Preparation method of twelve-ingredient bitong liniment for dispelling wind, removing dampness, activating blood, resolving stasis, reducing swelling and relieving pain

Abstract

This invention relates to a twelve-ingredient Bi Tong liniment for dispelling wind and dampness, promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain, and its uses. On one hand, this invention relates to a twelve-ingredient Bi Tong liniment, which is made from the following twelve medicinal herbs in the following weight ratios per 1000ml: 50-70g of *Smilax glabra*, 50-70g of *Asarum heterotropoides*, 25-35g of *Caulis Lysimachiae*, 10-20g of *Herba Clematis*, 8-12g of *Radix et Rhizoma Asarum*, 8-12g of *Radix Zanthoxyli*, 8-12g of *Radix Aconitum*, 20-30g of *Radix et Rhizoma Scutellariae*, 40-60g of *Radix Polygoni Cuscutae*, 15-25g of *Radix Sparganii*, 15-25g of *Rhizoma Curcumae*, and 15-25g of *Radix Curcumae*. In Miao medicine, this twelve-ingredient Bi Tong liniment has the function of lifting the shin and strengthening the legs; in traditional Chinese medicine, it has the functions of dispelling wind and dampness, promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. Clinically, it is often used for cold-dampness Bi syndrome, sprains, and muscle strains. The present invention's twelve-ingredient Bi Tong liniment has the excellent properties described in the specification, such as excellent biological effects.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to a medicine that has the functions of lifting the knee and strengthening the leg in Miao medicine and the functions of dispelling wind and dampness, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain in traditional Chinese medicine. It is often used clinically for cold-dampness arthralgia, sprains and muscle injuries, etc. In particular, it is a medicine called Twelve-Flavor Bi-Tong Liniment that is administered externally. Background Technology

[0002] Twelve-Ingredient Bi-Tong Liniment (National Drug Approval Number Z20025115) is an exclusive Miao medicine product of the applicant company, which has been used clinically for decades. As an OTC Class A drug, Twelve-Ingredient Bi-Tong Liniment is administered externally. In Miao medicine, it is believed to have the function of lifting and strengthening the legs. In traditional Chinese medicine, it is believed to have the functions of dispelling wind and dampness, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain. Clinically, it is often used for cold-dampness arthralgia, sprains and strains, etc.

[0003] According to the national drug standard WS-10106(ZD-0106)-2002-2012Z, the Twelve-Ingredient Pain Relief Liniment is made from 12 Chinese medicinal herbs in the following weight proportions:

[0004] 60g of *Smilax glabra*, 60g of *Erigeron breviscapus*, and 30g of *Eupatorium fortunei*.

[0005] 15g of *Penetrating Fragrance*, 10g of *Asarum heterotropoides* leaves, 10g of Sichuan peppercorns.

[0006] Aconitum carmichaelii 10g, Aconitum carmichaelii 25g, Polygonum multiflorum 50g

[0007] 20g of Polygonum cuspidatum, 20g of Sparganium stoloniferum, and 20g of Curcuma zedoaria.

[0008] The national drug standard WS-10106(ZD-0106)-2002-2012Z also records the preparation method of the Twelve-Ingredient Pain Relief Liniment. Based on a total of 330g of the aforementioned medicinal materials, the method includes the following steps: The twelve ingredients are extracted twice with 50% ethanol. The first extraction uses 2.5 times the amount of 50% ethanol for 10 days, and the second extraction uses 2 times the amount of 50% ethanol for 10 days, stirring twice daily. The extracts are filtered, the filtrates are combined, and 50% ethanol is added to 1000ml. The mixture is mixed well, allowed to stand for 14 days, filtered again, and then bottled. The resulting Twelve-Ingredient Pain Relief Liniment is believed to be a brownish-yellow liquid that develops sediment upon prolonged standing and has a fragrant odor.

[0009] Cold-dampness arthralgia refers to both cold arthralgia and damp arthralgia, illnesses caused by external invasion of cold and damp pathogens. Cold arthralgia, also known as painful arthralgia, is caused by prolonged exposure to cold and dampness in harsh climates or prolonged residence in such environments, leading to stagnation of blood and qi, and blood stasis. Its main manifestation is fixed, cold pain. Patients experience worsening pain upon exposure to cold and relief upon exposure to warmth. It may also be accompanied by symptoms such as limited flexion and extension of limbs and joints, and numbness. Damp arthralgia, also known as fixed arthralgia, is caused by insufficient vital energy, external invasion of dampness, and imbalance of the nutritive and defensive qi, leading to obstruction of the meridians and poor blood circulation. Patients mainly present with numbness of the skin and fixed, swollen, and painful joints. Generally, improving blood and qi circulation and promoting blood circulation can promote recovery.

[0010] Clinically, sprains and strains can lead to blood stasis and tendon injury, usually occurring in the early stages of injury. Symptoms include severe pain at the injury site, which worsens with movement, significant swelling, tenderness upon pressure, purplish-blue skin, thin white tongue coating, and a wiry, tense pulse. Traditional Chinese medicine treatment for sprains and strains typically focuses on promoting blood circulation, warming the meridians, regulating qi, relieving pain, and reducing swelling.

[0011] Although the Twelve-Ingredient Bi-Tong Liniment has shown reliable clinical efficacy, there is still a need to improve the quality of the product. For example, the quality of the drug can be evaluated by examining its blood-activating and stasis-removing effects, including using animal models of blood-activating and stasis-removing experiments to evaluate the biological effects of the Twelve-Ingredient Bi-Tong Liniment, in order to provide a Twelve-Ingredient Bi-Tong Liniment with one or more superior properties. Summary of the Invention

[0012] The purpose of this invention is to provide a Twelve-Ingredient Pain-Relieving Liniment with one or more superior properties, particularly a Twelve-Ingredient Pain-Relieving Liniment with superior biological properties, such as a Twelve-Ingredient Pain-Relieving Liniment with superior functions of dispelling wind and dampness, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain. It has been unexpectedly discovered that, through the method of this invention, one or more Twelve-Ingredient Pain-Relieving Liniments with superior effects as described in the context of this invention can be obtained, and this invention is based on such discoveries.

[0013] Therefore, the first aspect of the present invention provides a twelve-ingredient liniment, which is made from the following twelve medicinal materials: *Smilax glabra*, *Asarum sieboldii*, *Aristolochia debilis ... scoparia*, *Zanthoxylum bungeanum*, *Aconitum carmichaelii*, *Polygonum multiflorum*, *Polygonum cuspidatum*, *Sparganium stoloniferum*, and *Curcuma zedoaria*.

[0014] According to the first aspect of the present invention, the twelve-ingredient liniment is made from the following twelve medicinal materials in the following weight proportions per 1000ml: 50-70g of *Smilax glabra*, 50-70g of *Asarum heterotropoides*, 25-35g of *Cynanchum paniculatum*, 10-20g of *Hypericum perforatum*, 8-12g of *Asarum heterotropoides*, 8-12g of *Zanthoxylum bungeanum*, 8-12g of *Aconitum carmichaelii*, 20-30g of *Ligusticum striatum*, 40-60g of *Polygonum cuspidatum*, 15-25g of *Polygonum cuspidatum*, 15-25g of *Sparganium stoloniferum*, and 15-25g of *Curcuma zedoaria*.

[0015] According to the first aspect of the present invention, the Twelve-Flavor Pain Relief Liniment is made from the following twelve medicinal materials in the following weight ratio per 1000ml: 60g of *Artemisia scoparia*, 60g of *Asarum heterotropoides*, 30g of *Aristolochia debilis*, 15g of *Aristolochia debilis*, 10g of *Asarum heterotropoides*, 10g of *Zanthoxylum bungeanum*, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*.

[0016] According to the first aspect of the present invention, the Twelve-Flavor Pain Relief Liniment is made from the following twelve medicinal materials in the following weight ratio per 1000ml: 50g of Artemisia argyi, 70g of Asarum sieboldii, 25g of Aristolochia debilis, 20g of Aristolochia debilis, 8g of Asarum sieboldii, 12g of Zanthoxylum bungeanum, 8g of Aconitum carmichaelii, 30g of Polygonum multiflorum, 40g of Polygonum cuspidatum, 25g of Polygonum cuspidatum, 15g of Sparganium stoloniferum, and 25g of Curcuma zedoaria.

[0017] According to the first aspect of the present invention, the Twelve-Flavor Pain Relief Liniment is made from the following twelve medicinal materials in the following weight ratio per 1000ml: 70g of *Artemisia argyi*, 50g of *Asarum heterotropoides*, 35g of *Eupatorium fortunei*, 10g of *Clematis chinensis*, 12g of *Asarum heterotropoides*, 8g of *Zanthoxylum bungeanum*, 12g of *Aconitum carmichaelii*, 20g of *Ligusticum striatum*, 60g of *Polygonum cuspidatum*, 15g of *Polygonum cuspidatum*, 25g of *Sparganium stoloniferum*, and 15g of *Curcuma zedoaria*.

[0018] According to the first aspect of the present invention, the twelve-ingredient pain-relieving liniment is prepared per 1000 ml by a method comprising the following steps:

[0019] Add 1.5 times the total weight of 50% ethanol (pre-adjusted to pH 3-4, such as pH 3.0, pH 3.5, or pH 4.0) to the twelve herbs used to prepare 1000ml of liniment. Soak for 6 days with stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid.

[0020] Add 1.5 times the total weight of the medicinal materials in 50% ethanol (pre-adjusted to pH 3-4 with hydrochloric acid, such as pH 3.0, pH 3.5, or pH 4.0) and soak for 5 days, stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid.

[0021] Combine the two immersion solutions, filter, add sodium hydroxide to adjust the pH to 4.5-5.0, let stand for 4 days, and then filter.

[0022] After adjusting the pH of the filtrate to 6.0-6.5 with sodium hydroxide, add 50% ethanol to 1000ml, let it stand for 3 days, filter, and fill into bottles to obtain the final product.

[0023] According to the first aspect of the present invention, the twelve-ingredient liniment for relieving pain is made from the following twelve medicinal materials in the following weight ratios per 1000ml: 60g of *Smilax glabra*, 60g of *Asarum heterotropoides*, 30g of *Cynanchum paniculatum*, 15g of *Clematis chinensis*, 10g of *Asarum heterotropoides* leaf, 10g of *Zanthoxylum bungeanum*, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum multiflorum*, 20g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*; the preparation method is as follows:

[0024] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0025] According to the first aspect of the present invention, the twelve-ingredient liniment for relieving pain is made from the following twelve medicinal materials in the following weight ratios per 1000ml: 50g of *Smilax glabra*, 70g of *Asarum heterotropoides*, 25g of *Cynanchum paniculatum*, 20g of *Clematis chinensis*, 8g of *Asarum heterotropoides* leaf, 12g of *Zanthoxylum bungeanum*, 8g of *Aconitum carmichaelii*, 30g of *Ligusticum striatum*, 40g of *Polygonum multiflorum*, 25g of *Polygonum cuspidatum*, 15g of *Sparganium stoloniferum*, and 25g of *Curcuma zedoaria*; the preparation method is as follows:

[0026] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0027] According to the first aspect of the present invention, the twelve-ingredient liniment for relieving pain is made from the following twelve medicinal materials in the following weight ratios per 1000ml: 70g of *Smilax glabra*, 50g of *Asarum heterotropoides*, 35g of *Cynanchum paniculatum*, 10g of *Clematis chinensis*, 12g of *Asarum heterotropoides* leaf, 8g of Sichuan pepper, 12g of *Aconitum carmichaelii*, 20g of *Ligusticum striatum*, 60g of *Polygonum multiflorum*, 15g of *Polygonum cuspidatum*, 25g of *Sparganium stoloniferum*, and 15g of *Curcuma zedoaria*; the preparation method is as follows:

[0028] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 (pH 6.38) with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0029] Furthermore, a second aspect of the present invention provides a method for preparing a twelve-ingredient liniment, wherein the following twelve medicinal materials are used per 1000 ml of the liniment in the following weight proportions: 50-70g of *Smilax glabra*, 50-70g of *Asarum heterotropoides*, 25-35g of *Cynanchum paniculatum*, 10-20g of *Clematis chinensis*, 8-12g of *Asarum heterotropoides* leaf, 8-12g of *Zanthoxylum bungeanum*, 8-12g of *Aconitum carmichaelii*, 20-30g of *Ligusticum striatum*, 40-60g of *Polygonum cuspidatum*, 15-25g of *Polygonum cuspidatum*, 15-25g of *Sparganium stoloniferum*, and 15-25g of *Curcuma zedoaria*. The method comprises the following steps:

[0030] Add 1.5 times the total weight of the twelve medicinal herbs to 50% ethanol (pre-adjusted to pH 3-4 with hydrochloric acid, such as pH 3.0, pH 3.5, or pH 4.0) and soak for 6 days, stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid.

[0031] Add 1.5 times the total weight of the medicinal materials in 50% ethanol (pre-adjusted to pH 3-4 with hydrochloric acid, such as pH 3.0, pH 3.5, or pH 4.0) and soak for 5 days, stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid.

[0032] Combine the two immersion solutions, filter, add sodium hydroxide to adjust the pH to 4.5-5.0, let stand for 4 days, and then filter.

[0033] After adjusting the pH of the filtrate to 6.0-6.5 with sodium hydroxide, add 50% ethanol to 1000ml, let it stand for 3 days, filter, and fill into bottles to obtain the final product.

[0034] According to the method described in the second aspect of the present invention, the following twelve medicinal materials are used in the following weight proportions per 1000 ml of the liniment: 60g of *Artemisia scoparia*, 60g of *Asarum sieboldii*, 30g of *Eupatorium fortunei*, 15g of *Clematis chinensis*, 10g of *Asarum sieboldii*, 10g of *Zanthoxylum bungeanum*, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum cuspidatum*, 20g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*.

[0035] According to the method described in the second aspect of the present invention, the following twelve medicinal materials are used in the following weight proportions per 1000 ml of the liniment: 50g of *Artemisia scoparia*, 70g of *Asarum sieboldii*, 25g of *Eupatorium fortunei*, 20g of *Asarum heterotropoides*, 8g of *Asarum sieboldii*, 12g of *Zanthoxylum bungeanum*, 8g of *Aconitum carmichaelii*, 30g of *Ligusticum striatum*, 40g of *Polygonum multiflorum*, 25g of *Polygonum cuspidatum*, 15g of *Sparganium stoloniferum*, and 25g of *Curcuma zedoaria*.

[0036] According to the method described in the second aspect of the present invention, the following twelve medicinal materials are used in weight proportions to prepare 1000 ml of the liniment: 70 g of *Artemisia argyi*, 50 g of *Asarum heterotropoides*, 35 g of *Eupatorium fortunei*, 10 g of *Clematis chinensis*, 12 g of *Asarum heterotropoides*, 8 g of *Zanthoxylum bungeanum*, 12 g of *Aconitum carmichaelii*, 20 g of *Ligusticum striatum*, 60 g of *Polygonum cuspidatum*, 15 g of *Polygonum cuspidatum*, 25 g of *Sparganium stoloniferum*, and 15 g of *Curcuma zedoaria*.

[0037] According to the method described in the second aspect of the present invention, in preparing 1000 ml of the liniment, the following twelve medicinal materials are used in the following weight proportions: 60g of *Artemisia argyi*, 60g of *Asarum sieboldii*, 30g of *Cynanchum paniculatum*, 15g of *Clematis chinensis*, 10g of *Asarum sieboldii* leaf, 10g of *Zanthoxylum bungeanum*, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum multiflorum*, 20g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*; the method includes the following steps:

[0038] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0039] According to the method described in the second aspect of the present invention, in preparing 1000 ml of the liniment, the following twelve medicinal materials are used in the following weight proportions: 50g of *Artemisia argyi*, 70g of *Asarum sieboldii*, 25g of *Eupatorium fortunei*, 20g of *Clematis chinensis*, 8g of *Asarum sieboldii* leaf, 12g of *Zanthoxylum bungeanum*, 8g of *Aconitum carmichaelii*, 30g of *Ligusticum striatum*, 40g of *Polygonum multiflorum*, 25g of *Polygonum cuspidatum*, 15g of *Sparganium stoloniferum*, and 25g of *Curcuma zedoaria*; the method includes the following steps:

[0040] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0041] According to the method described in the second aspect of the present invention, the following twelve medicinal materials are used in the following weight ratios per 1000 ml of the liniment: 70g of *Artemisia argyi*, 50g of *Asarum sieboldii*, 35g of *Cynanchum paniculatum*, 10g of *Clematis chinensis*, 12g of *Asarum sieboldii*, 8g of *Zanthoxylum bungeanum*, 12g of *Aconitum carmichaelii*, 20g of *Ligusticum striatum*, 60g of *Polygonum multiflorum*, 15g of *Polygonum cuspidatum*, 25g of *Sparganium stoloniferum*, and 15g of *Curcuma zedoaria*; the method includes the following steps:

[0042] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 (pH 6.38) with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0043] Furthermore, a third aspect of the present invention provides the use of a composition in the preparation of a medicine for dispelling wind and dampness, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain, or in the preparation of a medicine for cold-dampness arthralgia and sprains, wherein the composition is made from the following twelve medicinal materials: *Smilax glabra*, *Asarum sieboldii*, *Aquilaria sinensis*, *Aquilaria agallocha*, *Aquilaria sinensis*, *Zanthoxylum bungeanum*, *Aconitum carmichaelii*, *Polygonum multiflorum*, *Polygonum cuspidatum*, *Sparganium stoloniferum*, and *Curcuma zedoaria*.

[0044] According to the third aspect of the invention, the composition is a liniment, which is made of twelve medicinal materials in the following weight ratios per 1000ml: 50-70g of *Artemisia scoparia*, 50-70g of *Asarum sieboldii*, 25-35g of *Aristolochia debilis*, 10-20g of *Aristolochia debilis*, 8-12g of *Asarum sieboldii*, 8-12g of *Zanthoxylum bungeanum*, 8-12g of *Aconitum carmichaelii*, 20-30g of *Ligusticum striatum*, 40-60g of *Polygonum cuspidatum*, 15-25g of *Polygonum cuspidatum*, 15-25g of *Sparganium stoloniferum*, and 15-25g of *Curcuma zedoaria*.

[0045] According to the third aspect of the invention, the composition is a liniment made of twelve medicinal herbs in the following weight proportions per 1000ml: 60g of *Artemisia scoparia*, 60g of *Asarum sieboldii*, 30g of *Eupatorium fortunei*, 15g of *Clematis chinensis*, 10g of *Asarum sieboldii*, 10g of *Zanthoxylum bungeanum*, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum cuspidatum*, 20g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*.

[0046] According to the third aspect of the invention, the composition is a liniment made of the following twelve medicinal materials in the following weight proportions per 1000ml: 50g of *Artemisia scoparia*, 70g of *Asarum sieboldii*, 25g of *Eupatorium fortunei*, 20g of *Asarum heterotropoides*, 8g of *Asarum sieboldii*, 12g of *Zanthoxylum bungeanum*, 8g of *Aconitum carmichaelii*, 30g of *Ligusticum striatum*, 40g of *Polygonum multiflorum*, 25g of *Polygonum cuspidatum*, 15g of *Sparganium stoloniferum*, and 25g of *Curcuma zedoaria*.

[0047] According to the third aspect of the invention, the composition is a liniment made of the following twelve medicinal materials in the following weight proportions per 1000ml: 70g of *Artemisia scoparia*, 50g of *Asarum sieboldii*, 35g of *Eupatorium fortunei*, 10g of *Clematis chinensis*, 12g of *Asarum sieboldii*, 8g of *Zanthoxylum bungeanum*, 12g of *Aconitum carmichaelii*, 20g of *Ligusticum striatum*, 60g of *Polygonum multiflorum*, 15g of *Polygonum cuspidatum*, 25g of *Sparganium stoloniferum*, and 15g of *Curcuma zedoaria*.

[0048] According to a third aspect of the invention, the composition is a liniment, which is prepared per 1000 ml by a method comprising the following steps:

[0049] Add 1.5 times the total weight of 50% ethanol (pre-adjusted to pH 3-4, such as pH 3.0, pH 3.5, or pH 4.0) to the twelve herbs used to prepare 1000ml of liniment. Soak for 6 days with stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid.

[0050] Add 1.5 times the total weight of the medicinal materials in 50% ethanol (pre-adjusted to pH 3-4 with hydrochloric acid, such as pH 3.0, pH 3.5, or pH 4.0) and soak for 5 days, stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid.

[0051] Combine the two immersion solutions, filter, add sodium hydroxide to adjust the pH to 4.5-5.0, let stand for 4 days, and then filter.

[0052] After adjusting the pH of the filtrate to 6.0-6.5 with sodium hydroxide, add 50% ethanol to 1000ml, let it stand for 3 days, filter, and fill into bottles to obtain the final product.

[0053] According to the third aspect of the invention, the composition is a liniment, which is made from twelve medicinal herbs in the following weight ratio per 1000ml: 60g of *Artemisia scoparia*, 60g of *Asarum sieboldii*, 30g of *Eupatorium fortunei*, 15g of *Clematis chinensis*, 10g of *Asarum sieboldii* leaf, 10g of *Zanthoxylum bungeanum*, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum multiflorum*, 20g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*; the preparation method is as follows:

[0054] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0055] According to the third aspect of the invention, the composition is a liniment, which is made from twelve medicinal herbs in the following weight ratio per 1000ml: 50g of *Artemisia scoparia*, 70g of *Asarum sieboldii*, 25g of *Eupatorium fortunei*, 20g of *Clematis chinensis*, 8g of *Asarum sieboldii*, 12g of *Zanthoxylum bungeanum*, 8g of *Aconitum carmichaelii*, 30g of *Ligusticum striatum*, 40g of *Polygonum multiflorum*, 25g of *Polygonum cuspidatum*, 15g of *Sparganium stoloniferum*, and 25g of *Curcuma zedoaria*; the preparation method is as follows:

[0056] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0057] According to the third aspect of the invention, the composition is a liniment, which is made from twelve medicinal herbs in the following weight ratio per 1000ml: 70g of *Artemisia scoparia*, 50g of *Asarum sieboldii*, 35g of *Eupatorium fortunei*, 10g of *Clematis chinensis*, 12g of *Asarum sieboldii* leaf, 8g of Sichuan pepper, 12g of *Aconitum carmichaelii*, 20g of *Ligusticum striatum*, 60g of *Polygonum multiflorum*, 15g of *Polygonum cuspidatum*, 25g of *Sparganium stoloniferum*, and 15g of *Curcuma zedoaria*; the preparation method is as follows:

[0058] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.0-6.5 (pH 6.38) with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

[0059] The twelve-ingredient Bi Tong liniment obtained by this invention exhibits excellent effects as described in the context of this invention. Detailed Implementation

[0060] The invention can be further described through the following embodiments; however, the scope of the invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the invention without departing from its spirit and scope. The materials and methods used in the experiments are described generally and / or specifically. Although many materials and methods of operation used to achieve the objectives of the invention are well known in the art, the invention is still described in as much detail as possible herein.

[0061] Example 1: Preparation of Twelve-Flavor Pain-Relieving Liniment

[0062] The prescription and preparation method in this embodiment are the same as those in the national standard.

[0063] prescription:

[0064] 60g of *Smilax glabra*, 60g of *Erigeron breviscapus*, and 30g of *Eupatorium fortunei*.

[0065] 15g of *Penetrating Fragrance*, 10g of *Asarum heterotropoides* leaves, 10g of Sichuan peppercorns.

[0066] Aconitum carmichaelii 10g, Aconitum carmichaelii 25g, Polygonum multiflorum 50g

[0067] 20g of Polygonum cuspidatum, 20g of Sparganium stoloniferum, and 20g of Curcuma zedoaria.

[0068] Preparation method:

[0069] The above twelve ingredients are extracted twice using 50% ethanol. The first extraction uses 2.5 times the volume of 50% ethanol for 10 days, and the second extraction uses twice the volume of 50% ethanol for 10 days, stirring twice daily. The extracts are filtered, the filtrates are combined, and approximately 192 ml of 50% ethanol is added to bring the total volume to 1000 ml. The mixture is then stirred, allowed to stand for 14 days, filtered, and bottled to obtain the medicine known as the Twelve-Ingredient Pain-Relieving Liniment. This preparation method takes a total of 34 days and uses approximately 1785 ml of 50% ethanol (density 0.93).

[0070] Example 2: Preparation of Twelve-Flavor Pain-Relieving Liniment

[0071] prescription:

[0072] 60g of *Smilax glabra*, 60g of *Erigeron breviscapus*, and 30g of *Eupatorium fortunei*.

[0073] 15g of *Penetrating Fragrance*, 10g of *Asarum heterotropoides* leaves, 10g of Sichuan peppercorns.

[0074] Aconitum carmichaelii 10g, Aconitum carmichaelii 25g, Polygonum multiflorum 50g

[0075] 20g of Polygonum cuspidatum, 20g of Sparganium stoloniferum, and 20g of Curcuma zedoaria.

[0076] Preparation method:

[0077] The above twelve ingredients are extracted twice using 50% ethanol. The first extraction uses twice the amount of 50% ethanol for 12 days, and the second extraction uses 1.5 times the amount of 50% ethanol for 12 days, stirring twice daily. The extracts are filtered, the filtrates are combined, and 50% ethanol is added to a final volume of 1000 ml. The mixture is then stirred, allowed to stand for 16 days, filtered again, and bottled to obtain the medicine known as the Twelve-Ingredient Pain-Relieving Liniment. This preparation method takes a total of 40 days and uses approximately 1820 ml of 50% ethanol (density 0.93).

[0078] Example 3: Preparation of Twelve-Flavor Pain-Relieving Liniment

[0079] prescription:

[0080] 60g of *Smilax glabra*, 60g of *Erigeron breviscapus*, and 30g of *Eupatorium fortunei*.

[0081] 15g of *Penetrating Fragrance*, 10g of *Asarum heterotropoides* leaves, 10g of Sichuan peppercorns.

[0082] Aconitum carmichaelii 10g, Aconitum carmichaelii 25g, Polygonum multiflorum 50g

[0083] 20g of Polygonum cuspidatum, 20g of Sparganium stoloniferum, and 20g of Curcuma zedoaria.

[0084] Preparation method:

[0085] The above twelve ingredients are extracted twice using 50% ethanol. The first extraction uses three times the volume of 50% ethanol for 8 days, and the second extraction uses 2.5 times the volume of 50% ethanol for 8 days, stirring twice daily. The extracts are filtered, the filtrates are combined, and 50% ethanol is added to a final volume of 1000 ml. The mixture is then stirred, allowed to stand for 12 days, filtered again, and bottled to obtain the medicine known as the Twelve-Ingredient Pain-Relieving Liniment. This preparation method takes a total of 28 days and uses approximately 1845 ml of 50% ethanol (density 0.93).

[0086] Example 4: Preparation of a Twelve-Flavor Pain-Relieving Liniment

[0087] prescription:

[0088] 50g of *Smilax glabra*, 70g of *Erigeron breviscapus*, and 25g of *Eupatorium fortunei*.

[0089] 20g of *Penetrating Fragrance*, 8g of *Sweet Potato Leaf Asarum*, 12g of Sichuan Pepper.

[0090] Aconitum carmichaelii 8g, Epimedium brevicornu 30g, Polygonum multiflorum 40g

[0091] Polygonum cuspidatum 25g, Sparganium stoloniferum 15g, Curcuma zedoaria 25g.

[0092] Preparation method:

[0093] The above twelve ingredients are extracted twice using 50% ethanol. The first extraction uses 2.5 times the volume of 50% ethanol for 10 days, and the second extraction uses twice the volume of 50% ethanol for 10 days, stirring twice daily. The extracts are filtered, the filtrates are combined, and 50% ethanol is added to a final volume of 1000 ml. The mixture is then stirred, allowed to stand for 14 days, filtered again, and bottled to obtain the medicine known as the Twelve-Ingredient Pain-Relieving Liniment. This preparation method takes a total of 34 days and uses approximately 1763 ml of 50% ethanol (density 0.93).

[0094] Example 5: Preparation of a Twelve-Flavor Pain-Relieving Liniment

[0095] prescription:

[0096] 70g of *Smilax glabra*, 50g of *Erigeron breviscapus*, and 35g of *Eupatorium fortunei*.

[0097] 10g of *Penetrating Fragrance*, 12g of *Sweet Potato Leaf Asarum*, 8g of Sichuan Pepper

[0098] Aconitum carmichaelii 12g, Antiaris pilosa 20g, Polygonum multiflorum 60g

[0099] Polygonum cuspidatum 15g, Sparganium stoloniferum 25g, Curcuma zedoaria 15g.

[0100] Preparation method:

[0101] The above twelve ingredients are extracted twice using 50% ethanol. The first extraction uses 2.5 times the volume of 50% ethanol for 10 days, and the second extraction uses twice the volume of 50% ethanol for 10 days, stirring twice daily. The extracts are filtered, the filtrates are combined, and 50% ethanol is added to a final volume of 1000 ml. The mixture is then stirred, allowed to stand for 14 days, filtered again, and bottled to obtain the medicine known as the Twelve-Ingredient Pain-Relieving Liniment. This preparation method takes a total of 34 days and uses approximately 1796 ml of 50% ethanol (density 0.93).

[0102] Example 6: Preparation of a Twelve-Flavor Pain-Relieving Liniment

[0103] prescription:

[0104] 60g of *Smilax glabra*, 60g of *Erigeron breviscapus*, and 30g of *Eupatorium fortunei*.

[0105] 15g of *Penetrating Fragrance*, 10g of *Asarum heterotropoides* leaves, 10g of Sichuan peppercorns.

[0106] Aconitum carmichaelii 10g, Aconitum carmichaelii 25g, Polygonum multiflorum 50g

[0107] 20g of Polygonum cuspidatum, 20g of Sparganium stoloniferum, and 20g of Curcuma zedoaria.

[0108] Preparation method:

[0109] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids obtained from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0 (pH 4.67). The mixture is then allowed to stand for 4 days and filtered again. Sodium hydroxide is added to the filtrate to adjust the pH to 6.0-6.5 (pH 6.23). Then, approximately 327 ml of 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the medicine known as the Twelve-Ingredient Pain Relief Liniment. The total preparation time for this method is 18 days, and the total amount of 50% ethanol (density 0.93) used is approximately 1392 ml.

[0110] Example 7: Preparation of Twelve-Flavor Pain-Relieving Liniment

[0111] Prescription (same as in Example 4):

[0112] 50g of *Smilax glabra*, 70g of *Erigeron breviscapus*, and 25g of *Eupatorium fortunei*.

[0113] 20g of *Penetrating Fragrance*, 8g of *Sweet Potato Leaf Asarum*, 12g of Sichuan Pepper.

[0114] Aconitum carmichaelii 8g, Epimedium brevicornu 30g, Polygonum multiflorum 40g

[0115] Polygonum cuspidatum 25g, Sparganium stoloniferum 15g, Curcuma zedoaria 25g.

[0116] Preparation method (basically the same as in Example 6):

[0117] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids obtained from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0 (pH 4.56). The mixture is then allowed to stand for 4 days and filtered again. Sodium hydroxide is added to the filtrate to adjust the pH to 6.0-6.5 (pH 6.17). Then, approximately 322 ml of 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the medicine known as the Twelve-Ingredient Pain Relief Liniment. The total preparation time for this method is 18 days, and the total amount of 50% ethanol (density 0.93) used is approximately 1380 ml.

[0118] Example 8: Preparation of a Twelve-Flavor Pain-Relieving Liniment

[0119] Prescription (same as in Example 4):

[0120] 70g of *Smilax glabra*, 50g of *Erigeron breviscapus*, and 35g of *Eupatorium fortunei*.

[0121] 10g of *Penetrating Fragrance*, 12g of *Sweet Potato Leaf Asarum*, 8g of Sichuan Pepper

[0122] Aconitum carmichaelii 12g, Antiaris pilosa 20g, Polygonum multiflorum 60g

[0123] Polygonum cuspidatum 15g, Sparganium stoloniferum 25g, Curcuma zedoaria 15g.

[0124] Preparation method (basically the same as in Example 6):

[0125] The above twelve ingredients are soaked in 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, 1.5 times their total weight of 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) is added and soaked for 5 days, stirring twice a day. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids obtained from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.5-5.0 (pH 4.84). The mixture is then allowed to stand for 4 days and filtered again. Sodium hydroxide is added to the filtrate to adjust the pH to 6.0-6.5 (pH 6.38). Then, approximately 331 ml of 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the medicine known as the Twelve-Ingredient Pain Relief Liniment. The total preparation time for this method is 18 days, and the total amount of 50% ethanol (density 0.93) used is approximately 1402 ml.

[0126] The pH values ​​of the liniments in Examples 1-8 were measured to be in the range of 6.3-6.8, and the pH values ​​of the liniments prepared in other examples of the present invention were also in the range of 6.2-7.0.

[0127] Experimental Example 1: Determination of Stilbene Glycoside Content in Twelve-Flavor Bi Tong Liniment by HPLC

[0128] 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (C20H22O9), commonly referred to as stilbene glycoside in this field, is one of the main components of Polygonum multiflorum, a plant in the Polygonaceae family. It is also the content detection component in the quality standard of the Twelve-Ingredient Bi-Tong Liniment. The National Drug Standard WS-10106(ZD-0106)-2002-2012Z stipulates that the content of Polygonum multiflorum in each 1ml of the liniment, calculated as 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (C20H22O9), shall not be less than 0.30mg.

[0129] This experimental example provides a method for determining the stilbene glycoside content in the Twelve Flavors Bi Tong Liniment of the present invention, and also provides a method for determining the content of the relevant liniment.

[0130] Determination method (this method is as follows):

[0131] According to the specifications of high performance liquid chromatography in Section 0512 of the General Chapter of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the operation should be carried out in the dark.

[0132] Chromatographic conditions and system suitability: Use an octadecylsilyl-bonded silica column (Ascentis® Express C18 column, 250 mm × 4.6 mm, 5 μm) as the packing material, acetonitrile-water (18:82) as the mobile phase, and a detection wavelength of 320 nm. The theoretical plate number, calculated based on the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside peak, should be no less than 5000 (actual plate number 12000).

[0133] Preparation of reference solution: Take an appropriate amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard (purchased from the National Institutes for Food and Drug Control), accurately weigh it, and add dilute ethanol to prepare a solution containing 20 μg per ml.

[0134] Preparation of the test solution: Accurately measure 5 ml of the Twelve-Flavor Bi Tong Liniment and place it in a 25 ml volumetric flask. Dilute with dilute ethanol to the mark and shake well. Accurately measure 5 ml of the solution and place it in a 25 ml volumetric flask. Dilute with dilute ethanol to the mark and shake well.

[0135] Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0136] According to the measurement,

[0137] The stilbene glycoside content in the twelve-ingredient Bi Tong liniment obtained in Examples 1-5 was 0.463 mg / ml, 0.438 mg / ml, 0.456 mg / ml, 0.368 mg / ml, and 0.539 mg / ml, respectively.

[0138] The stilbene glycoside content in the twelve-ingredient Bi Tong liniment obtained in Examples 6-8 was 0.728 mg / ml, 0.573 mg / ml (equivalent to 0.716 mg / ml when using 50g of Polygonum multiflorum), and 0.896 mg / ml (equivalent to 0.747 mg / ml when using 50g of Polygonum multiflorum).

[0139] In Examples 1-3, the amount of Polygonum multiflorum used was the same, resulting in a similar level of stilbene glycosides in the final product (average 0.452 mg / ml). However, in Examples 4-5, the different amounts of Polygonum multiflorum used led to significantly different results compared to Examples 1-3. Nevertheless, the amount of stilbene glycosides contributed by Polygonum multiflorum in Examples 1-5 was essentially the same based on the proportion of Polygonum multiflorum used. In Examples 6-8, when the amount of Polygonum multiflorum used was the same, the stilbene glycoside content in the final product was essentially the same (average 0.730 mg / ml), which was significantly higher than that in Examples 1-5, where the amount of Polygonum multiflorum used was comparable. This result indicates that the Twelve-Flavor Bi-tong Liniment prepared using the method in Examples 6-8 has a significantly higher stilbene glycoside content.

[0140] In all embodiments of the preparation of the Twelve-Flavor Pain-Relieving Liniment of the present invention, the Polygonum multiflorum medicinal material used was from the same batch. The inventors prepared the test solution according to the following procedure and determined the stilbene glycoside content in the Polygonum multiflorum medicinal material using the above-mentioned HPLC conditions: Approximately 0.2 g of Polygonum multiflorum medicinal material powder (passed through a No. 4 sieve) was accurately weighed and placed in a stoppered conical flask. 25 ml of dilute ethanol was accurately added, and the weight was measured. The mixture was heated under reflux for 30 minutes, cooled, and weighed again. The lost weight was replenished with dilute ethanol, shaken well, allowed to stand, and the supernatant was filtered. 5 ml of the filtrate was accurately measured and placed in a 50 ml volumetric flask. Diluted to the mark with dilute ethanol, and shaken well. The stilbene glycoside content in the Polygonum multiflorum medicinal material used in the present invention was determined to be 2.14%.

[0141] The inventors have discovered that when using the method of Example 6, the choice of solvent pH value affects the stilbene glycoside content in the final product, as shown in the following supplementary experiment a.

[0142] Supplementary Experiment a: Referring to the prescription and method of Example 6, the only difference was that the 50% ethanol used in the two maceration processes, which was pre-adjusted to pH 3.5 with hydrochloric acid, was replaced with six extracts pre-adjusted to pH 2.0, 2.5, 3.0, 4.0, 4.5, and 5.0 with hydrochloric acid. Six batches of the final product, Twelve-Flavor Pain Relief Liniment, were obtained. The amount of 50% ethanol used in the six batches of liniment (including the ethanol used in the two maceration processes and the ethanol added later) was in the range of 1370-1420 ml. The stilbene glycoside content of the six batches of liniment was determined using the method of Experiment 1 (maceration solvent pH from low to high). The results were: 0.282 mg / ml, 0.507 mg / ml, 0.693 mg / ml, 0.686 mg / ml, 0.524 mg / ml, and 0.492 mg / ml, respectively. This was combined with the pH values ​​used in Examples 6-8. 3.5 Results of the liniment obtained from the impregnation solvent show that when the pH value of the impregnation solvent is in the range of 3.0 to 4.0, the content of stilbene glycosides in the final product is significantly higher. Further decreasing or increasing the pH value will significantly reduce the stilbene glycoside content. As revealed in Experiment 4 below regarding the pharmacodynamics of the liniment, the biological effects of the two liniments from Examples 1 and 6 with different stilbene glycoside contents are significantly different. Therefore, it can be expected that using the method described in Example 6 and using an impregnation solvent with a pH of 3.0 to 4.0 will be beneficial for obtaining liniments with better biological effects.

[0143] Experimental Example 2: Determination of the Limit of Aconitine in Twelve-Flavor Bi-Tong Liniment

[0144] Take 60ml of the Twelve-Flavor Bi Tong Liniment, evaporate it to near dryness, add 25ml of water to dissolve it, add concentrated ammonia solution to adjust the pH value to 9-10, extract with 25ml of ether 4 times, combine the ether extracts, evaporate to dryness in a 50℃ water bath, dissolve the residue in anhydrous ethanol and make up to 5ml, shake well, and use it as the test solution.

[0145] Separately prepare aconitine reference standard by adding anhydrous ethanol to a solution with a concentration of 0.5 mg / ml, and use this solution as the reference standard.

[0146] Take 10 µl of the test solution, 8 µl of the reference solution, 6 µl of the reference solution, 4 µl of the reference solution, and 2 µl of the reference solution, and spot them separately on the same silica gel G thin-layer plate.

[0147] Using petroleum ether (30~60°C)-diethyl ether-acetone (5:3:2) as the developing solvent, the mixture was placed in a chromatography tank saturated with ammonia vapor, developed, removed, dried, and sprayed with modified potassium bismuth iodide reagent.

[0148] It is generally stipulated in the art that, in the chromatogram of the test sample, the spot appearing at the corresponding position as in the chromatogram of the reference sample should be smaller than the spot of 8 µl of the reference solution.

[0149] The spot sizes of Examples 1-3, 5-6, and 8 were all between the spot sizes of 4µl and 2µl of the reference solution. The spots of Examples 4 and 7 were smaller than the spot sizes of 2µl of the reference solution. This indicates that the amount of aconitine in the prepared liniments of Examples 1-5 and Examples 6-8 is essentially the same, and all conform to general guidelines in the art. Furthermore, the aconitine content in the liniments obtained by supplementary test a using two impregnation solvents at pH 3.0 and 4.0 was measured, showing that the spot sizes were all between the spot sizes of 4µl and 2µl of the reference solution.

[0150] Experimental Example 3: Precipitation Investigation of Twelve-Flavor Bi-tong Liniment

[0151] It is known that the Twelve-Flavor Bi Tong Liniment will precipitate after being left for a long time. This embodiment attempts to investigate the amount of precipitation.

[0152] Take a 200ml sample of the Twelve-Flavor Bi Tong Liniment and add it to a 200ml measuring cylinder. Let it stand at 4-6°C for 14 days. Then carefully aspirate 170ml of the liquid from the surface. Place the measuring cylinder and the remaining liquid at 45°C to air dry. Accurately stabilize the weight W1 of the measuring cylinder and the residue. Then wash the measuring cylinder, air dry it, and weigh the measuring cylinder W0. Calculate the amount of precipitate formed per 200ml using the above method, using W1-W0.

[0153] The precipitation amounts of Examples 1-5 were all in the range of 4.5-4.9g, for example, the precipitation amount of Example 1 was 4.68g. The precipitation amounts of Examples 6-8 were all in the range of 2.1-2.4g, for example, the precipitation amount of Example 6 was 2.26g. The precipitation amount of the Twelve-Flavor Pain Relief Liniment obtained by the method of Examples 6-8 was significantly lower under the above test conditions.

[0154] Furthermore, the inventors unexpectedly discovered that sedimentation treatment in two environments, pH 4.5-5.0 and pH 6.0-6.5, in Example 6 was beneficial for obtaining a liniment with low precipitation, as shown in Supplementary Experiment b below.

[0155] Supplementary test b:

[0156] Referring to the prescription and preparation method of Example 6, the only difference is that during the preparation process, the medicinal solutions obtained from the two soakings are combined, filtered, and left to stand for 7 days (maintaining pH of about 3.5). After filtration, sodium hydroxide is added to adjust the pH of the medicinal solution to 6.0~6.5 (pH 6.31), and then 50% ethanol is added to 1000ml. The solution is then filled to obtain the medicine b1 of the Twelve-Flavor Pain Relief Liniment.

[0157] Referring to the prescription and preparation method of Example 6, the only difference is that the medicinal solutions obtained from the two soakings are combined during the preparation process, filtered, and sodium hydroxide is added to adjust the pH of the medicinal solution to 6.0~6.5 (pH 6.24). After standing for 7 days, filtered again, 50% ethanol is added to make up to 1000ml, and then bottled to obtain preparation b2 of the Twelve-Flavor Pain Relief Liniment. The total amount of 50% ethanol used for preparation b1 and preparation b2 is 1385ml and 1402ml, respectively.

[0158] The precipitation amounts of reagents b1 and b2, as determined by the method in Test Example 3 of this invention, were 3.46 g and 4.83 g, respectively.

[0159] In addition, the stilbene glycoside content in reagents b1 and b2 was determined according to the method of Test Example 1 of the present invention, and the results were 0.638 mg / ml and 0.674 mg / ml, respectively.

[0160] Furthermore, the aconitine content in reagents b1 and b2 was determined according to the method of Test Example 2 of this invention. The results showed that the spot size of reagent b2 was between 4 µl and 2 µl of the reference solution, but the spot size of reagent b2 was between 8 µl and 4 µl of the reference solution and close to 8 µl. Supplementary Test b showed that the liniment (reagent b2) obtained by precipitation of the solution extracted with a low-pH solvent under near-neutral pH conditions had a higher precipitation amount; while the liniment (reagent b1) obtained by precipitation of the solution extracted with a low-pH solvent under near-neutral pH conditions had a lower precipitation amount, but a higher aconitine content, approaching an unacceptable limit. This indicates that the preparation process of Example 6 of this invention, which involves impregnation under low pH conditions followed by precipitation under medium pH conditions, not only improves the content of active ingredients such as stilbene glycosides in the final product but also results in less precipitation and lower aconitine content—a finding that was fundamentally unforeseen in the prior art.

[0161] Experimental Example 4: Pharmacological Study on the Effects of Twelve-Ingredient Bi-tong Liniment on Promoting Blood Circulation, Removing Blood Stasis, Reducing Swelling, and Relieving Pain

[0162] Numerous studies have reported on the pharmacodynamic effects of using animal models to investigate the blood-activating, stasis-removing, swelling-reducing, and pain-relieving properties of drugs. Examples include the works of Chen Qi (Methodology of Pharmacological Research of Traditional Chinese Medicine, 2nd ed. Beijing: People's Medical Publishing House, 2006: 298-299), Li Liming (Li Liming et al. Effects of Maifusheng on hemorheology and coagulation function in rats with acute blood stasis model. Journal of Guangzhou University of Traditional Chinese Medicine, 2016, 33(3): 353-357), and Liu Lifang (Liu Lifang et al. Effects of extracts from various parts of leech on rat blood stasis model induced by high molecular weight dextran. Chinese Patent Medicine, 2003, 25(7): 589-590). This experimental case also refers to the methods described in these studies to investigate the pharmacodynamic effects of the twelve-ingredient Bitong liniment prepared in this invention on the blood-activating, stasis-removing, swelling-reducing, and pain-relieving properties.

[0163] 1. Effects of drugs on xylene-induced increased capillary permeability in mouse skin

[0164] Forty male Kunming mice (SCXK(Gui)2023-0002, Guizhou Medical University (Experimental Animal Center), weighing 18-21g, were randomly divided into four groups of 10 mice each: a saline negative control group, a topical application group (0.4ml / cm³), and a treatment group (Example 1). 2 Example 6: Liniment group (0.4 ml / cm) 2 Positive control group (diclofenac diethylamine cream, diclofenac sodium, 0.4 mg API / cm³) 2 ).

[0165] One day before administration, each group had 3cm × 3cm of hair removed from both sides of the spine on the back of the animals using 6% sodium sulfide depilatory agent, and 2cm × 2cm of hair removed from the midline of the abdomen of the mice.

[0166] Apply each sample evenly to the entire hair removal area on the back according to the dosage, cover it with two layers of sterile gauze (for negative control, apply to the hair removal area after absorbing the sterile gauze), and then fix it with adhesive tape. Remove the covering after 8 hours, and remove the residual test drug with warm water. Administer once daily for 5 consecutive days.

[0167] Three hours after the last administration, 0.2 mL of 0.5% Evans blue saline solution was injected into each mouse via the tail vein. Immediately afterwards, 30 μL of xylene was dropped into the center of the shaved area on the mouse's abdomen. After 30 minutes, the mice were euthanized by cervical dislocation. Abdominal skin was harvested, inverted, and placed on a plate. The skin was graded into 10 levels based on the intensity of the blue spot color, with each level scored as 1 point (decimal scores were allowed). Each piece of skin was then minced using surgical scissors and added to a stoppered glass test tube. 10 mL of acetone-sodium sulfate solution (1% anhydrous sodium sulfate: acetone = 3:7) was added, and the tubes were placed in the dark. The tubes were gently shaken twice daily, morning and evening. After 3 days, the tubes were centrifuged at 2000 rpm for 10 minutes. The supernatant was measured at 590 nm, and the absorbance (OD) was zeroed using acetone-sodium sulfate solution without skin soaking. The inhibition rate was calculated using the following formula:

[0168] Inhibition rate = [(mean OD value of control group - mean OD value of treatment group) ÷ mean OD value of control group] × 100%.

[0169] The results are shown in Table 1.

[0170] Table 1: Effect of topical application on capillary permeability in mouse skin (mean ± SD)

[0171]

[0172] Note: ** indicates p < 0.01 compared to the negative control group, # indicates p < 0.05 compared to the liniment in Example 1.

[0173] The results showed that compared with the negative control group, both the color and OD value of the locus coeruleus decreased with statistical significance. However, compared with the liniment of Example 1, the two parameters of the liniment of Example 6 were significantly lower, indicating that the liniment of Example 6 had a significantly better inhibitory effect on the capillary permeability of the skin of mice induced by xylene.

[0174] 2. Effects of the drug on hemorheology of rats with acute blood stasis model

[0175] SD rats weighing 190 - 220 g (SCXK(Guizhou)2023 - 0002, Experimental Animal Center of Guizhou Medical University), 40 in total, half male and half female, were randomly divided into 5 groups, with 8 rats in each group, namely the negative control group, the model group, the liniment group of Example 1 (0.5 ml / cm 2 ), the liniment group of Example 6 (0.5 ml / cm 2 ), and the positive control group (Voltaren cream 0.5 mg API / cm 2 ). One day before drug administration, the skin was prepared by removing the hair on both sides of the spine of the animals with 6% sodium sulfide depilatory. The hair removal area was about 4 cm × 10 cm. Each sample was evenly applied to the entire hair removal area (the negative control group and the model group were applied with normal saline), covered with 2 layers of sterilized gauze on it, and then fixed with adhesive tape. After 8 h, the covering was removed, and the remaining samples were removed with warm water. The drug was administered once a day for 10 consecutive days. 4 h after the last drug administration, except for the negative control group, the other groups were subcutaneously injected with 0.1% epinephrine injection at 0.08 mL / 100 g body weight. After 2 h, the animals were immersed in ice water at 4°C for 5 min. After another 2 h, the same amount of epinephrine injection was injected again in the same way. After fasting (free drinking water) for 18 h, blood was taken to measure the hematocrit HCT value (L / L). Blood was collected in 1% heparin anticoagulant test tubes, and the whole blood viscosity (high shear 200 / s, medium shear 30 / s, low shear 5 / s) was measured with heparin anticoagulated blood. Then the blood was centrifuged and the plasma viscosity (mpas·s) was measured. The results are shown in Table 2.

[0176] Table 2: Effects of the liniment on hemorheology of rats (mean ± sd)

[0177]

[0178] Note: Compared with the model group, **p < 0.01, *p < 0.05.

[0179] The above results showed that compared with the negative control group, the whole blood viscosity and plasma viscosity of the model group were significantly increased (p < 0.01), indicating that the model was successfully established; compared with the model group, the whole blood viscosity and plasma viscosity of the positive control group and the liniment of Example 6 were significantly decreased (p < 0.01 or p < 0.05), while the whole blood viscosity and plasma viscosity of the liniment of Example 1 decreased, but the difference was less than that of the liniment of Example 6.

[0180] The results of the above-mentioned skin capillary permeability and acute blood stasis model show that the liniment of Example 6 of the present invention exhibits significantly better biological effects, which is related to the difference in chemical composition of the liniment obtained in Example 1.

[0181] 3. Effects of the drug on the microcirculation of the mouse auricle

[0182] Forty healthy Kunming mice, half male and half female, were randomly divided into four groups according to their body weight (18-21g): a model control group (deionized water), a positive control group (Xiaoshangtong liniment, H21022277, produced by Shenyang Junyuan Pharmaceutical Co., Ltd.), the liniment group of Example 1, and the liniment group of Example 6. Each group of animals was immersed in the respective test drug for 30 minutes, once daily for 7 consecutive days. One hour after the last administration, except for the model control group, the animals in the other groups were anesthetized by intraperitoneal injection of sodium pentobarbital 40mg / kg. Ear hair was clipped, and the diameter and flow pattern of the mouse auricular venules were observed on a microscope stage. A microcirculatory disorder model was established by injecting 10% dextran into the tail vein of each mouse at a dose of 0.2mg / mouse. The above indicators were immediately observed and recorded, and the changes in indicators before and after modeling were compared. Criteria for judging flow pattern changes: linear (linear-granular) flow is scored as follows: 4 points; dotted (granular-linear) flow is scored as follows: 3 points; granular (flocculent) flow is scored as follows: 2 points; and stagnant flow is scored as follows: flow pattern recovery time (s). The results are shown in Table 3.

[0183] Table 3: Effects of the drug on microcirculation in the mouse auricle (mean ± sd)

[0184]

[0185] Note: Compared with the model control group, **p<0.01, *p<0.05.

[0186] The results show that the liniment of Example 6 can significantly reduce the change value of microvenous flow in the auricle of mice with microcirculation disorders (p<0.05) and significantly shorten the flow recovery time (p<0.01), and its effect is significantly better than that of the liniment of Example 1.

[0187] The spirit of the present invention has been described in detail above through preferred embodiments. Those skilled in the art will understand that any modifications, equivalent variations, and alterations made to the above embodiments based on the technical essence of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing a twelve-ingredient liniment, wherein each 1000ml of the liniment is made from the following twelve medicinal materials in the following weight ratio: 50-70g of *Smilax glabra*, 50-70g of *Asarum heterotropoides*, 25-35g of *Cynanchum paniculatum*, 10-20g of *Clematis chinensis*, 8-12g of *Asarum heterotropoides* leaf, 8-12g of *Zanthoxylum bungeanum*, 8-12g of *Aconitum carmichaelii*, 20-30g of *Ligusticum striatum*, 40-60g of *Polygonum cuspidatum*, 15-25g of *Polygonum cuspidatum*, 15-25g of *Sparganium stoloniferum*, and 15-25g of *Curcuma zedoaria*; the method includes the following steps: Add 1.5 times the total weight of the twelve herbs to 50% ethanol (pre-adjusted to pH 3-4 with hydrochloric acid) to prepare 1000ml of liniment. Soak for 6 days with stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid. Add 1.5 times the total weight of the medicinal materials and soak in 50% ethanol pre-adjusted to pH 3-4 with hydrochloric acid for 5 days, stirring twice a day. After draining the liquid, squeeze the residue to fully drain the liquid. Combine the two immersion solutions, filter, add sodium hydroxide to adjust the pH to 4.5-5.0, let stand for 4 days, and then filter. After adjusting the pH of the filtrate to 6.0-6.5 with sodium hydroxide, add 50% ethanol to 1000ml, let it stand for 3 days, filter, and fill into bottles to obtain the final product.

2. According to the method of claim 1, each 1000ml of the Twelve-Flavor Pain-Relieving Liniment is made from the following twelve medicinal materials in the following weight ratio: 60g of *Artemisia argyi*, 60g of *Asarum heterotropoides*, 30g of *Cynanchum paniculatum*, 15g of *Hypericum perforatum*, 10g of *Asarum heterotropoides* leaf, 10g of Sichuan pepper, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum cuspidatum*, 20g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*.

3. According to the method of claim 1, each 1000ml of the Twelve-Flavor Pain Relief Liniment is made from the following twelve medicinal materials in the following weight ratio: 50g of *Smilax glabra*, 70g of *Asarum heterotropoides*, 25g of *Eupatorium fortunei*, 20g of *Hypericum perforatum*, 8g of *Asarum heterotropoides* leaf, 12g of Sichuan pepper, 8g of *Aconitum carmichaelii*, 30g of *Ligusticum striatum*, 40g of *Polygonum cuspidatum*, 25g of *Polygonum cuspidatum*, 15g of *Sparganium stoloniferum*, and 25g of *Curcuma zedoaria*.

4. According to the method of claim 1, each 1000ml of the Twelve-Flavor Pain-Relieving Liniment is made from the following twelve medicinal materials in the following weight ratio: 70g of *Smilax glabra*, 50g of *Asarum heterotropoides*, 35g of *Eupatorium fortunei*, 10g of *Hypericum perforatum*, 12g of *Asarum heterotropoides* leaf, 8g of Sichuan pepper, 12g of *Aconitum carmichaelii*, 20g of *Ligusticum striatum*, 60g of *Polygonum multiflorum*, 15g of *Polygonum cuspidatum*, 25g of *Sparganium stoloniferum*, and 15g of *Curcuma zedoaria*.

5. According to the method of claim 1, each 1000ml of the twelve-ingredient Bi-tong liniment is made from the following twelve medicinal materials in the following weight ratio: 60g of *Smilax glabra*, 60g of *Asarum heterotropoides*, 30g of *Cynanchum paniculatum*, 15g of *Clematis chinensis*, 10g of *Asarum heterotropoides* leaf, 10g of Sichuan pepper, 10g of *Aconitum carmichaelii*, 25g of *Ligusticum striatum*, 50g of *Polygonum multiflorum*, 20g of *Polygonum cuspidatum*, 20g of *Sparganium stoloniferum*, and 20g of *Curcuma zedoaria*; the method includes the following steps: The above twelve herbs are soaked in 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day, with 1.5 times their total weight. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, the herbs are soaked in 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 5 days, stirring twice a day, with 1.5 times their total weight. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.

67. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.23 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

6. According to the method of claim 1, each 1000ml of the twelve-ingredient Bi-tong liniment is made from the following twelve medicinal materials in the following weight ratio: 50g of *Smilax glabra*, 70g of *Asarum heterotropoides*, 25g of *Cynanchum paniculatum*, 20g of *Clematis chinensis*, 8g of *Asarum heterotropoides* leaf, 12g of *Zanthoxylum bungeanum*, 8g of *Aconitum carmichaelii*, 30g of *Ligusticum striatum*, 40g of *Polygonum multiflorum*, 25g of *Polygonum cuspidatum*, 15g of *Sparganium stoloniferum*, and 25g of *Curcuma zedoaria*; the method includes the following steps: The above twelve herbs are soaked in 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day, with 1.5 times their total weight. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, the herbs are soaked in 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 5 days, stirring twice a day, with 1.5 times their total weight. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.

56. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.17 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

7. According to the method of claim 1, each 1000ml of the twelve-ingredient Bi-tong liniment is made from the following twelve medicinal materials in the following weight ratio: 70g of *Smilax glabra*, 50g of *Asarum heterotropoides*, 35g of *Cynanchum paniculatum*, 10g of *Clematis chinensis*, 12g of *Asarum heterotropoides* leaf, 8g of *Zanthoxylum bungeanum*, 12g of *Aconitum carmichaelii*, 20g of *Ligusticum striatum*, 60g of *Polygonum multiflorum*, 15g of *Polygonum cuspidatum*, 25g of *Sparganium stoloniferum*, and 15g of *Curcuma zedoaria*; the method includes the following steps: The above twelve herbs are soaked in 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 6 days, stirring twice a day, with 1.5 times their total weight. After draining the liquid, the residue is squeezed to extract the liquid completely. Then, the herbs are soaked in 50% ethanol (pre-adjusted to pH 3.5 with hydrochloric acid) for 5 days, stirring twice a day, with 1.5 times their total weight. After draining the liquid, the residue is squeezed to extract the liquid completely. The liquids from the two soakings are combined, filtered, and sodium hydroxide is added to adjust the pH to 4.

84. The mixture is then allowed to stand for 4 days and filtered again. The filtrate is then adjusted to pH 6.38 with sodium hydroxide, and 50% ethanol is added to bring the total volume to 1000 ml. The mixture is allowed to stand for another 3 days, filtered, and bottled to obtain the final product.

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