Preparation of pharmaceutical compounds with antibacterial and Anti-inflammatory activities by using deep comelt solvent extract of chinese herbal composition

Abstract

A use of deep eutectic solvents extract of Chinese herbal composition, which is used for the preparation of pharmaceutical components with antibacterial activity, the extract is obtained by the deep eutectic solvents after ultrasonic extraction of Chinese herbal composition, the Chinese herbal composition is composed of Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, Anisomeles indica (L.) Kuntze. and Lonicera japonica. Thus, by using the deep eutectic solvents compatible with green chemistry, the active ingredients of the Chinese herbal composition can be effectively extracted for the preparation of the pharmaceutical compositions with antibacterial and anti-inflammatory activity, and organic solvents will not be used to meet the green chemical requirements, which has the advantages of non-volatile, low toxicity, biodegradable, sustainable regeneration, low price and simple.

Description

[Technical Field]

[0001] This invention relates to the extraction and application of active ingredients in a traditional Chinese medicine composition, and in particular to the use of the eutectic solvent extract of the traditional Chinese medicine composition for the preparation of pharmaceutical compositions with antibacterial and anti-inflammatory activities. [Previous Technology]

[0002] Traditional Chinese medicine (TCM) herbs possess significant medicinal value due to their rich bioactive components, with applications encompassing disease prevention, treatment, and health management. However, the extraction and application of TCM herbs still face multiple challenges, including how to extract their active ingredients efficiently and environmentally. The choice of extraction technology and solvent has a crucial impact on the extraction rate, extraction quality, and ultimate medical application effects.

[0003] Traditional extraction methods often rely on organic solvents, such as ethanol or chloroform. While these solvents offer advantages in efficiency, they also suffer from drawbacks such as high toxicity, volatility, and significant environmental impact. Furthermore, the recovery and treatment of organic solvents consume substantial amounts of energy, increasing production costs and posing a challenge to sustainable development. Therefore, green extraction technology has received considerable attention in recent years. Green extraction aims to reduce energy consumption and promote waste recycling by selecting environmentally friendly, renewable, and low-toxicity solvents, thereby achieving a win-win situation for both economic benefits and environmental protection.

[0004] Natural Deep Eutectic Solvents (NaDES) offer significant advantages as an innovative solvent in green extraction. NaDES is a deep eutectic mixture formed by combining two or more primary metabolites (such as sugars, organic acids, and amino acids) in a specific molar ratio. It achieves a low melting point through hydrogen bonding interactions and is characterized by renewability, low toxicity, and biodegradability. Compared to traditional organic solvents, NaDES not only significantly improves the extraction efficiency of plant active ingredients but also ensures the stability and storability of the extracted compounds. Its tunable polarity and high solubility for various hydrophilic and hydrophobic compounds make it a model of "designed solvents."

[0005] Studies have shown that the composition and molar ratio of NaDES directly affect its extraction efficiency, especially its physicochemical properties such as polarity, viscosity, and solubility, which play a crucial role in the extraction process. NaDES has been successfully applied in the extraction of active ingredients in cosmetics, pharmaceuticals, and food. For example, it can be used to efficiently extract phenolic compounds from plants and has proven to have broad application potential in sunscreens, food preservatives, and protein stabilizers.

[0006] Although NaDES has shown great application potential in many fields, research on topics such as skin care, medical materials and pharmaceuticals is still relatively limited. Therefore, further exploration of the application of NaDES in the above fields will not only help promote the sustainable development of related industries, but also provide new technical solutions for the efficient utilization of traditional Chinese medicine. [Summary of the Invention]

[0007] To overcome the above-mentioned technical problems, the purpose of this invention is to provide a deep eutectic solvent extract of a traditional Chinese medicine composition for the preparation of pharmaceutical compositions with antibacterial and anti-inflammatory activities. It is prepared by ultrasonic extraction of a traditional Chinese medicine composition (composed of Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, Euphorbia fischeriana, and Lonicera japonica) using a deep eutectic solvent that conforms to green chemistry. This effectively extracts the active ingredients in the traditional Chinese medicine composition for the preparation of pharmaceutical compositions with antibacterial activities, and avoids the use of organic solvents, thus meeting the requirements of green chemistry. It has the advantages of being non-volatile, low in toxicity, biodegradable, sustainable and renewable, inexpensive, and easy to prepare.

[0008] To achieve the above objectives, the present invention provides the use of a deep eutectic solvent extract of a traditional Chinese medicine composition for preparing pharmaceutical compositions with antibacterial and anti-inflammatory activities; wherein the extract is obtained by ultrasonic extraction and concentration of the traditional Chinese medicine composition in a deep eutectic solvent; the hydrogen bond acceptor (HBA) of the deep eutectic solvent is betaine (Bet), the hydrogen bond donor (HBD) of the deep eutectic solvent is selected from glycerol (Gly), lactic acid (LA) or mannose (MAN), and the traditional Chinese medicine composition is selected from a composition including Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, Gynostemma pentaphyllum and Lonicera japonica, or a composition including peppermint and Acer palmatum.

[0009] In this embodiment of the invention, the herbal composition comprises Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, and Anisomeles indica (L.) Kuntze., which are the whole herb (leaf, stem, and root) of the aforementioned herbs as medicinal parts. Lonicera japonica is derived from its flowers, leaves, and stems as medicinal parts. The herbal composition is prepared by drying the aforementioned medicinal parts at room temperature (40°C to 60°C) with hot air, and then mixing them with a deep eutectic solvent as a raw material for ultrasonic extraction. Preferably, the dried herbal composition can be further ground to form powder particles that can pass through a 20-60 mesh sieve to obtain a powdered herbal composition, thereby improving the efficiency of ultrasonic extraction when mixed with a deep eutectic solvent.

[0010] In this embodiment of the invention, the herbal composition contains peppermint (scientific name: Mentha spp.) taken from its whole plant (leaves, stems, and roots) as the medicinal part, and Australian tea tree (scientific name: Melaleuca alternifolia) taken from its leaves as the medicinal part. The herbal composition is prepared by drying the aforementioned medicinal parts at room temperature (40°C to 60°C) with hot air, and then mixing them with a deep eutectic solvent as a raw material for ultrasonic extraction. Preferably, the dried herbal composition can be further ground to form powder particles that can pass through a 20-mesh to 60-mesh sieve to obtain a powdered herbal composition, thereby improving the efficiency of ultrasonic extraction when mixed with a deep eutectic solvent.

[0011] In one embodiment of the present invention, the eutectic solvent may be betaine (abbreviated as Bet, molecular formula C5H11NO2) as the hydrogen bond acceptor, and may be glycerol (abbreviated as Gly, molecular formula C3H8O3), lactate (abbreviated as LA, molecular formula C12H22O11), or mannose (abbreviated as MAN, molecular formula C6H12O6) as the hydrogen bond donor. In one embodiment, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the eutectic solvent may be from 3:1 to 1:3. In another embodiment, the molar ratio of hydrogen bond acceptors to hydrogen bond donors in the eutectic solvent can be 3:1, 2:1, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 or 1:3; the molar ratio of hydrogen bond acceptors to hydrogen bond donors in the eutectic solvent can be within the range of any two of the above molar ratio values, but is not limited to this.

[0012] In one embodiment of the present invention, the eutectic solvent may be selected from betaine (Bet) as the hydrogen bond acceptor, and may be selected from glycerol (Gly), lactic acid (LA), or mannose (MAN) as the hydrogen bond donor. In one embodiment, the eutectic solvent is prepared by mixing betaine and glycerol in a molar ratio of 1:1.5 to 1:2.5; preferably, the molar ratio of betaine to glycerol is 1:2. In another embodiment, the eutectic solvent is prepared by mixing betaine and lactic acid in a molar ratio of 1:0.5 to 1:1.5; preferably, the molar ratio of betaine to lactic acid is 1:1. In yet another embodiment, the eutectic solvent is prepared by mixing betaine and mannose in a molar ratio of 1:0.5 to 1:1.5; preferably, the molar ratio of betaine to mannose is 1:1.

[0013] In one embodiment of the present invention, the solid-liquid weight ratio of the herbal composition to the deep eutectic solvent can be from 1:5 to 1:15. In one embodiment, the solid-liquid weight ratio of the herbal composition to the deep eutectic solvent can be 1:3, 1:6, 1:9, 1:10, 1:13, or 1:15, and the solid-liquid weight ratio of the herbal composition to the deep eutectic solvent can be within the range of any two of the above values, but is not limited thereto. Preferably, the solid-liquid weight ratio of the herbal composition to the deep eutectic solvent is 1:10.

[0014] In this embodiment of the invention, the ultrasonic extraction process can be completed using the ultrasonic extraction system and equipment of Republic of China Invention Patent No. TWI783508B, in order to achieve the purpose of low-temperature extraction and retention of more heat-sensitive phytochemicals.

[0015] In this embodiment of the invention, the extraction temperature for ultrasonic extraction can be from 25°C to 35°C. Preferably, the extraction temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C; the extraction temperature can be within the range formed by any two of the above values, but is not limited thereto.

[0016] In the embodiments of the present invention, the extraction time for ultrasonic extraction can be from 30 minutes to 60 minutes. Preferably, the extraction time can be 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes; the extraction time can be within the range of any two of the above values, but is not limited thereto.

[0017] In this embodiment of the invention, the ultrasonic extraction is performed under conditions where the ultrasonic power is from 1000 watts (W) to 3000 W and the ultrasonic frequency is from 20 kHz to 100 kHz. Preferably, the ultrasonic power can be 1000 W, 1200 W, 1400 W, 1600 W, 1800 W, 2000 W, 2200 W, 2400 W, 2600 W, 2800 W, or 3000 W, and the ultrasonic power can be within the range formed by any two of the above values, but is not limited thereto. Preferably, the ultrasonic frequency can be 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, or 100 kHz, and the ultrasonic frequency can be within the range formed by any two of the above values, but is not limited thereto.

[0018] In this embodiment of the invention, the pharmaceutical composition can be a pharmaceutical formulation or a liquid gel dressing. When the pharmaceutical composition is a liquid gel dressing, the eutectic solvent extract obtained from the herbal composition through eutectic solvent extraction can be further dissolved or dispersed in a carrier solvent along with other adjuvants, so that the eutectic solvent extract, adjuvants, and carrier solvent are mixed together to form a liquid gel dressing. Specifically, the carrier solvent can be water or hydrosol. The water can be selected from purified water, water for injection (WFI), distilled water, sterile water, or electrolyzed reduced water. The hydrosol can be Australian tea tree hydrosol.

[0019] In the embodiments of the present invention, based on a total weight of 100 wt% of the herbal composition, the herbal composition may include 20 wt% to 25 wt% of Artemisia argyi, 15 wt% to 20 wt% of Houttuynia cordata, 15 wt% to 20 wt% of Hedyotis diffusa, 9 wt% to 13 wt% of Eucommia ulmoides, and 27 wt% to 32 wt% of Lonicera japonica. Specifically, based on a total weight of 100 wt% of the herbal composition, the amount of Artemisia argyi added may be 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, or 25 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on a total weight of 100 wt% of the herbal composition, the amount of Houttuynia cordata added may be 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, or 20 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on a total weight of 100 wt% of the herbal composition, the amount of *Hedyotis diffusa* added can be 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, or 20 wt%, but is not limited to these; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on a total weight of 100 wt% of the herbal composition, the amount of *Needle Atractylodes macrocephala* added can be 9 wt%, 10 wt%, 11 wt%, 12 wt%, or 13 wt%, but is not limited to these; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on a total weight of 100 wt% of the herbal composition, the amount of *Lonicera japonica* added can be 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, or 32 wt%, but is not limited to these; each of the above specific values ​​can be considered as the endpoint of another range.

[0020] Preferably, based on the total weight of the herbal composition as 100wt%, the herbal composition may include 22.7wt% of Artemisia argyi, 18.2wt% of Houttuynia cordata, 18.2wt% of Hedyotis diffusa, 11.4wt% of Euphorbia fischeriana, and 29.5wt% of Lonicera japonica.

[0021] In an embodiment of the present invention, the pharmaceutical composition system includes a liquid gel dressing, wherein, based on 100 wt% of the liquid gel dressing, the liquid gel dressing includes 10 wt% to 20 wt% of a deep eutectic solvent extract, 5 wt% to 11 wt% of polyvinyl pyrrolidone (PVP), 1 wt% to 3 wt% of hydroxypropyl methylcellulose (HPMC), 0.1 wt% to 0.3 wt% of calcium chloride (CaCl2), 0.05 wt% to 0.15 wt% of disodium ethylenediaminetetraacetate (EDTA-2Na), and the balance being water. Specifically, based on 100 wt% of the liquid gel dressing, the amount of the deep eutectic solvent extract added can be 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, or 20 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on 100 wt% of the liquid gel dressing, the amount of polyvinylpyrrolidone (PVP) added can be 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, or 11 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on 100 wt% of the liquid gel dressing, the amount of hydroxypropyl methylcellulose (HPMC) added can be 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, or 3 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on 100 wt% of the liquid gel dressing, the amount of calcium chloride (CaCl2) added can be 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, or 0.3 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on 100 wt% of the liquid gel dressing, the amount of disodium ethylenediaminetetraacetate (EDTA-2Na) added can be 0.05 wt%, 0.1 wt%, or 0.15 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range.

[0022] Preferably, the liquid gel dressing comprises 15 wt% eutectic solvent extract, 8 wt% polyvinylpyrrolidone (PVP), 2 wt% hydroxypropyl methylcellulose (HPMC), 0.2 wt% calcium chloride (CaCl2), 0.1 wt% disodium ethylenediaminetetraacetate (EDTA-2Na), and the balance being water.

[0023] In an embodiment of the present invention, when the herbal composition is Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, Euphorbia fischeriana, and Lonicera japonica, the pharmaceutical composition system includes the liquid gel dressing and water additionally added to the liquid gel dressing. Assuming the liquid gel dressing comprises 100 wt%, the additional water is 20 wt% to 70 wt%. Specifically, the additional water may be 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 65 wt%, or 70 wt%, but is not limited thereto; each of the above specific values ​​can be considered as an endpoint of another range.

[0024] In the embodiments of the present invention, based on a total weight of 100 wt% of the herbal composition, the herbal composition includes 45 wt% to 55 wt% of peppermint and 45 wt% to 55 wt% of Australian tea tree. Specifically, based on a total weight of 100 wt% of the herbal composition, the amount of peppermint added can be 45 wt%, 47 wt%, 49 wt%, 50 wt%, 51 wt%, 53 wt%, or 55 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Specifically, based on a total weight of 100 wt% of the herbal composition, the amount of Australian tea tree added can be 45 wt%, 47 wt%, 49 wt%, 50 wt%, 51 wt%, 53 wt%, or 55 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range.

[0025] Preferably, based on the total weight of the herbal composition of 100wt%, the herbal composition may include 50wt% of peppermint and 50wt% of Australian tea tree.

[0026] In an embodiment of the present invention, the pharmaceutical composition system includes a liquid gel dressing, wherein, based on 100 wt% of the liquid gel dressing, the liquid gel dressing includes 10 wt% to 20 wt% of a deep eutectic solvent extract, 5 wt% to 11 wt% of polyvinylpyrrolidone (PVP), 1 wt% to 3 wt% of hydroxypropyl methylcellulose (HPMC), and the balance being Australian tea tree hydrosol. Specifically, based on 100 wt% of the liquid gel dressing, the amount of deep eutectic solvent extract added can be 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, or 20 wt%, but is not limited thereto; each of the above specific values ​​can be used as the endpoint of another range. Specifically, based on 100 wt% of the liquid gel dressing, the amount of polyvinylpyrrolidone (PVP) added can be 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, or 11 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range. Similarly, based on 100 wt% of the liquid gel dressing, the amount of hydroxypropyl methylcellulose (HPMC) added can be 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, or 3 wt%, but is not limited thereto; each of the above specific values ​​can be considered as the endpoint of another range.

[0027] Preferably, the liquid gel dressing comprises 15 wt% eutectic solvent extract, 8 wt% polyvinylpyrrolidone (PVP), 2 wt% hydroxypropyl methylcellulose (HPMC), and the remainder Australian tea tree syrup.

[0028] In an embodiment of the present invention, the pharmaceutical composition is a liquid gel dressing used to inhibit the growth activity of Propionibacterium acnes.

[0029] In an embodiment of the present invention, when the eutectic solvent is a combination of betaine and mannose (Bet-MAN), water of 55 wt% to 65 wt% of the weight of the liquid gel dressing is added to the liquid gel dressing to form a first diluent gel. The first diluent gel is used to inhibit the growth activity of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa.

[0030] In an embodiment of the present invention, the first diluted gel is used to inhibit inflammation and is non-cytotoxic to keratinocytes and THP-1 cells.

[0031] In an embodiment of the present invention, the first dilution gel is used to inhibit the expression of IL-1β and IL-8 in keratinocytes induced by Propionibacterium acnes.

[0032] In an embodiment of the present invention, the first dilution gel is used to inhibit the expression of IL-1β and IL-8 in THP-1 cells induced by Propionibacterium acnes.

[0033] In an embodiment of the present invention, when the eutectic solvent is a combination of betaine and lactic acid (Bet-LA), an additional 30 wt% to 40 wt% of water is added to the liquid gel dressing to form a second diluted gel. The second diluted gel is used to inhibit the growth activity of Propionibacterium acne, Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa.

[0034] Regarding the technology, means and other effects adopted by the present invention to achieve the above objectives, preferred embodiments are described in detail below with reference to the accompanying drawings.

Implementation Method

[0035] To facilitate understanding of the present invention, the following description is provided in conjunction with the inventive drawings and embodiments.

[0036] Preparation Example 1: Preparation Method of Deep Eutectic Solvent Extract of Traditional Chinese Medicine Composition

[0037] Referring to Table 1, this preparation example prepares the deep eutectic solvent extracts of the herbal compositions of Examples 1 to 3 (E1 to E3) according to the composition ratios in Table 1. First, the herbal compositions were obtained according to the composition ratios in Table 1 and dried, then ground and sieved through a 20-60 mesh sieve to form herbal composition powder (extract). In this preparation example, betaine (Bet) was mixed with glycerol (Gly), lactic acid (LA), or mannose (MAN) in the molar ratios in Table 1 to prepare the deep eutectic solvent. After preparing the herbal composition powder and the deep eutectic solvent, the herbal composition powder was added to the deep eutectic solvent at a solid-liquid weight ratio of 1:10 to form a mixture. Then, the mixture was subjected to ultrasonic extraction for 30 to 60 minutes under the conditions of extraction temperature of 25°C to 35°C, extraction power of 1000W to 3000W, and ultrasonic frequency of 20kHz to 100kHz. Afterward, the mixture was centrifuged at 3000rpm for 10 minutes to remove residue and filter. The extract was collected and concentrated at 50°C at a rate of 40wt% to 60wt% of water content per hour. 100 liters of extract were concentrated to 25 liters in 2 hours, achieving a 4-fold reduction in pressure concentration of the extract. Finally, the deep eutectic solvent extract of the herbal composition was obtained.

[0038] As shown in Table 1, the herbal composition of the deep eutectic solvent extract of the herbal composition in Example 1 (E1) is Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, Acanthopanax senticosus, and Lonicera japonica, and its deep eutectic solvent is betaine and mannose (molar ratio of 1:1). The herbal composition of the deep eutectic solvent extract of the herbal composition in Example 2 (E2) is Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, Acanthopanax senticosus, and Lonicera japonica, and its deep eutectic solvent is betaine and lactic acid (molar ratio of 1:1). The herbal composition of the deep eutectic solvent extract of the herbal composition in Example 3 (E3) is Peppermint and Camellia sinensis, and its deep eutectic solvent is betaine and glycerol (molar ratio of 1:2).

[0039] Table 1: Molar ratio of the composition of the eutectic solvent and weight percentage (wt%) of the extracts in Examples 1 to 3 (E1 to E3). Wherein "-" indicates that the component is not included. Example code E1 E2 E3 Eutectic solvent (Morby) hydrogen bond acceptor Betaine 1 1 1 hydrogen bond donor Mannose 1 - - Lactic acid (LA) - 1 - Glycerin (GLY) - - 2 Chinese herbal composition (extracted product) (wt%) mugwort 22.7 22.7 - Houttuynia cordata 18.2 18.2 - White Flower Snake Tongue Grass 18.2 18.2 - Fish needle grass 11.4 11.4 - honeysuckle 29.5 29.5 - Mint - - 50 Australian tea tree - - 50

[0040] Preparation Example 2: Preparation Method of Liquid Gel Dressing

[0041] Referring to Table 2, this preparation example is prepared by ultrasonically extracting the extracts (traditional Chinese medicine composition) of Examples 1 to 3 (E1 to E3) according to the composition ratio in Table 2, and then adding other excipients to prepare the liquid gel dressings of Examples 1A to 3A (E1A to E3A).

[0042] Table 2: Composition ratio of liquid gel dressings of Examples 1A to 3A (E1A to E3A) (by weight percentage). Wherein "-" indicates that the component is not included. Example code E1A E2A E3A Liquid gel dressing E1 Dry weight of extract 15wt% - - E2 Extract dry weight - 15wt% - E3 Extract dry weight - - 15wt% PVP 8wt% 8wt% 8wt% HPMC 2wt% 2wt% 2wt% Calcium chloride 0.2wt% 0.2wt% - EDTA-2Na 0.1wt% 0.1wt% - water margin margin - Australian tea tree hydrosol - - margin

[0043] Example 1A (E1A): With a total weight of 100wt% of liquid gel dressing, 15wt% of the extract of Example 1 (E1) was mixed with 8wt% of polyvinylpyrrolidone (PVP), 2wt% of hydroxypropyl methylcellulose (HPMC), 0.2wt% of calcium chloride, 0.1wt% of disodium ethylenediaminetetraacetate (EDTA-2Na) and the balance of water and stirred thoroughly to obtain the liquid gel dressing of Example 1A (E1A).

[0044] Example 2A (E2A): With a total weight of 100wt% of liquid gel dressing, 15wt% of the extract from Example 2 (E2) was mixed with 8wt% of polyvinylpyrrolidone (PVP), 2wt% of hydroxypropyl methylcellulose (HPMC), 0.2wt% of calcium chloride, 0.1wt% of disodium ethylenediaminetetraacetate (EDTA-2Na), and the remainder water and stirred thoroughly to obtain the liquid gel dressing of Example 2A (E2A).

[0045] Example 3A (E3A): With a total weight of 100wt% of liquid gel dressing, 15wt% of the extract of Example 3 (E3) was mixed with 8wt% of polyvinylpyrrolidone (PVP), 2wt% of hydroxypropyl methylcellulose (HPMC) and the remainder of Australian tea tree hydrosol and stirred thoroughly to prepare the liquid gel dressing of Example 3A (E3A).

[0046] Preparation Example 3: Preparation Method of Dilution Formulation for Liquid Gel Dressing

[0047] Referring to Table 3, this preparation example is based on the composition ratio in Table 3, and the liquid gel dressings of Examples 1A to 3A (E1A to E3A) are further prepared into diluted gel dressings of Examples 1B to 3B (E1B to E3B).

[0048] Example 1B (E1B): The total weight of the liquid gel dressing of Example 1A (E1A) was 100wt%, and an additional 60wt% of water was added to prepare the diluted gel dressing of Example 1B (E1B).

[0049] Example 2B (E2B): The total weight of the liquid gel dressing of Example 2A (E2A) was 100wt%, and an additional 35wt% of water was added to prepare the diluted gel dressing of Example 2B (E2B).

[0050] Table 3: Composition ratios (by weight percentage) of the diluted gel dressings of Examples 1B to 2B (E1B to E2B). Wherein "-" indicates that the component is not included. Example code E1B E2B Diluted gel dressing E1A Liquid Gel Dressing 100wt% - E2A Liquid Gel Dressing - 100wt% Water (add extra) 60wt% 35wt%

[0051] The above describes the composition ratio and preparation method of the deep eutectic solvent extract (E1 to E3), liquid gel dressing (E1A to E3A), and diluted gel dressing (E1B to E3B) of the herbal composition of the present invention. The effects of the products of the aforementioned examples on antibacterial and anti-inflammatory activity are explained below with reference to Figures 1A to 5B.

[0052] Test Example 1: Antibacterial Activity of Liquid Gel Dressing

[0053] Figures 1A to 1C show the experimental results of using the liquid gel dressings prepared in Examples 1A to 3A (E1A to E3A) to determine their anti-acne activity against Propionibacterium acnes. A blank test and kanamycin were also provided as control groups in this experimental example.

[0054] In this experimental example, after preparing the liquid gel dressings of Examples 1A to 3A, a culture medium for Propionibacterium acnes was prepared, and the bacterial solution was evenly inoculated onto the surface of the culture medium to form a bacterial layer. Next, a certain amount of liquid gel dressing (E1A, E2A, E3A) was dripped onto designated areas on the surface of the culture medium with the inoculated bacterial solution. Control groups were also set up, including: an equal volume of deionized water added to the culture medium as a blank test, and 20 µg / mL kanamycin solution added to the culture medium as a positive control. Afterwards, the treated culture medium was placed under suitable culture conditions for the growth of Propionibacterium acnes (e.g., 37°C, anaerobic environment) for a certain period of time (approximately 24-48 hours). After the culture was completed, the size of the inhibition zone around the sample was observed and recorded, and the size of the inhibition zone was compared to evaluate the antibacterial activity of each example.

[0055] As shown in Figures 1A to 1C, the order of antibacterial efficacy is as follows: Example 1A (Bet-MAN) exhibits the best antibacterial activity (Figure 1A), which is superior to the antibacterial effect of kanamycin; Example 2A (Bet-LA) exhibits the second best antibacterial activity (Figure 1B), and its antibacterial activity is only slightly less than that of kanamycin; Example 3A (Bet-GLY) has relatively lower antibacterial activity compared to the other examples, but still has a significant antibacterial effect (Figure 1C). It can be seen from the above that, when the herbal composition has the same composition ratio, the liquid gel dressing prepared using betaine and mannose (Bet-MAN) as a deep eutectic solvent can obtain excellent antibacterial effects; while the liquid gel dressing prepared using betaine and lactic acid (Bet-LA) as a deep eutectic solvent still has considerable antibacterial activity.

[0056] When the liquid gel dressings of Examples 1A to 3A are applied to human skin, they initially present a moist gel-like state as shown in Figure 6A. After about 5 to 8 seconds, the liquid gel dressing will form a dry film as shown in Figure 6B. It is worth noting that, as shown in Figure 7, comparing the liquid gel dressing prepared in Example 3A (E3A) with a liquid gel dressing used as a blank test (containing no glycerin, but with the same other components as E3A), the liquid gel dressing of E3A, after drying and forming a film, still adheres tightly to the skin at the periphery; in contrast, the liquid gel dressing of the blank test, after drying, shows a noticeable lifting at the periphery of the film, forming a white edge detached from the skin; thus, it is evident that the liquid gel dressing of E3A has significantly better skin affinity.

[0057] Experimental Example 2: Antibacterial Activity of Diluted Gel Dressing

[0058] Figures 2A to 2D show the experimental results of using the diluted gel dressings prepared in Examples 1B to 2B (E1B to E2B) to determine the inhibitory activity against *Propionibacterium acne* (Figure 2A), *Staphylococcus epidermidis* (Figure 2B), *Staphylococcus aureus* (Figure 2C), or *Pseudomonas aeruginosa* (Figure 2D). A blank test and kanamycin were also provided as control groups in this experimental example.

[0059] In this experimental example, after preparing the diluted gel dressings of Examples 1B to 2B, culture media containing *Propionibacterium acnes*, *Staphylococcus epidermidis*, *Staphylococcus aureus*, and *Pseudomonas aeruginosa* were prepared, and the bacterial suspensions were evenly inoculated onto the surface of the culture media to form a bacterial layer. Next, a certain amount of the diluted gel dressing (E1B, E2B) was dropped onto designated areas on the surface of the culture media inoculated with the bacterial suspensions. Control groups were also set up, including: an equal volume of deionized water added to the culture media as a blank test, and 20 µg / mL kanamycin solution added to the culture media as a positive control. The treated culture media were then placed under suitable culture conditions for *Propionibacterium acnes* growth (e.g., 37°C, anaerobic environment) and incubated for a certain period (approximately 24-48 hours). After incubation, the size of the inhibition zone around the sample was observed and recorded, and the size of the inhibition zone was compared to evaluate the antibacterial activity of each example.

[0060] Figure 2A shows the antibacterial effect against *Propionibacterium acnes*. Example 1B (E1B) showed a larger inhibition zone, indicating good antibacterial effect. Example 2B (E2B) showed a slightly smaller inhibition zone compared to E1B, but still exhibited some antibacterial activity. Kanamycin formed the largest inhibition zone, demonstrating the most significant effect. The blank test did not form an inhibition zone, indicating no antibacterial activity.

[0061] Figure 2B shows the antibacterial effect against Staphylococcus epidermidis. E1B formed a moderate inhibition zone, indicating good antibacterial activity. E2B had a slightly smaller inhibition zone than E1B, indicating slightly lower activity against Staphylococcus epidermidis. Kanamycin had a larger inhibition zone and was the best positive control. The blank test showed no inhibition zone, indicating no antibacterial effect.

[0062] Figure 2C shows the antibacterial effect against Staphylococcus aureus. E1B showed a larger inhibition zone, indicating good antibacterial effect. E2B's inhibition zone was slightly smaller than E1B's, but still showed some antibacterial activity. Kanamycin formed the largest inhibition zone, showing the most significant effect. The blank test did not form an inhibition zone, indicating no antibacterial activity.

[0063] Figure 2D shows the antibacterial effect against Pseudomonas aeruginosa. E1B showed a clear inhibition zone, but its activity was slightly weaker compared to other strains. E2B had a smaller inhibition zone, indicating the lowest antibacterial effect against Pseudomonas aeruginosa. Kanamycin still showed the largest inhibition zone, indicating the strongest antibacterial effect. The blank test showed no inhibition zone, indicating no antibacterial activity.

[0064] As can be seen from the above, when the herbal composition has the same composition ratio, the diluted gel dressing prepared by using betaine and mannose (Bet-MAN) as the deep eutectic solvent can obtain excellent antibacterial effect; while the diluted gel dressing prepared by using betaine and lactic acid (Bet-LA) as the deep eutectic solvent still has considerable antibacterial activity.

[0065] Experimental Example 3: Cytotoxicity Assessment of Diluted Gel Dressing

[0066] This test case investigates the effect of the deep eutectic solvent extract of the herbal composition in the diluted gel dressing of Example 1B (E1B) on the cell survival rate of keratinocytes (HaCaT cells) and macrophages (THP-1) at different concentrations, in order to determine whether it has cytotoxicity.

[0067] Figure 3A shows the effect on keratinocytes (HaCaT cells). As shown in Figure 3A, in the diluted gel dressing of Example 1B (E1B), when 100 mg of the herbal composition eutectic solvent extract per milliliter of eutectic solvent was added to the keratinocyte culture medium and diluted to a concentration range of 0.1 mg to 1 mg of the herbal composition eutectic solvent extract per milliliter of eutectic solvent, the survival rate of keratinocytes was consistently higher than 90%. When the concentration increased (from 0.25 mg / mL to 1 mg / mL), the cell survival rate decreased slightly, but the difference was not significant. It is evident that even at a high concentration of 1 mg / mL, the eutectic solvent herbal extract in the diluted gel dressing of Example 1B (E1B) exhibits very low cytotoxicity to keratinocytes, demonstrating its good safety profile and suitability for use in skin care products.

[0068] Figure 3B shows the effect on THP-1 macrophages. As shown in Figure 3B, in the diluted gel dressing of Example 1B (E1B), within the concentration range of 0.1 mg to 1 mg of the herbal composition eutectic solvent extract per milliliter of eutectic solvent, the macrophage survival rate remained between 85% and 90%. At a high concentration of 1 mg / mL, the cell survival rate was slightly lower than that of keratinocytes, but still maintained at a high level. It is evident that the herbal composition eutectic solvent extract contained in the diluted gel dressing of Example 1B (E1B) did not cause significant toxicity to macrophages within the tested concentration range, indicating that this composition is suitable for use in drugs or topical preparations with anti-inflammatory effects.

[0069] As observed from the above results, cell viability decreased slightly with increasing concentration, but the overall change was not significant, indicating that the cytotoxicity of this extract was not significantly correlated with concentration. The experimental results show that the deep eutectic solvent extract of the herbal composition in Example 1B exhibits high viability against both keratinocytes and macrophages, further confirming its good safety and application potential, making it particularly suitable for use in anti-inflammatory skin care products or immune-related treatments. These results fully support its potential as a low-toxicity, highly biocompatible candidate material, suitable for subsequent clinical and product development.

[0070] Experiment 4: Evaluation of the anti-inflammatory effect of diluted gel dressing

[0071] The diluted gel dressing of Example 1B (E1B) showed a good inhibitory effect on the expression of IL-1β and IL-8 inflammatory genes in keratinocytes and THP-1 macrophages induced by Propionibacterium acnes.

[0072] As shown in Figure 4A, under the condition of no added diluted gel dressing (concentration 0 mg / mL), the expression of IL-1β in keratinocytes significantly increased. However, as the dressing concentration increased to 0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, the expression of IL-1β gradually decreased, and a significant inhibitory effect was observed at a concentration of 0.5 mg / mL. Similarly, as observed in Figure 4B, the expression of IL-8 also showed a similar trend. Without the addition of diluted gel dressing, the expression of IL-8 significantly increased, and as the concentration increased, the expression of IL-8 gradually decreased, reaching a significant inhibitory effect at high concentrations. These results indicate that the deep eutectic solvent extract of the herbal composition in the diluted gel dressing of Example 1B can effectively inhibit the inflammatory response induced by Propionibacterium acnes, and has a good concentration-dependent inhibitory effect on the expression of IL-1β and IL-8 genes in keratinocytes and macrophages, further confirming its anti-inflammatory effect and potential for application in skin care products.

[0073] As can be observed from Figure 5A, under the condition of no added diluent gel dressing (concentration 0 mg / mL), the performance of IL-1β increased significantly. However, as the concentration of the diluted gel dressing increased to 0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL, the performance of IL-1β gradually decreased, reaching the best inhibitory effect at a concentration of 0.5 mg / mL. As can be seen from Figure 5B, the performance of IL-8 showed a similar trend. Without the addition of diluent gel dressing, the performance of IL-8 increased significantly. As the concentration of the diluted gel dressing increased, the performance of IL-8 decreased significantly, and the inhibitory effect was most significant at a concentration of 0.5 mg / mL. These results indicate that the deep eutectic solvent extract of the herbal composition in the diluted gel dressing of Example 1B can effectively inhibit the inflammatory response of macrophages induced by Propionibacterium acnes. The inhibition of IL-1β and IL-8 gene expression is concentration-dependent, further confirming its good anti-inflammatory properties and showing potential in anti-inflammatory treatment and related product applications.

[0074] In summary, the present invention uses a deep eutectic solvent to extract the active ingredients of a traditional Chinese medicine composition. This method has advantages such as low volatility, non-flammability, solubility for various plant compounds, chemical stability, adjustable viscosity, and a rapid preparation process. Furthermore, the strong hydrogen bonds between the components of the deep eutectic solvent and the extract can increase the yield of the extract. Moreover, the present invention uses a deep eutectic solvent combination of betaine with ingredients such as mannose, lactic acid, or glycerol, combined with ultrasonic extraction to obtain a deep eutectic solvent extract of the traditional Chinese medicine composition. This extract exhibits significant effects in antibacterial and anti-inflammatory properties. In addition, the ultrasonic extraction method makes the extraction process of the deep eutectic solvent extract of the traditional Chinese medicine composition rapid and simple, and allows for non-high-temperature extraction of the traditional Chinese medicine composition in a short time, thus preserving heat-sensitive phytochemicals. Therefore, the deep eutectic solvent extract of the traditional Chinese medicine composition of the present invention can be used to prepare pharmaceutical components for liquid gel dressings under the premise of complying with green chemistry. [Simplified Explanation of the Diagram]

[0075] Figures 1A to 1C show the antibacterial activity test results of the liquid gel dressings of Examples 1A to 3A of the present invention in Test Example 1; Figures 2A to 2D show the antibacterial activity test results of the diluted gel dressings of Examples 1B and 2B of the present invention in Test Example 2; Figures 3A and 3B show the cytotoxicity test results of the diluted gel dressing of Example 1B of the present invention in Test Example 3; Figures 4A and 4B show the anti-inflammatory activity test results of the diluted gel dressing of Example 1B of the present invention in Test Example 4; Figures 5A and 5B show the anti-inflammatory activity test results of the diluted gel dressing of Example 1B of the present invention in Test Example 4; Figures 6A and 6B are photographs of the initial gel state and the dried film state of the liquid gel dressings of the present invention after application; Figure 7 is a photograph of the liquid gel dressing of Example 3A of the present invention in use on human skin.

Claims

1. The use of a deep eutectic solvent extract of a traditional Chinese medicine composition for preparing a pharmaceutical composition with antibacterial and anti-inflammatory activities; the pharmaceutical composition is a liquid gel dressing used to inhibit the growth activity of Propionibacterium acnes; wherein, The eutectic solvent extract was obtained by ultrasonic extraction and concentration of the herbal composition using a eutectic solvent. The hydrogen bond acceptor (HBA) of the eutectic solvent is betaine (Bet), and the hydrogen bond donor (HBD) of the eutectic solvent is selected from glycerol (Gly), lactic acid (LA), or mannose (MAN). The herbal composition is selected from a combination including Artemisia argyi, Houttuynia cordata, Hedyotis diffusa, Gynostemma pentaphyllum, and Lonicera japonica, or from a combination including peppermint and Camellia sinensis.

2. For the purpose described in claim 1, the deep eutectic solvent is prepared by mixing betaine and glycerol in a molar ratio of 1:1.5 to 1:2.

5.

3. For the purpose described in claim 1, the deep eutectic solvent is prepared by mixing betaine and lactic acid in a molar ratio of 1:0.5 to 1:1.

5.

4. For the purpose described in claim 1, the deep eutectic solvent is prepared by mixing betaine and mannose in a molar ratio of 1:0.5 to 1:1.

5.

5. As described in claim 1, the herbal composition comprises, by weight of 100wt%, 20wt% to 25wt% of Artemisia argyi, 15wt% to 20wt% of Houttuynia cordata, 15wt% to 20wt% of Hedyotis diffusa, 9wt% to 13wt% of Euphorbia fischeriana, and 27wt% to 32wt% of Lonicera japonica.

6. As described in claim 5, the pharmaceutical composition system comprises a liquid gel dressing, wherein, Based on 100 wt% of the liquid gel dressing, the liquid gel dressing comprises a deep eutectic solvent extract obtained by dry weight of 10 wt% to 20 wt% of a traditional Chinese medicine composition, 5 wt% to 11 wt% of polyvinylpyrrolidone (PVP), 1 wt% to 3 wt% of hydroxypropyl methylcellulose (HPMC), 0.1 wt% to 0.3 wt% of calcium chloride (CaCl2), 0.05 wt% to 0.15 wt% of disodium ethylenediaminetetraacetate (EDTA-2Na), and the balance being water.

7. As claimed in claim 6, the pharmaceutical composition system includes the liquid gel dressing and water additionally added to the liquid gel dressing, wherein the additional water is 55 wt% to 65 wt% of 100 wt% of the liquid gel dressing.

8. As claimed in claim 1, the herbal composition comprises 45 wt% to 55 wt% of peppermint and 45 wt% to 55 wt% of Australian tea tree, based on a total weight of 100 wt%.

9. As described in claim 8, the pharmaceutical composition system comprises a liquid gel dressing, wherein, Based on 100 wt% of the liquid gel dressing, the liquid gel dressing comprises a deep eutectic solvent extract obtained by dry weight of 10 wt% to 20 wt% of a traditional Chinese medicine composition, 5 wt% to 11 wt% of polyvinylpyrrolidone (PVP), 1 wt% to 3 wt% of hydroxypropyl methylcellulose (HPMC), and the balance being Australian tea tree hydrosol.

10. The use as described in claim 1, wherein, The herbal composition is a powder of herbal composition that has been dried and pulverized through a 20-60 mesh sieve; the solid-liquid weight ratio of the herbal composition to the deep eutectic solvent can be 1:5 to 1:

15.

11. The use as described in claim 1, wherein, When the eutectic solvent is a combination of betaine and mannose (Bet-MAN), an additional 55 wt% to 65 wt% water is added to the liquid gel dressing to form a first dilution gel. This first dilution gel is used to inhibit the growth activity of Propionibacterium acne, Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa.

12. The use as described in claim 11, wherein, The first dilution gel is used to suppress inflammation and is non-cytotoxic to keratinocytes and THP-1 cells.

13. The use as described in claim 11, wherein, The first dilution gel is used to inhibit the expression of IL-1β and IL-8 in keratinocytes induced by Propionibacterium acnes.

14. The use as described in claim 11, wherein, The first dilution gel was used to inhibit IL-1β and IL-8 expression in THP-1 cells induced by Propionibacterium acnes.

15. The use as described in claim 1, wherein, When the eutectic solvent is a combination of betaine and lactic acid (Bet-LA), an additional 30 wt% to 40 wt% water is added to the liquid gel dressing to form a second dilution gel. This second dilution gel is used to inhibit the growth activity of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa.

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