cremastra appendiculata* to the alcohol solvent is 8-30 mL of solvent per gram of *Cremastra appendiculata*, preferably 8-12 mL of solvent per gram of *Cremastra appendiculata*, and more preferably 10 mL of solvent per gram of *Cremastra appendiculata*.

[0025] In some schemes, the extraction is performed ≥ 1 time, for example 2 or 3 times.

[0026] On the other hand, the present invention also discloses a pharmaceutical composition comprising a single herb of *Cremastra appendiculata* or the above-mentioned *Cremastra appendiculata* extract, and pharmaceutically acceptable excipients.

[0027] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0028] All reagents and raw materials used in this invention are commercially available, and the methods used in this invention, unless otherwise specified, are conventional methods.

[0029] The positive and progressive effects of this invention are as follows:

[0030] This invention provides the use of a single herb, *Cremastra appendiculata* or its extract, in the preparation of a medicament for treating osteoarthritis. This enriches the range of available medicaments for treating osteoarthritis and exhibits good therapeutic effects, such as improving gait in animal models of arthritis pain and significantly increasing the mechanical pain threshold. Furthermore, the improvement in the mechanical pain threshold is further enhanced with subsequent administration. Simultaneously, it significantly reduces the weight-bearing difference between the two hind limbs in animal models, demonstrating that *Cremastra appendiculata* or its extract has a therapeutic effect on osteoarthritis, particularly on pain and damage to the joints caused by osteoarthritis. Attached Figure Description

[0031] Figure 1 Effect of administration of 60% ethanol extract of *Cremastra appendiculata* on gait score in rats with pMMx+ACLT-induced knee arthritis.

[0032] Figure 2 Bar chart (D30) showing the effect of administration of 60% ethanol extract of *Cremastra appendiculata* on gait scores in rats with pMMx+ACLT-induced knee arthritis.

[0033] Figure 3 Effect of administration of 60% ethanol extract of *Cremastra appendiculata* on pain threshold in pMMX+ACLT-induced knee arthritis in rats

[0034] Figure 4 Effects of administration of 60% ethanol extract of *Cremastra appendiculata* at different time points on the pain threshold of pMMX+ACLT-induced knee arthritis in rats (A: D16, B: D23, C: D30). ).

[0035] Figure 5 Effects of administration of 60% ethanol extract of *Cremastra appendiculata* on pMMX+ACLT-induced knee arthritis and poor weight-bearing capacity of the hind limbs in rats

[0036] Figure 6 Effects of administration of 60% ethanol extract of *Cremastra appendiculata* on pMMX+ACLT-induced knee arthritis and poor weight-bearing capacity of the hind limbs in rats (A: D16, B: D23, C: D30). ). Detailed Implementation

[0037] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0038] Example

[0039] This embodiment investigates the therapeutic effect of *Cremastra appendiculata* extract on osteoarthritis pain and bone and joint damage induced by partial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT) in SD rats.

[0040] I. Test substance

[0041] Test substance: 60% ethanol extract of Pleione bulbocodioides (Franch.) Rolfe (Shan Ci Gu extract).

[0042] Source: Produced and provided by Shanghai Institute of Pharmaceutical Industry Co., Ltd.

[0043] Appearance: Solid powder.

[0044] Storage conditions: Store at 4℃.

[0045] Preparation method: Take the dried pseudobulb powder of Pleione bulbocodioides (Franch.) Rolfe, add solvent at a ratio of 1g of Pleione bulbocodioides to 10mL of 60% ethanol aqueous solution, reflux at 90-110℃ for 30min, filter, extract twice, combine the filtrates, recover the solvent, and dry to obtain Pleione bulbocodioides extract (60% ethanol extract of Pleione bulbocodioides).

[0046] II. Experimental Instruments and Materials

[0047] A. Name: Von Frey filaments, Manufacturer: IITC.

[0048] B. Name: Bipedal Balance Pain Tester, Manufacturer: Linton, Model: Incapacitance Testers.

[0049] C. Name: Respiratory Anesthesia Machine; Manufacturer: MIDMARK Group, USA; Model: VMR.

[0050] III. Experimental Animals and Their Rearing

[0051] 1. Animals

[0052] Species and Strain: Sprague-Dawley (SD) rats

[0053] Grade: Specific Pathogen Free (SPF) level

[0054] Sex: Male

[0055] Source: Shanghai Slack Experimental Animal Co., Ltd.

[0056] Quality Certificate Number of Experimental Animals: 20170005058153 / 20170005058675

[0057] Production License Number of Experimental Animals: SCXK(Shanghai) 2017-0005

[0058] Usage License Number of Experimental Animals: SYXK(Shanghai) 2020-0038

[0059] Number of Animals: 30

[0060] Body Weight of Animals at the Beginning of the Experiment: 200 - 250 g

[0061] Adaptation Time to the Environment: 5 - 7 days

[0062] Animal Numbering Method: Each cage is equipped with an identity card with information such as project number, experimental group, name of the experimental personnel, and animal species. The experimental rats are numbered by scribing on the tail root

[0063] 2. Environment

[0064] The environment in the animal house is maintained at a temperature of 23 ± 2°C, a humidity of 40 - 70%, and a 12-hour light-dark cycle. Five animals are housed in each cage, and the bedding (corn cob bedding, Suzhou Daichuan Trading Co., Ltd.) is changed twice a week

[0065] 3. Food and Drinking Water

[0066] During the experiment, the growth and reproduction feed for SPF-sized rats and mice Co is sterilized and purchased from Beijing Keao Xieli Feed Co., Ltd. The water for experimental animals is high-pressure sterilized filtered water

[0067] 4. Animal Selection and Fasting

[0068] The animals used in the experiment will maintain a healthy condition, and the animals will have free access to food and water during the experiment

[0069] IV. Experimental Methods

[0070] 1. Animal Grouping

[0071] One week after the surgical model was created, the animals were evenly divided into four groups according to the mechanical pain threshold measured by Vonfery: the model group, the sham surgery group, and the duloxetine group each had 8 animals, and the 60% ethanol extract of Pseudobulbus Cremastrae group each had 6 animals.

[0072] Table 1. Animal grouping and administration regimen

[0073]

[0074]

[0075] Note: Both the sham surgery group and the model group were given saline solution, Qd*3w means once a day for three consecutive weeks.

[0076] 2. Modeling and Methods

[0077] After acclimatization, rats were tested for normal pain threshold in the surgical side (right hind limb) knee joint using the Von Frey (Up-Down test) and bipedal balance test before modeling. Rats were then anesthetized with isoflurane, and the knee joint was shaved and disinfected. An incision was made near the medial side of the right knee, exposing the joint cavity with the knee in maximum flexion. The medial meniscus was separated, the medial 1 / 3 of the meniscus was removed, and the anterior cruciate ligament was cut. In the sham-operated group, only the skin was incised, the joint cavity was opened, and then sutured. The day of modeling was recorded as D0. One week after modeling (D7), rats were evenly grouped according to their Von Frey response values. After grouping, drug treatment was initiated via gavage (once daily).

[0078] The changes in knee pain threshold of each group of rats were measured using the Von Frey test on D1, D15, D16, D23 and D30; the weight-bearing capacity of the rats' lower limbs was measured using a bipedal balance analgesia device on D1, D16, D23 and D30; and the gait of the rats was scored according to the Coderre scoring method on D16, D23 and D30.

[0079] 3. Coderre method gait scoring

[0080] Coderre Gait Scoring Standard: Grade 0: Normal walking, 0 points; Grade I: Mild limping, with slight bending below the knee, 2 points; Grade II: Limping, with the affected limb able to touch the ground, 4 points; Grade III: Severe limping, with the affected limb off the ground and walking on three feet, 6 points.

[0081] 4. Mechanical pain threshold measurement

[0082] The up-down method was used for measurement: Rats were placed in a transparent enclosure with a sieve-like metal bottom, and allowed to adapt for approximately 15 minutes until their spontaneous exploration and grooming activities ceased. A Von Frey probe was used to vertically stimulate the middle of the sole of each rat's hind paw, causing it to slightly sway in an S-shape for 5 seconds, and the paw withdrawal response was observed. A positive response was defined as immediate and rapid withdrawal, leg lifting, or paw licking after stimulation, or immediate withdrawal after probe removal. Measurements were taken approximately 10 seconds apart; no response after probe stimulation was considered a negative response.

[0083] The rat paws were stimulated with a series of probes (0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15g). Starting with a 2g probe, if a withdrawal response occurred, it was recorded as X, and a weaker probe (1.4g) was used. If no withdrawal response occurred, it was recorded as O, and a stronger probe (4g) was used. The same method was continued for each subsequent test, using a continuous sequence of probes, avoiding skipping steps. Each response was recorded. When the first OX or XO response appeared, four more tests were performed to obtain a sequence, such as OXOXOX. For rats that did not show a withdrawal response even with a 15g probe, the 50% withdrawal threshold was recorded as 15g; if a withdrawal response still occurred with a 0.4g probe, the 50% withdrawal threshold was recorded as 0.4g. If neither the 15g nor 0.4g probes were used, the following formula was used for calculation:

[0084] 50% MWT(g) = (10 [Xf+kδ] ) / 10000

[0085] 50% MWT indicates that rats have approximately a 50% chance of foot withdrawal at this stimulus intensity. Xf = the value of the last Von Frey probe used (in logarithmic units). K value: Refer to the corresponding value for each gram probe in the instrument's accompanying table based on the measured sequence. δ is the average difference between the series of stimuli (in logarithmic units, 0.224 here).

[0086] The pain threshold elevation rate is calculated using the following formula:

[0087] Pain threshold improvement rate (%) = (50% MWT in the treatment group - 50% MWT in the model group) / 50% MWT in the model group × 100%.

[0088] 5. Measurement of the balance load-bearing value of both feet

[0089] The animal was placed inside the plexiglass enclosure of the bipedal balance pain device. After the animal completed its exploration behavior, the rat's forelimbs were placed on the inclined plane of the device, and its hind limbs were placed on the left and right balance plates respectively. When the animal was basically inactive, the button was pressed, and the average load-bearing force of the left and right hind limbs was measured over 5 seconds. Each animal was measured 3 times, with a 10-second interval between each measurement.

[0090] V. Results

[0091] 1. Observation of general clinical symptoms in rats after drug administration

[0092] No gross abnormalities were observed in any of the animals after a single gavage administration of the test substance.

[0093] 2. Effect of 60% ethanol extract of *Cremastra appendiculata* on body weight in pMMX+ACLT-induced inflammatory pain rats.

[0094] The results are shown in Table 2 below. The results show that after administration of each test substance, the body weight of the animals in each group increased over time. There were no significant differences between the groups.

[0095] Table 2. Effects of administration of 60% ethanol extract of *Cremastra appendiculata* on body weight of animals in each group (Mean ± SD)

[0096]

[0097] 3. Effects of 60% ethanol extract of *Cremastra appendiculata* on gait in rats with pMMx+ACLT-induced inflammatory pain.

[0098] The results are shown in Table 3 below. Figure 1-2 In the figure, Sham represents the sham surgery group, Model represents the model group, Dulovetine represents the duloxetine group, and SCG represents the 60% ethanol extract of Pseudobulbus Cremastrae var. sarcodactylis.

[0099] Table 3. Effects of administration of 60% ethanol extract of *Cremastra appendiculata* on gait scores of animals in each group.

[0100]

[0101] Note: ###P<0.001 vs sham surgery group, ##P<0.01 vs sham surgery group, *P<0.05 vs model group.

[0102] The results showed that rats in all surgical groups developed lameness, which lessened over time. Compared with the model group, the gait scores of rats in the positive control group (duloxetine) were all greater than 0.05 (P value), while the gait score of rats in the 60% ethanol extract group of *Pseudobulbus Cremastrae* showed a significant decrease at D30 due to inflammatory pain (P value less than 0.05), indicating that it could effectively improve the gait of rats with knee arthritis induced by pMMx+ACLT after three weeks of administration.

[0103] 4. Effect of 60% ethanol extract of *Cremastra appendiculata* on the mechanical pain threshold of pMMx+ACLT-induced inflammatory pain in rats.

[0104] The results are shown in Tables 4-5 and 5. Figure 3-4As shown in the figure, Sham represents the sham surgery group, Model represents the model group, Dulovetine represents the duloxetine group, and SCG represents the 60% ethanol extract of Pseudobulbus Cremastrae var. sarcodactylis.

[0105] Table 4. Effects of administration of 60% ethanol extract of *Cremastra appendiculata* on pain threshold in pMMX+ACLT-induced knee arthritis in rats

[0106]

[0107] Note: ###P<0.001 vs sham surgery group, *P<0.05, **P<0.01 vs model group.

[0108] Table 5. Effect of administration of 60% ethanol extract of *Cremastra appendiculata* on the rate of increase in pain threshold for pMMX+ACLT-induced inflammatory pain in rats.

[0109]

[0110]

[0111] The results showed that administration of 600 mg / kg of *Pseudobulbus Cremastrae* 60% ethanol extract increased the pain threshold of inflammatory pain in D16, D23, and D30 rats by 66.0%, 137.2%, and 268.6%, respectively. Administration of the positive control drug duloxetine at 5 mg / kg increased the pain threshold of inflammatory pain in D16, D23, and D30 rats by 53.2%, 90.1%, and 108.1%, respectively.

[0112] Pain threshold-time curves revealed that the improvement in mechanical pain threshold by the 60% ethanol extract of *Cremastra appendiculata* and the positive control drug increased with increasing dosing frequency. At D23 and D30, compared to the model group, both the 60% ethanol extract of *Cremastra appendiculata* and the duloxetine group significantly increased the 50% paw withdrawal threshold for pMMx+ACLT-induced inflammatory pain in SD rats. Trend analysis indicated that the 60% ethanol extract of *Cremastra appendiculata* was superior to duloxetine in improving the 50% paw withdrawal threshold for pMMx+ACLT-induced inflammatory pain in SD rats.

[0113] 5. Effect of 60% ethanol extract of *Cremastra appendiculata* on the difference in weight-bearing capacity of the hind limbs in rats with pMMX+ACLT-induced inflammatory pain.

[0114] The results are shown in Table 6 and Figure 5-6 As shown in the figure, Sham represents the sham surgery group, Model represents the model group, Dulovetine represents the duloxetine group, and SCG represents the 60% ethanol extract of Pseudobulbus Cremastrae var. sarcodactylis.

[0115] Table 6. Effects of administration of 60% ethanol extract of *Cremastra appendiculata* on pMMX+ACLT-induced knee arthritis and difference in hindlimb weight-bearing capacity in rats.

[0116]

[0117] Note: ###P<0.001 vs. sham surgery group, ***P<0.001 vs. model group.

[0118] The results showed that after gavage administration of 60% ethanol extract of Pseudobulbus Cremastrae to the animals, compared with the model group, the difference in weight-bearing between the two hind limbs in rats with knee joint inflammatory pain was significantly improved at D23 and D30. 60% ethanol extract of Pseudobulbus Cremastrae could significantly reduce the difference in weight-bearing between the two hind limbs in the model animals.

[0119] VI. Conclusion

[0120] In a pMMX (partial meniscectomy) plus ACLT (anterior cruciate ligament transection) induced knee arthritis pain model in SD rats, administration of a 60% ethanol extract of *Cremastra appendiculata* showed that it improved gait in the model animals; it significantly increased the mechanical pain threshold, and this improvement was further enhanced with repeated administration. Simultaneously, it significantly reduced the weight-bearing difference between the two hind limbs in the model animals.

[0121] In summary, this embodiment demonstrates that the test substance (60% ethanol extract of Pseudobulbus Cremastrae spp.) has a good therapeutic effect on osteoarthritis pain and bone and joint damage induced by partial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT) in SD rats by administering the test substance (60% ethanol extract of Pseudobulbus Cremastrae spp.) once daily for three consecutive weeks via gavage.

Claims

1. The use of *Cremastra appendiculata* extract as the sole active ingredient in the preparation of a medicament for treating knee osteoarthritis, characterized in that... The extract of *Cremastra appendiculata* is an alcohol solvent extract of *Cremastra appendiculata*. The *Pleione bulbocodioides* mentioned is *Pleione bulbocodioides* (Franch.) Rolfe; the alcohol solvent contains 60% alcohol by volume.

2. The use according to claim 1, characterized in that, The symptoms of knee osteoarthritis are pain and / or injury to the joints.

3. The use according to claim 1, characterized in that, The alcohol solvent is selected from methanol and / or ethanol.

4. The use according to claim 3, characterized in that, The alcohol solvent is ethanol.

5. The use according to claim 1, characterized in that, The preparation method of the *Cremastra appendiculata* extract is as follows: take *Cremastra appendiculata*, add the alcohol solvent for extraction, obtain the extract, and recover the solvent to obtain the extract.

6. The use according to claim 5, characterized in that, One or more of the following conditions must be met: (1) Extraction was performed by ultrasonic extraction or solvent reflux method, and the extraction time was 15 min-120 min; (2) Extraction is performed by ultrasonic extraction, wherein the ultrasonic power is 100-800W and the frequency is 20-60kHz; (3) Extraction was performed using ultrasonic extraction at a temperature of 5-50℃; (4) The aforementioned *Cremastra appendiculata* is the powder of the dried pseudobulb of the orchid *Cremastra appendiculata*. (5) The ratio of the material to the alcohol solvent is 8-30 mL of solvent per gram of *Cremastra appendiculata*. (6) The number of extractions is ≥ 1.

7. The use according to claim 6, characterized in that, One or more of the following conditions must be met: (1) Extraction is carried out by ultrasonic extraction or solvent reflux method, and the extraction time is 20min-40min, 50min-60min or 100min; (2) When using ultrasonic extraction, the extraction temperature is 10-40℃; (3) The ratio of the material to the alcohol solvent is 8-12 mL of solvent per gram of *Cremastra appendiculata*. (4) The extraction is performed 2 or 3 times.

8. The use according to claim 7, characterized in that, When using ultrasonic extraction, the extraction temperature is 20-30℃; And / or, the ratio of the *Cremastra appendiculata* to the alcohol solvent is 10 mL of solvent per gram of *Cremastra appendiculata* medicinal material.

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