A topical traditional Chinese medicine preparation for relieving pain and its preparation method

Abstract

This invention provides a topical traditional Chinese medicine preparation for pain relief and its preparation method, relating to the field of traditional Chinese medicine pharmaceutical technology. The preparation is made from *Saussurea involucrata*, *Eclipta prostrata*, *Eclipta prostrata*, *Rehmannia glutinosa*, *Ipomoea quamoclit*, *Kaempferia galanga*, *Phyllanthus emblica*, *Terminalia chebula*, *Gentiana macrophylla*, *Angelica sinensis*, *Paeonia suffruticosa*, *Lonicera japonica*, and *Codonopsis pilosula*. This preparation exhibits significant anti-inflammatory and analgesic effects, inhibiting the excessive secretion of LPS-induced pro-inflammatory factors TNF-α, IL-1β, and IL-6, and effectively reducing the abnormal release of NO under inflammatory conditions. Simultaneously, this preparation significantly inhibits carrageenan-induced rat paw edema and significantly reduces the content of TNF-α, IL-1β, and PGE2 in local tissues, effectively blocking the inflammatory cascade and exerting rapid swelling reduction and anti-inflammatory effects. This preparation possesses both good safety and multi-target, multi-pathway synergistic anti-inflammatory properties, inhibiting excessive secretion of inflammatory factors and effectively relieving acute inflammatory symptoms such as local redness, swelling, heat, and pain.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically to a topical traditional Chinese medicine preparation for relieving pain and its preparation method. Background Technology

[0002] Pain is a common clinical symptom, widely seen in various disease states such as rheumatoid arthritis, osteoarthritis, soft tissue injuries, neuropathic pain, and postoperative wounds. Currently, clinical treatment of pain mainly relies on nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids. Although these drugs have a rapid onset of action, long-term or high-dose use can easily lead to significant side effects such as tolerance, gastrointestinal damage, liver and kidney toxicity, and addiction, limiting their safety for long-term use.

[0003] Traditional Chinese medicine (TCM) has a long history and rich experience in treating pain externally. TCM believes that the core pathogenesis of pain is often "cold stagnation and blood stasis obstructing the meridians," or "pain arises from obstruction," often accompanied by inflammation-mediated local damage. Some patients also experience "pain due to deficiency of vital energy." To address these pathogenesis, physicians throughout history have skillfully employed methods such as promoting blood circulation and removing blood stasis, dispelling wind and dampness, clearing heat and detoxifying, and unblocking the meridians to relieve pain, resulting in numerous effective external prescriptions. Currently available external TCM analgesic preparations include Wusong Zhongtong Tincture, Yunnan Baiyao aerosol, and various TCM ointments and patches. However, existing topical Chinese medicine analgesic formulas still have many shortcomings: some formulas only use single blood-activating or cold-dispelling herbs, failing to form a scientific combination of principal, assistant, and adjuvant herbs, thus failing to achieve the synergistic effects of warming and dispelling cold, activating blood circulation and removing blood stasis, anti-inflammatory and analgesic, and strengthening the body's resistance; while some formulas take multiple effects into account, the proportions of herbs are unreasonable, either being too warming and drying, easily damaging the skin or spleen and stomach, or too cold, resulting in slow analgesic effects, seriously limiting their clinical application and promotion. Therefore, there is an urgent need to develop a topical Chinese medicine preparation with scientific compatibility, a clear mechanism of action, and good safety to overcome the shortcomings of existing technologies, such as single efficacy and obvious side effects, and to provide a better treatment option for pain-related diseases. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a topical traditional Chinese medicine preparation for relieving pain and its preparation method.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a topical traditional Chinese medicine preparation for relieving pain, which is made from the following raw materials in parts by weight: 13-30 parts of snow lotus, 10-30 parts of *Ligusticum striatum*, 3-15 parts of *Eclipta prostrata*, 8-25 parts of *Rehmannia glutinosa*, 5-25 parts of *Aristolochia debilis*, 5-25 parts of *Kaempferia galanga*, 3-21 parts of *Phyllanthus emblica*, 3-21 parts of *Terminalia chebula*, 8-25 parts of *Gentiana macrophylla*, 3-21 parts of *Evodia rutaecarpa*, 8-25 parts of *Paeonia suffruticosa*, 8-25 parts of *Lonicera japonica*, and 10-30 parts of *Codonopsis pilosula*.

[0009] Furthermore, the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 18-26 parts of snow lotus, 15-25 parts of *Eclipta prostrata*, 4-10 parts of *Eclipta prostrata*, 13-21 parts of *Rehmannia glutinosa*, 10-20 parts of *Aristolochia debilis*, 10-20 parts of *Kaempferia galanga*, 8-16 parts of *Phyllanthus emblica*, 8-16 parts of *Terminalia chebula*, 13-20 parts of *Gentiana macrophylla*, 8-16 parts of *Angelica sinensis*, 13-21 parts of *Paeonia suffruticosa*, 13-21 parts of *Lonicera japonica*, and 15-25 parts of *Codonopsis pilosula*.

[0010] Furthermore, the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 22 parts of snow lotus, 20 parts of *Eclipta prostrata*, 7 parts of *Eclipta prostrata*, 18 parts of *Rehmannia glutinosa*, 15 parts of *Aristolochia debilis*, 15 parts of *Kaempferia galanga*, 12 parts of *Phyllanthus emblica*, 12 parts of *Terminalia chebula*, 17 parts of *Gentiana macrophylla*, 12 parts of *Evodia rutaecarpa*, 18 parts of *Paeonia suffruticosa*, 18 parts of *Lonicera japonica*, and 20 parts of *Codonopsis pilosula*.

[0011] This invention provides a method for preparing a topical traditional Chinese medicine preparation for relieving pain. The traditional Chinese medicine preparation is prepared by adding pharmaceutically acceptable excipients according to conventional pharmaceutical methods, and is made into a topical preparation, including ointments, creams, gels, sprays, aerosols, plasters, and liniments.

[0012] Furthermore, the preparation method of the traditional Chinese medicine ointment includes the following steps:

[0013] (1) Preparation of Chinese herbal extracts: Weigh each Chinese herbal raw material according to the above weight proportions, add 8 times the amount of 75% ethanol for ultrasonic extraction for 2 h, and filter; add 6 times the amount of 75% ethanol to the filter residue again for ultrasonic extraction for 1.5 h, and filter; combine the two filtrates, concentrate under reduced pressure to a thick paste with a relative density of 1.10-1.35, and set aside;

[0014] (2) Preparation of oil phase: Stearic acid, glyceryl monostearate, liquid paraffin and lanolin are placed in a beaker, heated in a water bath to 75-80℃, stirred until completely melted, and kept warm for later use;

[0015] (3) Aqueous phase preparation: Dissolve the thick paste of Chinese herbal extract, glycerin, triethanolamine and ethylparaben obtained in step (1) in purified water, heat in a water bath at 75-80℃ and stir evenly, and keep warm for later use;

[0016] (4) Emulsification and molding: Under continuous stirring, the oil phase is slowly added to the water phase, and high-speed homogenization emulsification is continued at 75-80℃ for 10-15 min to form a primary emulsion; then heating is stopped, stirring is continued and cooling is carried out to room temperature, and the ointment is obtained after solidification.

[0017] This invention provides the application of the aforementioned traditional Chinese medicine preparation in the preparation of a drug for relieving pain.

[0018] The pharmacological effects of each traditional Chinese medicine in the herbal preparation of this invention are as follows:

[0019] Snow lotus: The whole plant, including roots, of *Saussurea involucrata* and *Saussurea involucrata*, both belonging to the Asteraceae family. It has a sweet and bitter taste and is warm in nature. It enters the liver and kidney meridians. It has the effects of tonifying the kidneys and strengthening yang, regulating menstruation and stopping bleeding. It is used for snow blindness, toothache, rheumatoid arthritis, impotence, irregular menstruation, metrorrhagia, and leukorrhea; externally, it is used to treat traumatic bleeding.

[0020] Rehmannia root: The dried tuberous root of Rehmannia glutinosa, a plant in the Scrophulariaceae family. It is sweet in taste and cold in nature. It enters the heart, liver, and kidney meridians. It has the effects of clearing heat and cooling the blood, nourishing yin and promoting body fluid production. It is used for heat entering the blood level, febrile rashes, hematemesis and epistaxis, yin deficiency due to febrile diseases, red tongue with thirst, constipation due to fluid depletion, yin deficiency fever, steaming bone fever, and internal heat with thirst.

[0021] Gentian root: The dried aerial parts of Artemisia annua, a plant in the Asteraceae family. It has a bitter taste and is cold in nature. It enters the lung and liver meridians. It has the effects of clearing heat and resolving phlegm, relieving cough and asthma, detoxifying and promoting diuresis, cooling the blood and stopping bleeding. It is used for cough due to lung heat, excessive phlegm and wheezing, sore throat, canker sores, damp-heat jaundice, epistaxis, hematochezia, metrorrhagia, and traumatic bleeding.

[0022] Small-leaved lotus: The dried, mature fruit of *Berberis vulgaris*, a plant in the Berberidaceae family. It has a sweet taste and neutral properties. It is used to regulate menstruation and promote blood circulation. It is indicated for amenorrhea due to blood stasis, dystocia, stillbirth, and retained placenta.

[0023] Kaempferia galanga: The dried rhizome of Kaempferia galanga, a plant in the ginger family. It has a pungent taste and warm properties. It enters the stomach meridian. It has the effects of regulating qi and warming the middle jiao, promoting digestion, and relieving pain. It is used for chest and diaphragm distension, cold pain in the epigastrium and abdomen, and indigestion.

[0024] Nine-dragon vine: The stem of the legume *Dracaena sanderiana*. It has a pungent and bitter taste, and is neutral in nature. It enters the liver and kidney meridians. It has the effects of dispelling wind and dampness, promoting qi and blood circulation. It is used for rheumatic pain, traumatic injuries, hemiplegia, stomach pain, malnutrition, and dysentery.

[0025] Medicinal Use: The dried whole herb of *Swertia scabra*, a plant in the Gentianaceae family. It is bitter and cold in nature. It enters the liver, stomach, and large intestine meridians. It has the effects of clearing damp-heat and strengthening the stomach. It is used for damp-heat jaundice, hypochondriac pain, dysentery with abdominal pain, and loss of appetite.

[0026] Hand-shaped Ginseng: The tuber of *Gynostemma pentaphyllum* and *Gynostemma scabra*, both belonging to the Orchidaceae family. It has a sweet taste and neutral properties. It enters the lung, spleen, and stomach meridians. It has the effects of relieving cough and asthma, tonifying the kidneys and spleen, regulating qi and blood, and relieving pain. It is used for cough and asthma due to lung deficiency, emaciation due to overwork, neurasthenia, soreness and weakness of the lower back and legs due to kidney deficiency, impotence, spermatorrhea, frequent urination, chronic hepatitis, chronic diarrhea, blood loss, leukorrhea, insufficient lactation, and traumatic injuries.

[0027] Phyllanthus emblica: The dried, ripe fruit of the plant Phyllanthus emblica, belonging to the Euphorbiaceae family. It has a sweet, sour, and astringent taste, and is cool in nature. It enters the lung and stomach meridians. It has the effects of clearing heat and cooling the blood, promoting digestion and strengthening the stomach, and generating fluids and relieving cough. It is used for blood heat and blood stasis, indigestion, abdominal distension, cough, sore throat, and dry mouth.

[0028] Terminalia chebula: The dried, ripe fruit of Terminalia chebula, a plant in the Combretaceae family. It has a sweet and astringent taste and is neutral in nature. It possesses the functions of clearing heat and detoxifying, astringing and nourishing blood, and harmonizing other herbs. It is used for various heat-related syndromes, diarrhea, jaundice, liver and gallbladder diseases, and post-illness weakness.

[0029] *Lysimachia christinae*: The dried aerial parts of *Lysimachia christinae*, a plant in the Lamiaceae family. It has a sweet and bitter taste, and is neutral in nature. It enters the Liver meridian. It has the effects of promoting blood circulation and stopping bleeding, dispelling wind and relieving pain. It is used for traumatic injuries, external bleeding, rheumatic pain, and jaundice.

[0030] Honeysuckle vine: The dried stems and branches of *Lonicera japonica*, a plant in the Caprifoliaceae family. It has a sweet taste and cold properties. It enters the lung and stomach meridians. It has the effects of clearing heat and detoxifying, dispelling wind and unblocking the meridians. It is used for febrile diseases, dysentery due to heat toxicity, carbuncles and sores, rheumatic arthralgia, and red, swollen, hot, and painful joints.

[0031] Moutan bark: The dried root bark of the peony plant (Ranunculaceae family). It tastes bitter and pungent, and is slightly cold in nature. It enters the heart, liver, and kidney meridians. It has the effects of clearing heat and cooling the blood, promoting blood circulation and removing blood stasis. It is used for heat entering the blood level, febrile diseases with rashes, hematemesis and epistaxis, night fever and morning coolness, steaming bone fever without sweating, amenorrhea and dysmenorrhea, traumatic injuries, carbuncles and boils.

[0032] (III) Beneficial Effects

[0033] This invention provides a topical traditional Chinese medicine preparation for relieving pain and its preparation method. The preparation is made from snow lotus, *Ligusticum striatum*, *Ligusticum chuanxiong*, *Rehmannia glutinosa*, *Ipomoea quamoclit*, *Kaempferia galanga*, *Phyllanthus emblica*, *Terminalia chebula*, *Gentiana macrophylla*, *Angelica sinensis*, *Paeonia suffruticosa*, *Lonicera japonica*, and *Codonopsis pilosula*. This invention follows the principle of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine formulation, using snow lotus and *Ligusticum striatum* as the principal herbs. Snow lotus has the effects of warming the kidneys and strengthening yang, dispelling cold and dampness, and promoting blood circulation; *Ligusticum striatum* has the effects of promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. Snow lotus warms yang to dispel cold stagnation, while *Ligusticum striatum* promotes blood circulation to remove blood stasis. The two herbs, one warming and one promoting circulation, work together to target the two core causes of pain, "cold" and "blood stasis," achieving a combined effect of warming, promoting blood circulation, and effectively relieving pain. The formula uses four herbs as assistant herbs: *Erigeron simonii*, *Rehmannia glutinosa*, *Aristolochia debilis*, and *Kaempferia galanga*. *Erigeron simonii* dispels wind and dampness, invigorates blood and relieves pain, and detoxifies and resolves phlegm; *Rehmannia glutinosa* clears heat and cools blood, nourishes yin and generates fluids; *Aristolochia debilis* dispels wind and dampness, invigorates blood and relieves pain, and unblocks meridians; and *Kaempferia galanga* warms the middle jiao and dispels cold, promotes qi circulation and relieves pain, and aids digestion. These four herbs enhance the analgesic effect of the principal herb by dispelling dampness, nourishing yin, unblocking meridians, and promoting qi circulation, respectively, while also regulating the balance of cold and heat in the body and protecting the spleen and stomach. The formula uses Phyllanthus emblica, Terminalia chebula, Gentiana macrophylla, Angelica dahurica, and Paeonia suffruticosa as adjuvant herbs. Phyllanthus emblica clears heat and cools the blood, promotes digestion and strengthens the stomach, generates fluids and relieves cough; Terminalia chebula clears heat and detoxifies, astringes and nourishes the blood; Gentiana macrophylla clears heat and detoxifies, promotes diuresis and invigorates blood; Angelica dahurica clears heat and promotes diuresis, strengthens the spleen and stomach, detoxifies and relieves pain; Paeonia suffruticosa clears heat and cools the blood, invigorates blood and removes blood stasis. Each of the five adjuvant herbs performs its function, refining the analgesic effect and restraining the imbalance of the principal and assistant herbs, thus protecting the body's vital energy. Lonicera japonica and Panax quinquefolius are used as guiding herbs. Lonicera japonica clears heat and detoxifies, dispels wind and unblocks the meridians; Panax quinquefolius tonifies qi and blood, generates fluids and relieves cough, and benefits the kidneys and strengthens the spleen. The two herbs work together, one active and one passive, one clearing and one tonifying, guiding the other herbs directly to the affected area and harmonizing their properties, ensuring that the entire formula eliminates pathogens without harming the body's vital energy.

[0034] This study systematically evaluated the anti-inflammatory and analgesic effects of the traditional Chinese medicine preparation of this invention through a combination of in vitro and in vivo methods. The results showed that the traditional Chinese medicine preparation significantly inhibited the excessive secretion of LPS-induced pro-inflammatory factors TNF-α, IL-1β, and IL-6, and effectively reduced the abnormal release of NO under inflammatory conditions, suggesting that it can alleviate oxidative stress and inflammatory damage by regulating the iNOS / NO pathway and the inflammatory factor network. Simultaneously, the preparation significantly inhibited carrageenan-induced paw edema in rats and significantly reduced the levels of TNF-α, IL-1β, and PGE2 in local tissues, effectively blocking the inflammatory cascade and exerting rapid swelling reduction and anti-inflammatory effects. The traditional Chinese medicine preparation of this invention possesses both good safety and multi-target, multi-pathway synergistic anti-inflammatory properties, effectively inhibiting the excessive secretion of inflammatory factors and effectively relieving acute inflammatory symptoms such as local redness, swelling, heat, and pain, providing solid experimental evidence for its development and application as a topical anti-inflammatory and analgesic drug. Attached Figure Description

[0035] Figure 1 For comparison of cell viability in each group.

[0036] Figure 2The degree of paw swelling was compared among the groups of rats.

[0037] Figure 3 Comparison of expression levels of inflammation-related factors in the plantar tissues of rats in each group; Note: Compared with the control group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] A topical traditional Chinese medicine preparation for relieving pain, the preparation being made from the following raw materials in parts by weight: 22 parts of snow lotus, 20 parts of *Eclipta prostrata*, 7 parts of *Eclipta prostrata*, 18 parts of *Rehmannia glutinosa*, 15 parts of *Aristolochia debilis*, 15 parts of *Kaempferia galanga*, 12 parts of *Phyllanthus emblica*, 12 parts of *Terminalia chebula*, 17 parts of *Gentiana macrophylla*, 12 parts of *Evodia rutaecarpa*, 18 parts of *Paeonia suffruticosa*, 18 parts of *Lonicera japonica*, and 20 parts of *Codonopsis pilosula*.

[0041] The preparation method of the traditional Chinese medicine ointment includes the following steps:

[0042] (1) Preparation of Chinese herbal extract: Weigh each Chinese herbal raw material according to the above weight proportions, add 8 times the amount of 75% ethanol for ultrasonic extraction for 2 h, and filter; add 6 times the amount of 75% ethanol to the filter residue again for ultrasonic extraction for 1.5 h, and filter; combine the two filtrates, concentrate under reduced pressure to a thick paste with a relative density of 1.15, and set aside for later use;

[0043] (2) Preparation of oil phase: Stearic acid, glyceryl monostearate, liquid paraffin and lanolin are placed in a beaker, heated in a water bath to 75-80℃, stirred until completely melted, and kept warm for later use;

[0044] (3) Aqueous phase preparation: Dissolve the thick paste of Chinese herbal extract, glycerin, triethanolamine and ethylparaben obtained in step (1) in purified water, heat in a water bath at 75-80℃ and stir evenly, and keep warm for later use;

[0045] (4) Emulsification and molding: Under continuous stirring, the oil phase is slowly added to the water phase, and high-speed homogenization emulsification is continued at 75-80℃ for 10-15 min to form a primary emulsion; then heating is stopped, stirring is continued and cooling is carried out to room temperature, and the ointment is obtained after solidification.

[0046] Example 2

[0047] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 13 parts of snow lotus, 10 parts of *Duyiwei*, 3 parts of *Xiaoyelian*, 8 parts of *Shengdi*, 5 parts of *Jiulongteng*, 5 parts of *Shankao*, 3 parts of *Yuganzi*, 3 parts of *Maohezi*, 8 parts of *Jinlongdancao*, 3 parts of *Dangyao*, 8 parts of *Danpi*, 8 parts of *Lonicera japonica*, and 10 parts of *Shouzhangshen*.

[0048] Example 3

[0049] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 28 parts of snow lotus, 28 parts of *Duyiwei*, 12 parts of *Xiaoyelian*, 22 parts of *Shengdi*, 22 parts of *Jiulongteng*, 22 parts of *Shankao*, 18 parts of *Yuganzi*, 18 parts of *Maohezi*, 22 parts of *Jinlongdancao*, 19 parts of *Dangyao*, 22 parts of *Danpi*, 23 parts of *Lonicera japonica*, and 28 parts of *Shouzhangshen*.

[0050] Example 4

[0051] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 15 parts of snow lotus, 12 parts of *Eclipta prostrata*, 5 parts of *Eclipta prostrata*, 10 parts of *Rehmannia glutinosa*, 7 parts of *Ipomoea quamoclit*, 7 parts of *Kaempferia galanga*, 5 parts of *Phyllanthus emblica*, 5 parts of *Terminalia chebula*, 10 parts of *Gentiana macrophylla*, 5 parts of *Evodia rutaecarpa*, 10 parts of *Paeonia suffruticosa*, 10 parts of *Lonicera japonica*, and 12 parts of *Codonopsis pilosula*.

[0052] Example 5

[0053] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 30 parts of snow lotus, 30 parts of *Duyiwei*, 15 parts of *Xiaoyelian*, 25 parts of *Shengdi*, 25 parts of *Jiulongteng*, 25 parts of *Shankao*, 21 parts of *Yuganzi*, 21 parts of *Maohezi*, 25 parts of *Jinlongdancao*, 21 parts of *Dangyao*, 25 parts of *Danpi*, 25 parts of *Lonicera japonica*, and 30 parts of *Shouzhangshen*.

[0054] Example 6

[0055] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 20 parts of snow lotus, 18 parts of *Duyiwei*, 10 parts of *Xiaoyelian*, 15 parts of *Shengdi*, 11 parts of *Jiulongteng*, 12 parts of *Shankao*, 9 parts of *Yuganzi*, 10 parts of *Maohezi*, 15 parts of *Jinlongdancao*, 9 parts of *Dangyao*, 15 parts of *Danpi*, 15 parts of *Lonicera japonica*, and 18 parts of *Shouzhangshen*.

[0056] Example 7

[0057] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 25 parts of snow lotus, 20 parts of *Duyiwei*, 10 parts of *Xiaoyelian*, 20 parts of *Shengdi*, 18 parts of *Jiulongteng*, 18 parts of *Shankao*, 16 parts of *Yuganzi*, 16 parts of *Maohezi*, 19 parts of *Jinlongdancao*, 15 parts of *Dangyao*, 20 parts of *Danpi*, 21 parts of *Lonicera japonica*, and 25 parts of *Shouzhangshen*.

[0058] Example 8

[0059] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 18 parts of snow lotus, 15 parts of *Duyiwei*, 8 parts of *Xiaoyelian*, 12 parts of *Shengdi*, 9 parts of *Jiulongteng*, 9 parts of *Shankao*, 7 parts of *Yuganzi*, 7 parts of *Maohezi*, 12 parts of *Jinlongdancao*, 7 parts of *Dangyao*, 12 parts of *Danpi*, 12 parts of *Lonicera japonica*, and 15 parts of *Shouzhangshen*.

[0060] Experimental Example 1

[0061] 1. Experimental drugs

[0062] Test substance 1: Snow lotus 22 g, Du Yi Wei 20 g, Xiao Ye Lian 7 g, Sheng Di 18 g, Jiu Long Teng 15 g, Shan Jia 15 g, Yang Gan Zi 12 g, Mao He Zi 12 g, Jin Long Dan Cao 17 g, Dang Yao 12 g, Dan Pi 18 g, Lonicera japonica 18 g, Shou Zhang Shen 20 g.

[0063] Test substance 2: 20 g of *Lysimachia christinae*, 7 g of *Lysimachia christinae*, 18 g of *Rehmannia glutinosa*, 15 g of *Gynostemma pentaphyllum*, 15 g of *Kaempferia galanga*, 12 g of *Eupatorium fortunei*, 12 g of *Terminalia chebula*, 18 g of *Paeonia suffruticosa*, 18 g of *Lonicera japonica*, and 20 g of *Codonopsis pilosula*.

[0064] Test substance 3: Snow lotus 22 g, Rehmannia glutinosa 18 g, 9 dragon vine 15 g, galangal 15 g, Phyllanthus emblica 12 g, Terminalia chebula 12 g, Gentiana scabra 17 g, Angelica dahurica 12 g, Lonicera japonica 18 g, Panax ginseng 20 g.

[0065] Test substance 4: Snow lotus 22 g, Du Yi Wei 20 g, Xiao Ye Lian 7 g, Shan Jia 15 g, Jin Long Dan Cao 17 g, Dang Yao 12 g, Dan Pi 18 g, Lonicera japonica 18 g, and Shou Zhang Shen 20 g.

[0066] Test substance 5: Snow lotus 22 g, Du Yi Wei 20 g, Xiao Ye Lian 7 g, Sheng Di 18 g, Jiu Long Teng 15 g, Yang Gan Zi 12 g, Mao He Zi 12 g, Jin Long Dan Cao 17 g, Dang Yao 12 g, Dan Pi 18 g.

[0067] 2 Experimental Methods

[0068] 2.1 Preparation of extracts

[0069] Weigh out each herb corresponding to test substances 1-5, wash them, add 8 times the total mass of the herbs to pure water, and extract by sonication for 2 h. After filtration, add 6 times the total mass of the herbs to pure water again and extract by sonication for 1.5 h. Filter again, combine the two filtrates, and concentrate them in a rotary evaporator to a crude drug concentration of 1 g / mL. After cooling to room temperature, filter and sterilize using a 0.22 μm microporous membrane, dispense into sterile centrifuge tubes, and freeze at -20℃ for later use. Before use, bring the solution to room temperature and dilute to the corresponding concentration with DMEM high-glucose medium according to experimental requirements.

[0070] 2.2 Cell Culture and Model Establishment

[0071] RAW264.7 cells were revived in DMEM high-glucose medium (containing 10% FBS and 1% penicillin-streptomycin mixture) and cultured at 37°C with 5% CO2 until the 3rd or 4th passage. Cells were then cultured at a rate of 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates and cultured for 24 h. After cell adhesion, the prepared extracts (final concentration 200 μg / mL) were added to each well of groups 1-5 of the test substance. The blank group and the model group were added with an equal volume of DMEM high-glucose medium. One h later, LPS was added to each well for stimulation at a final concentration of 100 ng / mL. The blank control group was not given LPS, but only DMEM high-glucose medium. The cells were cultured for 24 h.

[0072] 2.3 CCK-8 assay for cell viability

[0073] Cell viability was determined using a CCK-8 assay kit. After the experiment, the supernatant was discarded, and 100 μL of DMEM high-glucose medium containing 10% CCK-8 was added to each well. After incubation in the dark for 2 h, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0074] 2.4 ELISA method for detecting TNF-α, IL-1β, and IL-6 levels in cell culture supernatant

[0075] The expression levels of inflammation-related factors in the cell culture supernatant of each group were determined using an ELISA kit. After cell culture, the cell culture supernatant of each group was collected, and the expression levels of TNF-α, IL-1β, and IL-6 in the cell supernatant of each group were determined strictly according to the instructions of the TNF-α, IL-1β, and IL-6 ELISA kit.

[0076] 2.5 Determination of NO generation

[0077] The NO content in cell supernatant was determined using the Griess reagent method. First, equal volumes of Griess reagent A (1% sulfonamide) and B (0.1% NED) were mixed, and then an equal volume of the cell supernatant was added. After reacting at room temperature in the dark for 10 minutes, the absorbance was read at 540 nm using a microplate reader. NO2 was calculated using a sodium nitrate standard curve. - concentration.

[0078] 2.6 Statistical Methods

[0079] SPSS 26.0 software was used for statistical analysis. Experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups. If the variances were homogeneous, pairwise comparisons were further performed using the LSD test; if the variances were unequal, Dunnett's T3 test was used. A p-value < 0.05 was considered statistically significant.

[0080] 3 Results

[0081] 3.1 Effects of various drugs on cell viability

[0082] Depend on Figure 1 It was found that the cell viability in the model group decreased to about 50%, indicating that LPS successfully induced inflammatory damage in RAW264.7 cells. Meanwhile, the cell viability in test group 1-5 remained consistently above 95%, with no significant difference compared to the control group (P>0.05), indicating that the herbal preparation of this invention has no significant toxic effect on RAW264.7 macrophages and has good safety.

[0083] 3.2 Effects of various drugs on the levels of inflammation-related factors

[0084] As shown in Table 1, compared with the blank group, the expression levels of inflammation-related factors TNF-α, IL-1β, and IL-6 in the cell supernatant of the model group were significantly increased (P<0.01), indicating that LPS successfully induced an inflammatory response in RAW264.7 cells. Compared with the model group, the expression levels of the above three inflammatory factors in the cell supernatant of test substance groups 1-5 were significantly decreased (P<0.05 or P<0.01), with the decrease in test substance group 1 being the most significant (P<0.01), indicating that the traditional Chinese medicine preparation of the present invention can effectively reduce cellular inflammatory response by inhibiting the overexpression of inflammatory factors.

[0085] Table 1. Comparison of levels of inflammation-related factors in the supernatant of each group (pg / mL)

[0086]

[0087] Note: Compared with the blank group, # P<0.05, ##P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0088] 3.3 Effects of various drugs on NO production

[0089] As shown in Table 2, compared with the blank group, the NO content in the cell supernatant of the model group was significantly increased (P<0.01); compared with the model group, the NO content in the cell supernatant of test substance 1-5 groups all decreased to varying degrees, among which the decrease trend of test substance 1 group was the most significant (P<0.01). This indicates that the traditional Chinese medicine preparation of the present invention can effectively inhibit the production and release of NO in RAW264.7 cells under inflammatory conditions, and further reduce inflammatory damage by regulating the NO-mediated inflammatory pathway.

[0090] Table 2 Comparison of NO generation in supernatant of each group

[0091]

[0092] Note: Compared with the blank group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0093] 4. Conclusion

[0094] This study established an in vitro inflammation model using LPS-induced RAW264.7 macrophages and systematically evaluated the safety and anti-inflammatory activity of the traditional Chinese medicine (TCM) preparation of this invention. Results showed that the preparation had no significant cytotoxicity to RAW264.7 cells within the tested concentration range, confirming its good biocompatibility. Simultaneously, the preparation significantly inhibited the excessive secretion of LPS-induced pro-inflammatory factors TNF-α, IL-1β, and IL-6, and significantly reduced the abnormal release of NO under inflammatory conditions, further intervening in the iNOS / NO inflammatory pathway and alleviating oxidative stress and tissue damage. Further analysis revealed that the above effects were most pronounced in group 1, indicating a synergistic effect among the various TCM components in the TCM preparation of this invention, further confirming the rationality of the formulation. In summary, the TCM preparation of this invention not only has good safety but also possesses multi-target, multi-pathway synergistic anti-inflammatory characteristics, capable of inhibiting the overexpression of inflammatory factors and regulating NO-mediated inflammatory pathways from the source, providing reliable in vitro experimental support for its topical treatment of pain-related inflammatory diseases.

[0095] Experimental Example 2

[0096] 1. Experimental drugs

[0097] 22 g of snow lotus, 20 g of *Ligusticum striatum*, 7 g of *Eclipta prostrata*, 18 g of *Rehmannia glutinosa*, 15 g of *Ipomoea quamoclit*, 15 g of *Kaempferia galanga*, 12 g of *Phyllanthus emblica*, 12 g of *Terminalia chebula*, 17 g of *Gentiana macrophylla*, 12 g of *Angelica sinensis*, 18 g of *Paeonia suffruticosa*, 18 g of *Lonicera japonica*, and 20 g of *Codonopsis pilosula*. Weigh each ingredient according to the above weight ratios and prepare the cream according to the preparation method described in Example 1.

[0098] 2 Experimental Methods

[0099] 2.1 Model Construction and Drug Administration

[0100] Healthy SPF-grade SD rats, after one week of acclimatization, were randomly divided into five groups using a random number table: a control group, a model group, a low-dose traditional Chinese medicine preparation group, a high-dose traditional Chinese medicine preparation group, and a positive control group, with 10 rats in each group. Except for the control group, rats in the other groups were injected subcutaneously into the right hind paw to establish a paw swelling and inflammation model, while rats in the control group were injected subcutaneously into the right hind paw with an equal volume of physiological saline.

[0101] Thirty minutes after inducing inflammation, local administration was initiated in each group of rats, with the administration sites being the swollen areas on the right hind paw and plantar surface. The low-dose group received 0.5 g / kg body weight of the herbal cream, the high-dose group received 1 g / kg body weight of the herbal cream, the positive control group received 0.2 g / rat diclofenac gel, and the blank control and model groups received an equal volume of physiological saline. Administration was twice daily for three consecutive days. During the administration period, the rats' mental state, food and water intake, and changes in plantar swelling were closely monitored, and any abnormalities were promptly recorded.

[0102] 2.2 Detection Indicators

[0103] 2.2.1 Foot swelling

[0104] The thickness of the right hind paw of rats was measured using vernier calipers before inflammation and at 1 h, 2 h, 4 h, and 6 h after inflammation. The paw swelling degree was calculated at each time point. Paw swelling degree = (paw thickness after inflammation - paw thickness before inflammation) / paw thickness before inflammation × 100%.

[0105] 2.2.2 Inflammatory factors in the plantar tissue

[0106] After drug administration, rats were anesthetized by intraperitoneal injection of sodium pentobarbital. Tissue from the swollen area of ​​the right hind paw was quickly harvested, rinsed in pre-cooled PBS, blotted dry, and weighed. Tissue homogenization buffer was added at a 1:9 (w / v) ratio, and the mixture was homogenized on ice using a tissue homogenizer under ice bath conditions. The tissue homogenate was centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected. The levels of TNF-α, IL-1β, and PGE2 in the tissue homogenate supernatant were detected using ELISA.

[0107] 2.3 Statistical Methods

[0108] SPSS 26.0 software was used for statistical analysis. All data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. If homogeneity of variance was satisfied, the LSD method was used for pairwise comparisons between groups; if the variances were not homogeneous, Dunnett's T3 method was used. A p-value < 0.05 was considered statistically significant.

[0109] 3 Results

[0110] 3.1 Comparison of paw edema in rats of different groups

[0111] Depend on Figure 2 It was found that, compared with the blank group, the paw swelling of rats in the model group continued to increase within 0-4 h after modeling, reaching a peak at 4 h (P<0.01), and then gradually decreased, but was still significantly higher than that in the blank group. Compared with the model group, all treatment groups could inhibit the development of paw swelling to varying degrees. Among them, the high-dose group and the positive control group of the traditional Chinese medicine preparation of this invention showed significant anti-swelling effects from 2 h (P<0.05), and reached the maximum inhibitory effect at 4 h (P<0.01). The low-dose group of the test substance also showed a certain inhibitory trend after 4 h, but the difference was not statistically significant (P>0.05). The above results indicate that the traditional Chinese medicine preparation of this invention can alleviate local tissue edema caused by acute inflammation in a dose-dependent manner, and has a good anti-inflammatory and swelling-reducing effect.

[0112] 3.2 Comparison of inflammatory factor levels in the paw tissues of rats in different groups

[0113] As shown in Figure 3, compared with the blank group, the levels of inflammatory factors TNF-α, IL-1β, and PGE2 in the plantar tissue of rats in the model group were significantly increased (P<0.01); compared with the model group, the levels of TNF-α, IL-1β, and PGE2 in the plantar tissue of each treatment group decreased to varying degrees. Among them, the high-dose group and the positive control group showed the most significant inhibitory effects (P<0.01), and the low-dose group also showed a significant decreasing trend (P<0.05), indicating that the traditional Chinese medicine preparation of this invention can significantly inhibit the release of multiple key inflammatory mediators in the inflamed plantar tissue, and its anti-inflammatory mechanism involves multi-target and multi-pathway synergistic regulation.

[0114] 4. Conclusion

[0115] This study used a carrageenan-induced acute paw edema model in rats to systematically evaluate the in vivo anti-inflammatory and anti-edema effects of the traditional Chinese medicine preparation of this invention. The results showed that the preparation significantly inhibited the development of paw edema in rats and significantly reduced the levels of key pro-inflammatory mediators TNF-α, IL-1β, and PGE2 in inflamed tissues, effectively blocking the inflammatory cascade. In summary, the traditional Chinese medicine preparation of this invention exhibits good anti-inflammatory and anti-edema effects in vivo, showing a dose-dependent characteristic. It can not only rapidly relieve local swelling caused by acute inflammation but also regulate the release of multiple inflammatory factors at their source, providing solid experimental evidence for its development and application as a topical anti-inflammatory and analgesic drug.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A topical traditional Chinese medicine preparation for relieving pain, characterized in that, The traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 13-30 parts of snow lotus, 10-30 parts of *Eclipta prostrata*, 3-15 parts of *Eclipta prostrata*, 8-25 parts of *Rehmannia glutinosa*, 5-25 parts of *Aristolochia debilis*, 5-25 parts of *Kaempferia galanga*, 3-21 parts of *Phyllanthus emblica*, 3-21 parts of *Terminalia chebula*, 8-25 parts of *Gentiana macrophylla*, 3-21 parts of *Evodia rutaecarpa*, 8-25 parts of *Paeonia suffruticosa*, 8-25 parts of *Lonicera japonica*, and 10-30 parts of *Codonopsis pilosula*.

2. The topical traditional Chinese medicine preparation for relieving pain according to claim 1, characterized in that, The traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 18-26 parts of snow lotus, 15-25 parts of *Eclipta prostrata*, 4-10 parts of *Eclipta prostrata*, 13-21 parts of *Rehmannia glutinosa*, 10-20 parts of *Aristolochia debilis*, 10-20 parts of *Kaempferia galanga*, 8-16 parts of *Phyllanthus emblica*, 8-16 parts of *Terminalia chebula*, 13-20 parts of *Gentiana macrophylla*, 8-16 parts of *Evodia rutaecarpa*, 13-21 parts of *Paeonia suffruticosa*, 13-21 parts of *Lonicera japonica*, and 15-25 parts of *Codonopsis pilosula*.

3. A topical traditional Chinese medicine preparation for relieving pain according to claim 1, characterized in that, The traditional Chinese medicine preparation is made from the following raw materials in parts by weight: 22 parts of snow lotus, 20 parts of *Eclipta prostrata*, 7 parts of *Eclipta prostrata*, 18 parts of *Rehmannia glutinosa*, 15 parts of *Aristolochia debilis*, 15 parts of *Kaempferia galanga*, 12 parts of *Phyllanthus emblica*, 12 parts of *Terminalia chebula*, 17 parts of *Gentiana macrophylla*, 12 parts of *Evodia rutaecarpa*, 18 parts of *Paeonia suffruticosa*, 18 parts of *Lonicera japonica*, and 20 parts of *Codonopsis pilosula*.

4. The method for preparing the external application traditional Chinese medicine preparation according to any one of claims 1 to 3, characterized in that, The traditional Chinese medicine preparations are manufactured using conventional pharmaceutical methods, with the addition of pharmaceutically acceptable excipients, to produce external preparations, including ointments, creams, gels, sprays, aerosols, plasters, and liniments.

5. The preparation method according to claim 4, characterized in that, The preparation method of the traditional Chinese medicine ointment includes the following steps: (1) Preparation of Chinese herbal extracts: Weigh each Chinese herbal raw material according to the above weight proportions, add 8 times the amount of 75% ethanol for ultrasonic extraction for 2 h, and filter; add 6 times the amount of 75% ethanol to the filter residue again for ultrasonic extraction for 1.5 h, and filter; combine the two filtrates, concentrate under reduced pressure to a thick paste with a relative density of 1.10-1.35, and set aside; (2) Preparation of oil phase: Stearic acid, glyceryl monostearate, liquid paraffin and lanolin are placed in a beaker, heated in a water bath to 75-80℃, stirred until completely melted, and kept warm for later use; (3) Aqueous phase preparation: Dissolve the thick paste of Chinese herbal extract, glycerin, triethanolamine and ethylparaben obtained in step (1) in purified water, heat in a water bath at 75-80℃ and stir evenly, and keep warm for later use; (4) Emulsification and molding: Under continuous stirring, the oil phase is slowly added to the water phase, and high-speed homogenization emulsification is continued at 75-80℃ for 10-15 min to form a primary emulsion; then heating is stopped, stirring is continued and cooling is carried out to room temperature, and the ointment is obtained after solidification.

6. The use of the traditional Chinese medicine preparation according to any one of claims 1 to 3 in the preparation of a medicament for relieving pain.

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