A herb extract for treating kidney stones, its preparation method and application

Abstract

This invention provides an extract of *Cimicifuga foetida*, made from *Cimicifuga foetida* produced in Guizhou Province, using an intermittent warm percolation method for water extraction. The extract contains 10%-25% cimicifugoside, 3%-15% iso-cimicifugoside, and 20%-50% plantago asiatica glycoside. The preparation method and applications are also disclosed. The *Cimicifuga foetida* extract provided by this invention has a high content of active ingredients and significant anti-inflammatory and litholytic pharmacological activities. It can be used as a raw material for the treatment and adjuvant treatment of cholecystitis, gallstones, hydronephrosis, kidney stones, nephritis, bladder stones, and uric acid stones. It can also be used as an intermediate raw material for the separation and purification of plantago asiatica glycoside, cimicifugoside, and iso-cimicifugoside chemical standards or reference standards. The preparation method provided by this invention effectively solves the problem of extraction and enrichment of active ingredients in *Cimicifuga foetida*, and has advantages such as low production cost, ease of large-scale production, and stable and controllable quality.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, and in particular to an extract of Incarvillea arguta (Royle) Royle, its preparation method, and its application. Background Technology

[0002] Gallstones are a "disease of affluence," primarily involving urinary tract stones, liver stones, and gallstones. When they occur, the pain is excruciating; worsening conditions can lead to cholecystitis, hydronephrosis, and even gallbladder cancer. Gout is also often accompanied by uric acid stones. With improved living standards, the incidence of gallstones is gradually increasing. Current treatments for gallstones include surgery, extracorporeal shock wave lithotripsy (e.g., ultrasound), and medication. Medication can reduce patient suffering and is less expensive, thus alleviating the mental and financial burden on patients. Currently, there are many medications for treating gallstones, such as "Paishi Granules," "Danle Capsules (Tablets)," "Jinqiancao Granules," and "Shenshitong Granules," all of which have some effect. However, their formulations are complex, with many ingredients and unclear mechanisms of action, making quality control difficult. Therefore, developing highly effective, safe, and quality-controlled drugs for treating gallstones is a current research hotspot in this field.

[0003] Incarvillea argute(Royle)Royle, a medicinal herb commonly used by ethnic minorities in Guizhou, has the effects of clearing heat and detoxifying, promoting qi circulation and removing blood stasis, and promoting diuresis and expelling stones. It is used to treat cholecystitis, gallstones, kidney stones, hydronephrosis, bladder stones and other diseases (Bao Jun, Ran Maoxiong, Research and Development of Miao Medicine in Guizhou, Guizhou Science and Technology Press, 2002; Guizhou Provincial Drug Administration, ed., Quality Standards of Traditional Chinese Medicine and Ethnic Medicine in Guizhou Province, 2003). When we systematically studied the chemical composition of *Gnaphalium affine*, we found that the main chemical components of *Gnaphalium affine* produced in Guizhou Province are plantago ether glycoside, 5-hydroxy-4′,7-dimethoxyflavone, oleanolic acid, and other compounds, which are quite different from the chemical composition of *Gnaphalium affine* (also known as *Gnaphalium affine*) produced in northern my country (1. Yu Zhengwen, Zhu Haiyan, Yang Xiaosheng, et al., “Study on the chemical composition of *Gnaphalium affine* and its effect on promoting the differentiation of PC-12 cells (I)”, Chinese Journal of Traditional Chinese Medicine, 2005, 30(17):1335~1338; 2. Zhou Daying, Yang Xiaosheng, et al., “Study on the chemical composition of *Gnaphalium affine* produced in Guizhou”, Natural Product Research and Development, 2007, 5(19):807~808). This can explain why *Gnaphalium affine* (*Gnaphalium affine*) produced in Guizhou Province is used to treat calculi, while it is used to treat hepatitis in other provinces. In addition, the study found that the water extract and alcohol extract of *Gynostemma pentaphyllum* had comparable effects in dissolving stones, anti-inflammatory and analgesic properties in vitro, indicating that the active ingredients should be relatively polar compounds that can be dissolved in both ethanol and water.

[0004] Literature review revealed that drugs like "Jieshiqing Granules," which use *Gynostemma pentaphyllum* as the main ingredient for treating kidney stones, are compound preparations, and the effective components for treating kidney stones are not clearly identified. CN101693731A discloses that plantago ether glycosides extracted and purified from *Gynostemma pentaphyllum* possess anti-inflammatory and analgesic activity and in vitro stone-dissolving effects. However, further in-depth research by the inventors revealed that plantago ether glycosides are not the only component in *Gynostemma pentaphyllum* with a clear therapeutic effect on kidney stones. Summary of the Invention

[0005] This invention discloses an extract of *Gnaphalium affine* rich in plantain glycoside, cimicifuga glycoside, and iso-cimicifuga glycoside, as well as its preparation method and applications. Addressing the shortcomings of existing water-boiling and alcohol precipitation techniques, this invention employs intermittent warm maceration percolation for water extraction, combined with macroporous resin adsorption to enrich the active ingredients. This solves the problems of extracting effective components and separating impurities from *Gnaphalium affine*. The extract has a high content of active ingredients, and the synergistic effect between these components gives it significant anti-inflammatory, analgesic, litholytic, and litholytic effects.

[0006] To achieve the above technical objectives, the technical solution of the present invention is as follows:

[0007] A *Gnaphalium affine* extract is prepared by water extraction using intermittent warm percolation method from *Gnaphalium affine* produced in Guizhou Province. The extract contains 10%-25% *Cimicifuga foetida* glycosides, 3%-15% *Cimicifuga foetida* glycosides, and 20%-50% *Plantago asiatica* glycosides.

[0008] The aforementioned method for preparing the extract of *Gnaphalium affine* involves pulverizing dried *Gnaphalium affine*, adding 4-12 times its weight of deionized water, and extracting with water at 45-80°C using an intermittent warm percolation method until the exudate becomes lighter in color. The exudate is then filtered, and the filtrates are combined and allowed to cool to room temperature before adsorption by macroporous resin column chromatography. The elution is first performed with 5-10 column volumes of deionized water, followed by 4-8 column volumes of 20%-80% ethanol. The ethanol eluent is collected, the ethanol is removed, and the residue is dried to obtain the *Gnaphalium affine* extract.

[0009] In the aforementioned method for preparing the extract of *Gnaphalium affine*, the intermittent warm immersion percolation method is used for water extraction 2-4 times, with each extraction lasting 2-4 hours; the weight ratio of macroporous resin to the raw herb *Gnaphalium affine* is 1:1-4:1.

[0010] The aforementioned method for preparing the extract of *Gnaphalium affine* involves pulverizing dried *Gnaphalium affine* and passing it through a 20-100 mesh sieve. Then, 6-9 times the weight of the *Gnaphalium affine* in deionized water is added. The mixture is extracted three times at 60-80°C using an intermittent warm percolation method, with each extraction lasting 3 hours. The percolate is filtered, and the filtrates are combined. After being cooled to room temperature, the mixture is adsorbed using macroporous resin column chromatography. The weight ratio of macroporous resin to the raw *Gnaphalium affine* is 1.5:1-3:1. The mixture is first eluted with 8 column volumes of deionized water, then eluted with 5-6 column volumes of 35%-65% ethanol. The ethanol eluent is collected, concentrated under reduced pressure to remove the ethanol, and the residue is dried to obtain the *Gnaphalium affine* extract.

[0011] The aforementioned method for preparing the extract of *Gnaphalium affine* refers to *Gnaphalium affine* from Guizhou Province, specifically the whole herb or above-ground parts; the warm maceration percolation water extraction method employs a percolation device with a controllable temperature jacket; and the macroporous resin is of type D101, D100, DA-201, H103, S-8, HPD-100, HPD-400, HPD-600, DM-301, or AB-8.

[0012] In the aforementioned method for preparing the extract of *Gnaphalium affine*, the residue is dried by vacuum drying, microwave vacuum drying, spray drying, or freeze drying.

[0013] As mentioned above, the use of *Gynostemma pentaphyllum* extract in the preparation of drugs for treating kidney stones.

[0014] The aforementioned application of the herb extract for treating gallstones refers to gallstones, cholecystitis with gallstones, hydronephrosis, kidney stones, ureteral stones, bladder stones, or uric acid stones.

[0015] As mentioned above, the extract of *Gnaphalium affine* is used as a raw material to prepare chemical standards or reference standards for plantago ether glycosides, cimicifuga glycosides, and heterocimicifuga glycosides.

[0016] The aforementioned application of the extract of *Gnaphalium affine* involves fractional processing using atmospheric pressure silica gel column chromatography, followed by purification using reduced pressure / pressurized reversed-phase column chromatography, preparative high-performance liquid chromatography, or high-performance countercurrent chromatography. The separation material is silica gel or reversed-phase material RP-8 / RP-18. The eluent is one or more of petroleum ether, chloroform, ethyl acetate, acetone, methanol, ethanol, acetonitrile, and water.

[0017] The *Gnaphalium affine* extract provided by this invention is controlled by the content of cimicifugoside, isocifugoside, and plantain ether glycoside, and the content of these three compounds is detected by high performance liquid chromatography. The extract exhibits the following pharmacological activities: in vitro litholytic activity, protective effect against acetic acid-induced writhing response in mice, inhibitory effect against xylene-induced ear swelling in mice, and inhibitory effect against acetic acid-induced increase in peritoneal capillary permeability in mice, indicating that the extract has strong anti-inflammatory, analgesic, litholytic, and litholytic effects.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention discloses an extract of *Gnaphalium affine*, its preparation method, and its application. Literature review revealed that drugs such as "Jieshiqing Granules," which use *Gnaphalium affine* as the main ingredient to treat kidney stones, are compound preparations, but the active pharmaceutical ingredients for treating kidney stones are not clearly defined. CN101693731A discloses that *Gnaphalium affine* contains plantago ether glycosides, which have anti-inflammatory, analgesic, and in vitro stone-dissolving effects. Based on in-depth research on *Gnaphalium affine*, this invention discovered that in addition to plantago ether glycosides, the active pharmaceutical ingredients for treating kidney stones in *Gnaphalium affine* also include cimicifuga glycosides and iso-cimicifuga glycosides. An extract of *Gnaphalium affine* (effective fraction) rich in the active pharmaceutical ingredients cimicifuga glycosides, iso-cimicifuga glycosides, and plantago ether glycosides was obtained, exhibiting significant anti-inflammatory, analgesic, stone-dissolving, and stone-expelling effects.

[0020] 1. This invention employs an intermittent warm immersion percolation water extraction technique, which can effectively extract effective substances such as cimicifuga glycosides, heterocimicifuga glycosides, and plantain ether glycosides from *Gnaphalium affine*, while avoiding the loss of medicinal components due to excessively high extraction temperatures. The process is simple, low-cost, and easily scalable for large-scale production.

[0021] 2. The extract prepared by the method of the present invention is not concentrated in the conventional way, but is directly adsorbed, enriched and purified by macroporous resin, which has the characteristics of energy saving and consumption reduction and easy to scale up preparation.

[0022] 3. The preparation method of this invention solves the problem of extraction and enrichment of effective components in *Gnaphalium affine*. The resulting *Gnaphalium affine* extract has a high content of effective components, with the total content of the three main active ingredients exceeding 50%, which meets the requirements of Class 5 new Chinese medicine for the amount of controllable components. Furthermore, the synergistic effect among the main active ingredients, namely, *Cimicifuga foetida* glycoside, *Cimicifuga foetida* glycoside, and *Plantago asiatica* glycoside, makes the anti-inflammatory and analgesic activity and in vitro litholytic effect of the extract of this invention significantly better than that of *Plantago asiatica* glycoside monomer.

[0023] 4. The extract of *Heliotropium indicum* provided by this invention has great potential for the treatment and adjuvant treatment of stone diseases, such as gallstones, cholecystitis with stones, hydronephrosis, kidney stones, ureteral stones, bladder stones, and uric acid stones. Detailed Implementation

[0024] Example 1: A method for preparing an extract of *Gnaphalium affine*:

[0025] Take 1 kg of dried whole herb of *Gynostemma pentaphyllum* from Guizhou, crush it and pass it through a 20-mesh sieve. Place it in a percolation device with a temperature-controlled jacket, add 12 times its weight of deionized water and soak it three times at 80°C for 3 hours each time. Filter, combine the filtrates, and let them stand at room temperature. Adsorb the solution using 3 kg of D101 macroporous resin (resin to raw herb ratio = 3:1). First wash with 10 column volumes of deionized water, then elute with 6 column volumes of 60% ethanol. Collect the ethanol eluent, concentrate under reduced pressure to recover the ethanol, and vacuum dry the residue to obtain *Gynostemma pentaphyllum* extract, which contains 15% cimicifuga glycoside, 10% iso-cimicifuga glycoside, and 35% plantain ether glycoside.

[0026] Example 2: A method for preparing an extract of *Gnaphalium affine*:

[0027] Take 1 kg of the above-ground parts of *Gnaphalium affine* from Guizhou, chop it, and place it in a percolation device with a temperature-controlled jacket. Add 7 times its weight of deionized water and soak at 70°C for 4 times, 2 hours each time. Filter, combine the filtrates, and let them stand at room temperature. Then, adsorb and enrich the extract through a 2 kg DA-201 macroporous resin column (resin dosage: raw drug dosage = 2:1). First, wash with 6 column volumes of deionized water, then elute with 6 column volumes of 50% ethanol. Collect the ethanol eluent, concentrate under reduced pressure to recover the ethanol, and freeze-dry the residue to obtain *Gnaphalium affine* extract, which contains 20% cimicifuga glycoside, 12% iso-cimicifuga glycoside, and 38% plantain ether glycoside.

[0028] Example 3: A method for preparing an extract of *Gnaphalium affine*:

[0029] Take 1 kg of dried aerial parts of the medicinal herb *Cynanchum paniculatum* from Guizhou, pulverize it, and pass it through a 100-mesh sieve. Use an intermittent warm soaking percolation method, add 9 times its weight of deionized water, and soak at 60℃ three times, 2 hours each time. Filter, combine the filtrates, and let them stand at room temperature. Adsorb the residue onto 1.5 kg of HPD-100 macroporous resin (resin dosage: raw herb dosage = 1.5:1). First wash with 8 column volumes of deionized water, then elute with 5 column volumes of 40% ethanol. Collect the ethanol eluent, recover the ethanol, and vacuum dry the residue to obtain the extract of *Cynanchum paniculatum*, which contains 15% cimicifuga glycoside, 8% iso-cimicifuga glycoside, and 30% plantain ether glycoside.

[0030] Example 4: A method for preparing an extract of *Gnaphalium affine*:

[0031] Take 1 kg of dried whole herb of *Cynanchum paniculatum*, chop it, and use an intermittent warm soaking percolation method. Add 6 times its weight of deionized water and soak twice at 80℃ for 4 hours each time. Filter, combine the filtrates, and let them stand at room temperature. Adsorb the solution using 1 kg of S-8 macroporous resin (resin dosage: raw herb dosage = 1:1). First wash with 5 column volumes of deionized water, then elute with 4 column volumes of 35% ethanol. Collect the ethanol eluent, recover the ethanol, and microwave-dry the residue under vacuum to obtain *Cynanchum paniculatum* extract, which contains 10% cimicifuga glycoside, 5% iso-cimicifuga glycoside, and 45% plantain ether glycoside.

[0032] Example 5: A method for preparing an extract of *Gnaphalium affine*:

[0033] Take 1 kg of dried aerial parts of the medicinal herb *Cynanchum paniculatum* from Guizhou, pulverize it, and pass it through a 60-mesh sieve. Use an intermittent warm soaking percolation method, add 4 times its weight of deionized water, and soak at 45°C 4 times, 4 hours each time. Filter, combine the filtrates, and place them at room temperature. Adsorb the residue onto 4 kg of H103 macroporous resin (resin:crude drug = 4:1). First wash with 7 column volumes of deionized water, then elute with 8 column volumes of 65% ethanol. Collect the ethanol eluent, recover the ethanol, and dry the residue under reduced pressure and vacuum to obtain the extract of *Cynanchum paniculatum*, which contains 18% cimicifuga glycoside, 12% iso-cimicifuga glycoside, and 41% plantain ether glycoside.

[0034] Example 6: Preparation of plantago ether glycoside, phycocyanin-like glycoside, and isophycocyanin monomeric compounds

[0035] The extract (effective component) of *Gnaphalium affine* obtained in Example 2 was separated by 200-mesh silica gel column chromatography with chloroform / methanol (8:1) as eluent to obtain fraction 1. The silica gel column was then eluted with methanol, and the resulting fraction was further eluted by polyamide column chromatography with methanol / water as eluent to obtain fraction 2. Fraction 1 was then eluted by reversed-phase silica gel R-18 column chromatography with methanol / water (2:1) as eluent to obtain the monomeric plantago ether glycoside. Fraction 2 was further purified by HPLC separation with acetonitrile / water (3:1) as eluent to obtain monomeric cimicifuga glycoside and iso-cimicifuga glycoside. The spectral data are as follows:

[0036] Plantago ether glycoside: Molecular formula is C 16 H 24 O 9, ESI m / z: 623, 1 H-NMR (400MHz, CD3OD) δ: (9.24, 1H, s), (7.40, 1H, s), (5.86, 1H, s), (3.62–5.86, glucose protons), (0.94, 3H, d, J = 6.9Hz). 13 C-NMR(100MHz,CD3OD)δ:192.6(C-10),164.3(C-3),126.3(C-4),99.8(C-1'),96.8(C-1),78.5(C-5'),77.4(C-3' ),74.3(C-2'),73.4(C-5),71.7(C-4'),62.8(C-6'),52.3(C-9),38.9(C-6),35.1(C-8),33.1(C-7),16.4(C-11).

[0037] Cimicifugoside: Molecular formula is C 29 H 36 O 15,ESI m / z: 623, 1 ¹H-NMR (400 MHz, CD₃OD) δ: (7.59, 1H, d, J = 15.9 Hz), (7.05, d, 1H, J = 1.9 Hz), (6.88, 1H, m), (6.77, 1H, d, J = 8.1 Hz), (6.69, 1H, d, J = 2.0 Hz), (6.67, 1H, d, J = 8.0 Hz), (6.56, 1H, m), (6.27, 1H, d, J = 15.9 Hz), (5.18, 1H, d, J = 1.5 Hz, rhamnose-1), 4.91 (1H, dd, J = 9.5, 9.5 Hz, glucose-4), (4.37, 1H, d, J = 8.0 Hz), (4.04, 1H, ddd, J = 9.0, 8.0, 7.0), (3.91, 1H, dd, J = 3.6 1.6 Hz), (3.81, 1H, dd, J = 9.5, 9.5 Hz), (3.72, 1H, ddd, J = 9.0, 8.0, 7.0 Hz), (3.51 - 3.63, 5H, m), (3.80, 1H, dd, J = 13.9, 5.8 Hz), (3.28, 1H, dd, J = 9.4, 9.3 Hz), (3.28, 1H, dd, J = 8.1, 7.1 Hz), (1.08, 3H, d, J = 6.1 Hz), 13 ¹³C (100 MHz, CD₃OD) δ: AgLycome moiety: 131.4 (C-1), 116.9 (C-2), 146.1 (C-3), 144.6 (C-4), 116.2 (C-5), 121.3 (C-6), 72.3 (C-α), 36.5 (C-β); CaffeoyL moiety: 127.4 (C-1), 115.0 (C-2), 147.8 (C-3), 149.6 (C-4), 116.5 (C-5), 123.1 (C-6), 115.8 (C-α), 148.0 (C-β), 168.2 (C-γ); GLucose moiety: 104.3 (C-1), 76.3 (C-2), 81.8 (C-3), 70.3 (C-4), 76.2 (C-5), 62.3 (C-6); Rhamnose moiety: 103.0 (C-1), 72.3 (C-2), 72.2 (C-3), 73.9 (C-4), 70.5 (C-5), 18.7 (C-6).

[0038] Acteoside: Molecular formula C 29 H 36 O 15, ESI m / z: 623, 1H-NMR (400MHz, CD3OD) δ: (7.54, 1H, d, J = 15.0Hz), (7.02, d, 1H, J = 2.0Hz), (6. 88,1H,dd,J=8.0Hz), (6.75,1H,d,J=10.0Hz), (6.65,1H,d,J=5.0Hz), (6.62, 1H,d,J=8.0Hz),(6.53,1H,dd,J=8.0Hz),(6.28,1H,d,J=15.0Hz),(5.16,1H, brs), (4.31,1H,d,J=8.0Hz), (2.46,2H,t,J=7.6Hz), (1.23,3H,d,J=6.0Hz).

[0039] 13 C(100MHz, CD3OD)δ:AgLycome moiety:131.3(C-1),115.0(C-2),146.1(C-3),144.6(C-4),116.3(C-5),121.2(C-6),72.2(C-α),36.6(C-β); CaffeoyL moiety:127.6(C-1),115.0(C-2),146.8(C-3),149.6(C-4),116.5(C-5),123.1(C-6),114.8(C-α),148.0(C-β),168.2(C-γ); GLucose moiety:104.3(C-1),75.7(C-2),83.8(C-3),70.3(C-4),76.5(C-5),64.3(C-6); Rhamnose Moiety:103.0(C-1),72.3(C-2),72.2(C-3),73.9(C-4),70.1(C-5),17.8(C-6).

[0040] The three monomeric compounds obtained can be used as chemical standards or reference standards for psyllium ether glycoside, cimicifuga glycoside, and iso-cimicifuga glycoside.

[0041] The method for determining the content of medicinal components in the extract of *Gnaphalium affine* according to this invention is as follows:

[0042] Chromatographic conditions and system suitability tests were conducted using WeLchrom-C18 as the stationary phase; methanol-water as the mobile phase; isocratic elution for 20 min (A 60% - B 40%); flow rate of 1 mL / min; column temperature of 30℃; and detection wavelengths of 241 nm and 333 nm.

[0043] Preparation of reference solutions: Take appropriate amounts of citric acid, isocitric acid, and psyllium ether reference standards, accurately weigh them, and add them to the mobile phase to prepare solutions of 350 μg / mL, 100 μg / mL, and 20 μg / mL, respectively.

[0044] Preparation of the test solution: Accurately weigh about 3g of *Gnaphalium affine* extract powder and place it in a stoppered conical flask. Add 30mL of purified water and incubate at 70℃ twice for 40min each time. Filter, combine the filtrates, concentrate, and transfer the concentrate to a 25mL volumetric flask. Dilute to the mark with purified water and shake well to obtain the mother liquor. Accurately measure 1.0mL of the mother liquor and place it in a 10mL volumetric flask. Dilute to the mark with water, shake well, filter, and collect the filtrate to obtain the test solution.

[0045] According to the analysis, the mass content of phycocyanin-like glycosides, isophycocyanin-like glycosides, and plantago asiatica glycosides in the extract of *Gnaphalium affine* of this invention is 10%–25%, 3%–15%, and 20%–50%, respectively.

[0046] Experimental Example: Anti-inflammatory and litholytic effect of *Lithospermum erythrorhizon* extract

[0047] 1. Tail-flicking test of *Gynostemma pentaphyllum* extract in mice

[0048] Mice were screened using the tail pain threshold. One-third of the mouse's tail was immersed in a 48°C constant-temperature water bath. The time from when the mouse's tail entered the water to when it retracted was defined as the tail-flick latency (TFL), i.e., the mouse pain threshold. Mice that did not flick their tails within 10 seconds were discarded. Sixty qualified Kunming mice (half male and half female) were randomly divided into six groups of ten mice each: a blank control group, an aspirin 0.1 g / kg group, and groups receiving *Gnaphalium affine* extract (Example 2) at 0.13, 0.26, and 0.52 g / kg. All mice were administered the drugs by gavage, except for the blank control group which received the corresponding dose of distilled water. The drugs were administered once daily for three consecutive days. The pain threshold of each group was measured three times before administration, with 10-minute intervals between each measurement, and the average value was taken as the baseline pain threshold. Tail-flick time was measured 15, 30, 45, and 60 minutes after the last administration to observe changes in the pain threshold.

[0049] The results are shown in Table 1. Compared with the blank control group, the pain threshold of mice in the aspirin group was increased at each time point after administration, and the analgesic effect was obvious. 30 min after administration, the pain threshold of mice in the high-dose and medium-dose groups of *Gynostemma pentaphyllum* extract was increased, and the analgesic effect was obvious. 45 min after administration, the pain threshold of mice in the low-dose group of *Gynostemma pentaphyllum* extract was increased, and the analgesic effect was relatively obvious.

[0050] Table 1. Results of analgesia experiments (mouse tail-flick test). n=10)

[0051]

[0052] Note: Compared with the control group * P<0.05, ** P<0.01.

[0053] 2. Pain-inducing experiment using formaldehyde from *Gnaphalium affine* extract.

[0054] Sixty qualified Kunming mice (half male and half female) were randomly divided into six groups of ten mice each: a blank control group, an aspirin 0.1 g / kg group, and groups receiving *Gnaphalium affine* extract (Example 2) at doses of 0.13, 0.26, and 0.52 g / kg. All mice were administered the drugs by gavage, except for the blank control group which received the corresponding dose of distilled water. The drugs were administered once daily for three consecutive days. After the last administration, each mouse was subcutaneously injected with 5% formaldehyde solution (10 μL / mouse) into its left hind paw. The mouse was immediately placed in a glass container, and its behavioral responses were observed at 0-5 min (phase I) and 10-25 min (phase II). The responses were scored as follows: 0 points for walking freely; 1 point for limping or foot-supporting while stationary; 2 points for lifting the foot; and 3 points for licking, biting, or shaking the foot. The cumulative scores for both phases were recorded and weighted.

[0055] The results are shown in Table 2. Compared with the blank control group, the aspirin group and the high-dose group of *Gynostemma pentaphyllum* extract reduced the formaldehyde-induced pain score in the first time phase, showing significant analgesic effects. The aspirin group and the high-dose group of *Gynostemma pentaphyllum* extract reduced the formaldehyde-induced pain score in the second time phase, showing significant analgesic effects. The medium-dose group of *Gynostemma pentaphyllum* extract reduced the formaldehyde-induced pain score, showing relatively significant analgesic effects. In both the first and second time phases, the low-dose group of *Gynostemma pentaphyllum* extract did not reduce the formaldehyde-induced pain score, showing no significant analgesic effect.

[0056] Table 2 Results of analgesic experiments (formaldehyde-induced pain test) n=10)

[0057]

[0058] Note: Compared with the control group * P<0.05, ** P<0.01.

[0059] 3. Inhibitory effect of *Gynostemma pentaphyllum* extract on xylene-induced ear swelling in mice.

[0060] Sixty qualified Kunming mice (half male and half female) were randomly divided into six groups of ten mice each: a blank control group, an anti-inflammatory and choleretic group (1.5 g / kg), and groups receiving *Gnaphalium affine* extract (Example 2) at doses of 0.13, 0.26, and 0.52 g / kg. All mice were administered the drugs by gavage, except for the distilled water group which received the corresponding dose of distilled water. The drugs were administered once daily for six consecutive days. Forty minutes after the last administration, 0.03 mL of xylene was applied to the right auricle of each mouse to induce inflammation, while the left ear served as a control without xylene application. Thirty minutes later, the mice were euthanized by cervical dislocation. Both ears were cut off along the auricle, and ear pieces were removed from the same location on both ears using a 6 mm diameter punch. The ears were immediately weighed using an electronic balance, and the weight of each ear was recorded. The difference in weight between the left and right ears was used as the degree of swelling, and the swelling inhibition rate was calculated.

[0061] Swelling inhibition rate = (average auricular swelling in the distilled water group - average auricular swelling in the drug-treated group) / average auricular swelling in the distilled water group × 100%.

[0062] The results are shown in Table 3. Compared with the blank control group, the anti-inflammatory and choleretic tablet group, the high-dose group of *Gynostemma pentaphyllum* extract, and the medium-dose group of *Gynostemma pentaphyllum* extract inhibited xylene-induced auricular swelling and showed significant anti-inflammatory effects, with inhibition rates of 46.86%, 45.21%, and 41.89%, respectively. The low-dose group of *Gynostemma pentaphyllum* extract also inhibited xylene-induced auricular swelling and showed relatively significant anti-inflammatory effects, with an inhibition rate of 23.83%.

[0063] The dosage of the plantago asiatica extract group described in CN101693731A, after conversion, is 0.6 g / kg, and its swelling inhibition rate is 30.4%. It is evident that the high-dose and medium-dose groups of plantago asiatica extract showed significantly better inhibitory effects on xylene-induced auricular swelling than the 0.6 g / kg plantago asiatica extract group in CN101693731A.

[0064] Table 3. Effects of xylene on auricular swelling ( n=10)

[0065]

[0066] Note: Compared with the control group * P<0.05, ** P<0.01.

[0067] 4. Cholagogue experiment using extract from *Gnaphalium affine*

[0068] Sixty qualified Kunming mice (half male and half female) were randomly divided into six groups of ten mice each: a blank control group, a group receiving 1.5 g / kg of anti-inflammatory and choleretic tablets, and groups receiving 0.13, 0.26, and 0.52 g / kg of *Gnaphalium affine* extract (Example 2). All mice were administered the drugs by gavage, except for the distilled water group which received the corresponding dose of distilled water. The drugs were administered once daily for three consecutive days. Mice were fasted for 12 hours prior to the experiment but allowed free access to water. Forty minutes after the last administration, the bile duct and gallbladder were separated, the bile duct was ligated, and the gallbladder was removed and weighed.

[0069] The results are shown in Table 4. Compared with the blank control group, the gallbladder weight of mice in each experimental group, the high, medium and low dose groups of the herbal medicine and the anti-inflammatory and choleretic tablet group can all reduce the gallbladder weight and promote bile excretion, with obvious choleretic effects.

[0070] Table 4. Effects on gallbladder weight in mice ( n=10)

[0071]

[0072] Note: Compared with the control group * P<0.05, ** P<0.01.

[0073] 5. Stone-dissolving experiment using extract from *Gnaphalium affine*

[0074] Multiple gallstones of similar size, shape, intact surface, and weight from the same patient were collected, washed with clean water, and dried to constant weight. Using the test tube dissolution method, the gallstones were randomly assigned to groups. The stones were inserted into fine-mesh nylon mesh bags, immersed in test tubes containing dissolution solution, and allowed to stand at room temperature. The bags were shaken 3-4 times daily, and the dissolution solution was changed every other day. The presence of any broken stones was recorded. The replacement solution was stored in brown bottles. Changes in gallstone weight and composition of the replacement solution in the brown bottles were used as detection indicators. The experiment included a blank control group, a gallstone extract group, and high-concentration, medium-concentration, and low-concentration groups of gallstone extract (Example 2). Each group had 5 test tubes, each containing 5 mL of solution. The dissolution period was 14 days. After dissolution, the gallstones were removed, dried to constant weight, and the weight was recorded. The dissolution rate was calculated, and the amounts of total bilirubin and total cholesterol in the replacement solution in the brown bottles were measured.

[0075] Stone dissolution rate = (Weight of stones before the test - Weight of stones after the test) / Weight of stones before the test × 100%

[0076] As shown in Table 5, ① Gallstone dissolution rate: Comparing the gallstone dissolution rates of each experimental group with the blank control group, all experimental groups effectively dissolved gallstones, with significant dissolution effects. ② Total bilirubin: Comparing the total bilirubin levels of the experimental groups with the blank control group, total bilirubin was detectable in the Danle group, the high-concentration group of *Gnaphalium affine* extract, and the medium-concentration group, with significant effects. The low-concentration group of *Gnaphalium affine* extract showed no significant effect. ③ Total cholesterol: Comparing the total cholesterol levels of the experimental groups with the blank control group, total cholesterol was detectable in the high-concentration group, the medium-concentration group, and the low-concentration group of *Gnaphalium affine* extract, with significant effects.

[0077] The in vitro litholytic experiment results described in CN101693731A show that the litholytic rate of plantago asiatica glycoside at a concentration of 50 mg / mL is 15.44%, which is basically consistent with the litholytic rate of 15.56% of the high-concentration group of *Plantago asiatica* extract (1.02 mg / mL). It can be seen that when the amount of plantago asiatica glycoside in CN101693731A is more than 49 times that of *Plantago asiatica* extract, the litholytic effects of the two are the same. In other words, this invention achieves the same litholytic effect as plantago asiatica glycoside when the amount of *Plantago asiatica* extract is 1 / 49 of that of plantago asiatica glycoside, and its litholytic effect is significantly better than that of plantago asiatica glycoside, which can greatly reduce the amount of drug used.

[0078] Table 5 Results of in vitro litholysis experiments ( n=5)

[0079]

[0080]

[0081] Note: Compared with the control group * P<0.05, ** P<0.01.

[0082] As the experimental results above show, the *Cimicifuga foetida* extract prepared by this invention, which is rich in cimicifuga-like glycosides, heterocimicifuga-like glycosides, and plantain ether glycosides, has strong anti-inflammatory and analgesic activities and significant in vitro litholytic effects.

Claims

1. Application of *Hedyotis diffusa* extract in the preparation of litholytic drugs for treating calculi; wherein the calculi are gallstones, kidney stones, ureteral stones, bladder stones, or uric acid stones; the *Hedyotis diffusa* extract is made from *Hedyotis diffusa* produced in Guizhou Province. The dried *Hedyotis diffusa* is pulverized, and 4-12 times its weight of deionized water is added. Water extraction is performed at 45-80℃ using an intermittent warm percolation method until the exudate becomes lighter in color. The exudate is filtered, the filtrates are combined, and after being cooled to room temperature, macroporous resin column chromatography is used for adsorption. First, 5-10 column volumes of deionized water are used for elution, followed by 4-8 column volumes of 20%-80% ethanol. The ethanol eluent is collected, the ethanol is removed, and the residue is dried to obtain the *Hedyotis diffusa* extract; the *Hedyotis diffusa* extract contains 10%-25% cimicifuga glycosides, 3%-15% isocitric acid glycosides, and 20%-50% plantain glycosides.

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