Artemisia brachypoda extract as well as extraction process and application thereof

Abstract

The invention provides a short-pedicel aconite root extract as well as an extraction process and application thereof, and belongs to the technical field of medicinal plant processing. According to the technical scheme provided by the invention, active ingredients in the short-pedicel aconite root can be effectively enriched, the purity and the quality stability of the extract are remarkably improved, meanwhile, impurities are effectively removed through optimized process steps, and the production process is simplified; the obtained extract has clear component characteristics and remarkable biological activity, lays a solid foundation for developing related medicines, and has important application value in the aspects of modernization and quality control of traditional Chinese medicines.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant processing technology, and in particular to an extract of Artemisia argyi, its extraction process, and its application. Background Technology

[0002] *Aconitum carmichaelii*, the dried rhizome of a plant in the Ranunculaceae family, is mainly distributed in high-altitude areas of southwestern my country and is an important component of traditional Chinese medicine. This herb has a long history of folk use, often treating rheumatic pain, injuries, and other ailments; however, its application and dosage control have relied heavily on inherited experience. With the advancement of modern pharmacological research, researchers have discovered that *Aconitum carmichaelii* contains various bioactive components, with diterpenoid alkaloids being the main active substances. These components form the basis of its pharmacological activity but also raise potential safety concerns.

[0003] In the modernization of traditional Chinese medicine, how to efficiently extract target active ingredients from natural medicinal materials has always been an important issue. Traditional extraction methods include water extraction and alcohol extraction. Although these methods are simple to operate, they often suffer from low extraction efficiency and a large amount of impurities remaining. For medicinal materials like Saussurea involucrata, which have clearly defined active ingredients but are highly toxic, traditional extraction methods are unable to achieve precise extraction and purification of the effective components, resulting in difficulties in ensuring the quality uniformity and stability of the final product.

[0004] In recent years, with the development of separation and purification technologies, some new extraction methods have been applied to the field of traditional Chinese medicine. For example, ultrasound-assisted extraction and microwave-assisted extraction have shown certain advantages in improving extraction efficiency. However, these methods often require specialized equipment, are costly, and their application in large-scale production remains limited. Furthermore, simply pursuing extraction efficiency while neglecting the specificity and safety of the components is insufficient to meet the needs of modern pharmaceutical production.

[0005] Regarding quality control, existing technologies for the standards of *Saussurea involucrata* extract are incomplete, lacking a systematic quality evaluation system. This leads to significant differences in the content and proportion of active ingredients between different batches of products, directly affecting their pharmacological activity and the safety of clinical applications. Therefore, developing a preparation method that can balance extraction efficiency, component specificity, and process stability has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an extract of Artemisia selengensis, its extraction process, and its application, thus providing an effective means in this field.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing an extract of Artemisia selengensis, comprising the following steps: Degreased medicinal residue was obtained by defatting the powder of Artemisia argyi with petroleum ether. The defatted drug residue was extracted by heating and reflux with an ethanol solution to obtain an extract; The extract was concentrated under reduced pressure to obtain a fluid extract. The fluid extract was extracted with petroleum ether, and the petroleum ether layer was discarded to obtain the remaining aqueous layer. The remaining aqueous layer was extracted sequentially with ethyl acetate and n-butanol, and the n-butanol extracts were combined. The n-butanol extract was concentrated under reduced pressure to near dryness to obtain a concentrate. The concentrate was vacuum dried to obtain an extract of Artemisia argyi.

[0008] Preferably, the boiling range of the petroleum ether is 60-90°C.

[0009] Preferably, the degreasing process is carried out at room temperature, and the degreasing is performed twice, with each degreasing process lasting 18-30 hours.

[0010] Preferably, the ethanol solution is a 70% ethanol solution, and the amount used is 8-10 times the volume-to-weight ratio.

[0011] Preferably, the temperature of the heating and reflux extraction is 70-85℃, the number of extractions is 2-3 times, and the extraction time for each extraction is 1-2 hours.

[0012] Preferably, the extract is concentrated under reduced pressure at a temperature of 60°C and a vacuum of -0.08 MPa until the relative density of the fluid extract is 1.15-1.22.

[0013] Preferably, the fluid extract is extracted once with an equal volume of petroleum ether, and the remaining aqueous layer is extracted three times with an equal volume of ethyl acetate and three times with an equal volume of n-butanol.

[0014] Preferably, the extraction of ethyl acetate and n-butanol is performed three times each; The reduced pressure concentration conditions for the n-butanol extract were a temperature of 60°C and a vacuum of -0.08 MPa. The vacuum drying conditions are a temperature of 50°C and a vacuum degree of -0.09 MPa. After drying to constant weight, the material is pulverized and passed through an 80-mesh sieve.

[0015] The present invention also provides an extract of Artemisia argyi prepared by the above preparation method.

[0016] This invention also provides the application of the above-mentioned Saussurea involucrata extract in the preparation of anti-inflammatory drugs. The technical effects and advantages of this invention are as follows: The technical solution provided by this invention can effectively enrich the active ingredients in Artemisia selengensis, significantly improve the purity and quality stability of the extract, and effectively remove impurities through optimized process steps, simplifying the production process. The obtained extract has clear component characteristics and significant biological activity, laying a solid foundation for the development of related drugs and having important application value in the modernization and quality control of traditional Chinese medicine. Detailed Implementation

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0018] Example 1 Take 1.0 kg of *Artemisia annua* powder and add it to 5 L of petroleum ether (boiling range 60-90℃). Soak and defatt the powder twice at room temperature, 24 hours each time. After filtration, discard the petroleum ether solution and allow the residue to evaporate naturally at room temperature. Transfer the defatted residue to a multi-functional extraction tank and add 8 times (v / w) of 70% ethanol solution. Turn on the reflux device and extract twice at 75℃ for 1.5 hours each time. Combine the two extracts and filter through a 200-mesh filter cloth. Concentrate the combined filtrate under reduced pressure at 60℃ and -0.08 MPa, recovering the ethanol, until a fluid extract with a relative density of 1.15 is obtained, approximately 500 mL. Place this fluid extract in a separatory funnel, add an equal volume of petroleum ether, and extract by vigorous shaking. After standing and separating the layers, discard the petroleum ether layer. Extract the remaining aqueous layer three times each with equal volumes of ethyl acetate and n-butanol, and combine all the n-butanol extracts. The combined n-butanol extract was concentrated again under reduced pressure at 60°C and -0.08 MPa to near dryness. The concentrate was then transferred to a vacuum drying oven and dried at 50°C and -0.09 MPa to constant weight. After pulverizing, it was passed through an 80-mesh sieve to obtain a brownish-yellow to brownish-brown refined extract powder of Artemisia argyi, and the yield was recorded.

[0019] Example 2 Take 1.0 kg of *Artemisia annua* powder and add it to 5 L of petroleum ether (boiling range 60-90℃). Soak and defatt the powder twice at room temperature, 24 hours each time. After filtration, discard the petroleum ether solution and allow the residue to evaporate naturally at room temperature. Transfer the defatted residue to a multi-functional extraction tank and add 8 times (v / w) of 70% ethanol solution. Turn on the reflux device and extract twice at 75℃ for 1.5 hours each time. Combine the two extracts and filter through a 200-mesh filter cloth. Concentrate the combined filtrate under reduced pressure at 60℃ and -0.08 MPa to recover the ethanol until a fluid extract with a relative density of 1.20 is obtained, approximately 500 mL. Place this fluid extract in a separatory funnel, add an equal volume of petroleum ether, and extract by vigorous shaking. After standing and separating the layers, discard the petroleum ether layer. Extract the remaining aqueous layer three times each with equal volumes of ethyl acetate and n-butanol, and combine all the n-butanol extracts. The combined n-butanol extract was concentrated again under reduced pressure at 60°C and -0.08 MPa to near dryness. The concentrate was then transferred to a vacuum drying oven and dried at 50°C and -0.09 MPa to constant weight. After pulverizing, it was passed through an 80-mesh sieve to obtain a brownish-yellow to brownish-brown refined extract powder of Artemisia argyi, and the yield was recorded.

[0020] Example 3 Take 1.0 kg of *Artemisia annua* powder and add it to 5 L of petroleum ether (boiling range 60-90℃). Soak and defatt the powder twice at room temperature, 24 hours each time. After filtration, discard the petroleum ether solution and allow the residue to evaporate naturally at room temperature. Transfer the defatted residue to a multi-functional extraction tank and add 8 times (v / w) of 70% ethanol solution. Turn on the reflux device and extract twice at 80℃ for 1.5 hours each time. Combine the two extracts and filter through a 200-mesh filter cloth. Concentrate the combined filtrate under reduced pressure at 60℃ and -0.08 MPa to recover the ethanol until a fluid extract with a relative density of 1.18 is obtained, approximately 500 mL. Place this fluid extract in a separatory funnel, add an equal volume of petroleum ether, and extract by vigorous shaking. After standing and separating the layers, discard the petroleum ether layer. Extract the remaining aqueous layer three times each with equal volumes of ethyl acetate and n-butanol, and combine all the n-butanol extracts. The combined n-butanol extract was concentrated again under reduced pressure at 60°C and -0.08 MPa to near dryness. The concentrate was then transferred to a vacuum drying oven and dried at 50°C and -0.09 MPa to constant weight. After pulverizing, it was passed through an 80-mesh sieve to obtain a brownish-yellow to brownish-brown refined extract powder of Artemisia argyi, and the yield was recorded.

[0021] Example 4 Take 1.0 kg of *Artemisia annua* powder and add it to 5 L of petroleum ether (boiling range 60-90℃). Soak and defatt the powder twice at room temperature, 24 hours each time. After filtration, discard the petroleum ether solution and allow the residue to evaporate the solvent naturally at room temperature. Transfer the defatted residue to a multi-functional extraction tank and add 10 times (v / w) of 70% ethanol solution. Turn on the reflux device and extract twice at 75℃ for 2 hours each time. Combine the two extracts and filter through a 200-mesh filter cloth. Concentrate the combined filtrate under reduced pressure at 60℃ and -0.08 MPa to recover the ethanol until a fluid extract with a relative density of 1.22 is obtained, approximately 500 mL. Place this fluid extract in a separatory funnel, add an equal volume of petroleum ether, and extract by vigorous shaking. After standing and separating the layers, discard the petroleum ether layer. Extract the remaining aqueous layer three times each with equal volumes of ethyl acetate and n-butanol, and combine all the n-butanol extracts. The combined n-butanol extract was concentrated again under reduced pressure at 60°C and -0.08 MPa to near dryness. The concentrate was then transferred to a vacuum drying oven and dried at 50°C and -0.09 MPa to constant weight. After pulverizing, it was passed through an 80-mesh sieve to obtain a brownish-yellow to brownish-brown refined extract powder of Artemisia argyi, and the yield was recorded.

[0022] Example 5 Take 1.0 kg of *Artemisia annua* powder and add it to 5 L of petroleum ether (boiling range 60-90℃). Soak and defatt the powder twice at room temperature, 24 hours each time. After filtration, discard the petroleum ether solution and allow the residue to evaporate naturally at room temperature. Transfer the defatted residue to a multi-functional extraction tank and add 8 times (v / w) of 70% ethanol solution. Turn on the reflux device and extract three times at 75℃ for 1 hour each time. Combine the extracts and filter through a 200-mesh filter cloth. Concentrate the combined filtrate under reduced pressure at 60℃ and -0.08 MPa to recover the ethanol until a fluid extract with a relative density of 1.16 is obtained, approximately 500 mL. Place this fluid extract in a separatory funnel, add an equal volume of petroleum ether, and extract by vigorous shaking. After standing and separating the layers, discard the petroleum ether layer. Extract the remaining aqueous layer three times each with equal volumes of ethyl acetate and n-butanol, and combine all the n-butanol extracts. The combined n-butanol extract was concentrated again under reduced pressure at 60°C and -0.08 MPa to near dryness. The concentrate was then transferred to a vacuum drying oven and dried at 50°C and -0.09 MPa to constant weight. After pulverizing, it was passed through an 80-mesh sieve to obtain a brownish-yellow to brownish-brown refined extract powder of Artemisia argyi, and the yield was recorded.

[0023] Experimental Example 1. Preparation process 1.1 Raw Materials and Reagents The raw material used in the experiment was commercially available dried rhizomes of *Aconitum brachypodum* Diels, which were pulverized and passed through a 40-mesh sieve. Reagents used included ethanol, ethyl acetate, n-butanol, and petroleum ether, all of which were of analytical grade; the water used in the experiment was ultrapure water.

[0024] 1.2 The extraction and purification steps are described in Example 1.

[0025] 2. Performance Testing Methods To comprehensively evaluate the quality of the extract obtained by this process, the following detection method was established.

[0026] 2.1 Appearance and Yield Observe the color and morphology of the extract. Calculate the extract yield using the following formula: Yield (%) = (Dry weight of extract / Dry weight of raw material) × 100%.

[0027] 2.2 Determination of total alkaloid content by ultraviolet-visible spectrophotometry The total alkaloid content was determined by acid dye colorimetric method using aconitine as a standard.

[0028] Accurately weigh 5.00 mg of aconitine reference standard and place it in a 50 mL volumetric flask. Dissolve and dilute to volume with chloroform to prepare a 0.10 mg / mL stock solution. Accurately pipette 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the stock solution into a separatory funnel, and add chloroform to a final volume of 10 mL.

[0029] Add 5 mL of bromocresol green buffer (pH 4.0) and 5 mL of chloroform to each separatory funnel, shake vigorously for 3 minutes, allow to stand for separation, and then separate the chloroform layer solution. Measure the absorbance at 417 nm. Plot a standard curve with aconitine concentration (μg / mL) on the x-axis and absorbance on the y-axis to obtain the regression equation.

[0030] Accurately weigh approximately 10.00 mg of this product, perform the same procedure as described above, and determine the absorbance. Substitute the absorbance into the standard curve to calculate the total alkaloid content. Perform the determination in parallel for 6 times.

[0031] 2.3 Determination of aconitine, neoaconitine, and hypoaconitine content by high performance liquid chromatography The chromatographic conditions are as follows: The chromatographic column was a C18 column (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile-0.1% triethylamine solution (gradient elution: 0–15 min, 30% → 45% acetonitrile; 15–30 min, 45% → 55% acetonitrile); the flow rate was 1.0 mL / min; the detection wavelength was 235 nm; the column temperature was 30 °C; and the injection volume was 10 μL.

[0032] To prepare the reference solution, accurately weigh appropriate amounts of aconitine, neoaconitine, and hypoaconitine reference standards, and add methanol to prepare a mixed solution containing 20 μg of each reference standard per 1 mL.

[0033] To prepare the test solution, accurately weigh about 20.00 mg of the product, place it in a 10 mL volumetric flask, add methanol to dissolve it by sonication and make up to volume, filter it through a 0.45 μm microporous membrane, and collect the filtrate for later use.

[0034] Accurately pipette 10 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of the three diester alkaloids by peak area using the external standard method.

[0035] 3. Test Results The three batches of pilot samples (batch numbers: SX-230501, SX-230502, SX-230503) were tested according to the above method, and the results are as follows.

[0036] 3.1 Appearance, yield and total alkaloid content All three batches of samples were brownish-yellow powders with a slightly bitter odor. The yield and total alkaloid content determination results are shown in Table 1.

[0037] Table 1. Yield and total alkaloid content of *Saussurea involucrata* extract (n=6)

[0038] 3.2 Content of three diester-type alkaloids The results of HPLC determination of the contents of three major diester alkaloids are shown in Table 2.

[0039] Table 2. Content of three diester alkaloids in *Saussurea involucrata* extract (%, n=3)

[0040] 4. Activity detection results To evaluate the bioactivity of this extract, an in vitro anti-inflammatory experiment was conducted.

[0041] 4.1 Experimental Methods A lipopolysaccharide-induced inflammation model of mouse macrophages (RAW264.7) was established. Cells were divided into five groups: normal control group, model group (LPS, 1 μg / mL), positive control group (dexamethasone, 10 μM), low-dose group of the extract of this invention (25 μg / mL), and high-dose group of the extract of this invention (100 μg / mL). After pretreatment of cells with the drug for 1 hour, all groups except the normal control group were stimulated with LPS for 24 hours. Cell culture supernatant was collected, and nitric oxide (NO) release was determined using the Griess method. The experiment was independently repeated three times.

[0042] 4.2 Experimental Results The inhibitory effects of NO in the cell supernatant of each group are shown in Table 3. Compared with the normal control group, the NO release in the model group was significantly increased. Compared with the model group, the extract of this invention significantly inhibited LPS-induced NO production at concentrations of 25 μg / mL and 100 μg / mL in a dose-dependent manner, and its inhibitory effect was superior to that of the traditional water extract control group at the same concentration (the inhibition rate of the traditional water extract at 100 μg / mL was 28.47% ± 3.12%).

[0043] Table 3. Inhibitory effect of Saussurea involucrata extract on LPS-induced NO release from RAW264.7 cells (x ± s, n=3)

[0044] Note: a indicates P<0.01 compared with the normal control group; b indicates P<0.01 compared with the model group.

[0045] This invention effectively enriches active ingredients and removes impurities. Performance testing results show that the obtained extract has a high total alkaloid content, with the main diester-type alkaloid components clearly identified. In vitro anti-inflammatory activity experiments confirmed that the extract can significantly inhibit the release of the inflammatory factor NO, and the effect is superior to that of traditional preparation methods. In summary, this process is stable and feasible, the quality of the obtained extract is controllable, it has significant anti-inflammatory activity, and it has good application prospects.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an extract of *Saussurea involucrata*, characterized in that, Includes the following steps: Degreased medicinal residue was obtained by defatting the powder of Artemisia argyi with petroleum ether. The defatted drug residue was extracted by heating and reflux with an ethanol solution to obtain an extract; The extract was concentrated under reduced pressure to obtain a fluid extract. The fluid extract was extracted with petroleum ether, and the petroleum ether layer was discarded to obtain the remaining aqueous layer. The remaining aqueous layer was extracted sequentially with ethyl acetate and n-butanol, and the n-butanol extracts were combined. The n-butanol extract was concentrated under reduced pressure to near dryness to obtain a concentrate. The concentrate was vacuum dried to obtain an extract of Artemisia argyi.

2. The preparation method according to claim 1, characterized in that, The boiling range of the petroleum ether is 60-90℃.

3. The preparation method according to claim 2, characterized in that, The degreasing process is carried out at room temperature, and the degreasing is performed twice, with each degreasing process lasting 18-30 hours.

4. The preparation method according to claim 1, characterized in that, The ethanol solution is a 70% ethanol solution, and the amount used is 8-10 times the volume-to-weight ratio.

5. The preparation method according to claim 4, characterized in that, The temperature for the heating and reflux extraction is 70-85℃, the number of extractions is 2-3 times, and the extraction time for each extraction is 1-2 hours.

6. The preparation method according to claim 1, characterized in that, The extract was concentrated under reduced pressure at a temperature of 60°C and a vacuum of -0.08 MPa until the relative density of the fluid extract was 1.15-1.

22.

7. The preparation method according to claim 1, characterized in that, The fluid extract was extracted once with an equal volume of petroleum ether, and the remaining aqueous layer was extracted three times with an equal volume of ethyl acetate and three times with an equal volume of n-butanol.

8. The preparation method according to claim 7, characterized in that, The ethyl acetate and n-butanol were each extracted three times; The reduced pressure concentration conditions for the n-butanol extract were a temperature of 60°C and a vacuum of -0.08 MPa. The vacuum drying conditions are a temperature of 50°C and a vacuum degree of -0.09 MPa. After drying to constant weight, the material is pulverized and passed through an 80-mesh sieve.

9. The extract of Artemisia argyi prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the extract of Saussurea involucrata according to claim 9 in the preparation of anti-inflammatory drugs.

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