A method for preparing various active components from datura leaf

Abstract

This invention discloses a method for preparing multiple active components from Datura leaves, belonging to the field of phytochemistry. The method includes the following steps: extracting dried Datura leaves by maceration with 95% ethanol, and recovering the total ethanol extract under reduced pressure; extracting the total ethanol extract with 0.1% hydrochloric acid solution and filtering; passing the filtrate through a 732 cation exchange resin column, collecting the eluent, and simultaneously eluting the components adsorbed on the resin with 95% ethanol and 4N ammonia-ethanol solution, followed by chloroform extraction to obtain alkaloid components; concentrating the eluent and then passing it through an HPD-BJQH macroporous adsorption resin column, eluting sequentially with water, 30% ethanol, and 95% ethanol, collecting the eluents, and concentrating under reduced pressure to obtain glycoside components, flavonoid components, and solanolactone components. The components obtained by this invention exhibit good non-overlapping properties and are suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of phytochemistry, and more specifically to a method for preparing multiple active components from datura leaves. Background Technology

[0002] Datura (Datura stramonium L.) is a plant belonging to the genus Datura in the Solanaceae family. Widely distributed throughout my country, it is an important medicinal plant resource. Datura leaves contain various bioactive components, mainly including hyoscyamine alkaloids (such as scopolamine and atropine), flavonoids, solanolactones, and glycosides. Modern pharmacological studies have shown that solanolactones in Datura exhibit significant cytotoxic activity, showing inhibitory effects on tumor cells; its alkaloid components possess antispasmodic, analgesic, and anticholinergic pharmacological effects; and its flavonoids possess various biological activities such as antioxidant and anti-inflammatory properties. Therefore, the systematic isolation and purification of various active components from Datura leaves is of great significance for the in-depth development of its medicinal value.

[0003] Currently, existing technologies for the extraction and separation of active ingredients from datura leaves mainly suffer from the following problems:

[0004] First, existing extraction and separation methods mostly target single components, neglecting the systematic separation of multiple components. Current research largely focuses on the extraction of alkaloids from Datura stramonium, such as using acid-water extraction followed by purification with cation exchange resin to obtain total alkaloids. However, in addition to alkaloids, Datura stramonium leaves also contain abundant flavonoids, solanolactones, and glycosides, which also have significant medicinal value. Current technologies fail to achieve the simultaneous separation and utilization of these multiple active components, resulting in a waste of resources.

[0005] Secondly, there is serious cross-contamination among the components in existing separation processes. In conventional processes for extracting alkaloids from Datura leaves, the residue or eluent after acidic water extraction is often discarded directly, failing to effectively recover neutral or acidic components such as flavonoids and solanolactones. More importantly, even when using column chromatography, the similar polarity or interactions between the components often lead to cross-elution between alkaloids and components such as flavonoids and solanolactones, resulting in low purity of the obtained components that are difficult to meet the quality requirements for subsequent pharmacodynamic studies or drug development. For example, when using macroporous adsorption resins to separate Datura leaf extracts, the lack of a targeted gradient elution program often results in small amounts of flavonoids in the water-eluted fraction and glycosides remaining in the ethanol-eluted fraction, severely affecting the separation efficiency and subsequent utilization value of each component.

[0006] Third, existing resin coupling processes lack effective control over component cross-contamination. Although cation exchange resins and macroporous adsorption resins are commonly used materials for separating plant active ingredients, current technologies often employ simple sequential operations, failing to fully consider the impact of changes in the composition of components in the effluent after cation exchange resin treatment on the separation efficiency of macroporous adsorption resins. In particular, when cation exchange resin treatment is incomplete, small amounts of residual alkaloids in the effluent can co-adsorb or cross-elute with flavonoids, solanolone, and other components on the macroporous adsorption resin, introducing alkaloid impurities into the subsequently separated components and causing cross-contamination between components.

[0007] Fourth, existing gradient elution process parameters lack targeted optimization. Gradient elution with macroporous adsorption resins is a common method for separating components of different polarities in plant extracts, but systematic gradient elution conditions for glycosides, flavonoids, and solanolactones in Datura stramonium leaf extract have not been reported. Existing gradient elution programs mostly use a general water-ethanol gradient, which is not optimized based on the specific polarity differences and elution behavior of each component in Datura stramonium leaf. This results in blurred boundaries between elution sections, incomplete component separation, and significant component overlap between adjacent elution sections.

[0008] Fifth, existing technologies lack sufficient verification of the separation effectiveness of each component. Most current reports on the separation of active ingredients from Datura leaves focus only on the yield of the target component, lacking systematic analysis and evaluation of the purity of each separated component and the degree of cross-contamination between them. This lack of verification of separation effectiveness makes it difficult to guarantee the reliability of existing processes and hinders the standardization and industrialization of these processes.

[0009] Therefore, developing a method that can simultaneously isolate and prepare multiple active components from Datura leaves, with clear separation and no cross-contamination between the components, is of great significance for improving the comprehensive utilization efficiency of Datura leaf resources, obtaining high-purity active components, and meeting the quality requirements of drug development and natural product research. Summary of the Invention

[0010] To overcome the shortcomings of existing technologies, this invention provides a method for preparing multiple active components from datura leaves. The components obtained by this method have good non-overlapping properties and are suitable for widespread application.

[0011] The technical solution is as follows:

[0012] This invention provides a method for preparing multiple active components from datura leaves, comprising the following steps:

[0013] Step 1: Take dried datura leaves and extract them by maceration with ethanol. The solvent is recovered from the extract under reduced pressure to obtain the total ethanol extract of datura leaves.

[0014] Step 2: Extract the total ethanol extract of Datura leaves obtained in Step 1 with an acidic aqueous solution, filter, and obtain the filtrate;

[0015] Step 3: Pass the filtrate obtained in Step 2 through a cation exchange resin column, collect the effluent, and simultaneously elute and recover the components adsorbed on the cation exchange resin to obtain the alkaloid component.

[0016] Step 4: After recovering and concentrating the effluent collected in Step 3, pass it through a macroporous adsorption resin column and elute it sequentially with water and ethanol aqueous solution. Collect each eluent and concentrate it under reduced pressure to obtain glycoside, flavonoid and solanolactone components.

[0017] Furthermore, the ethanol mentioned in step 1 is 95% ethanol by volume, and the amount of ethanol used during maceration and extraction is 6 to 12 times the weight of the datura leaves, with each maceration and extraction lasting 2 to 5 days.

[0018] Furthermore, the amount of ethanol used during maceration extraction was 8 times the weight of the datura leaves, and the maceration extraction time was 3 days each time.

[0019] Furthermore, the acidic aqueous solution mentioned in step 2 is a 0.1% hydrochloric acid solution, the amount of which is 3 to 8 times the volume of the concentrated total extract of Datura stramonium leaf ethanol obtained in step 1, and the extraction is performed 3 to 8 times.

[0020] Furthermore, the amount of the acidic aqueous solution used is 5 times the volume of the concentrated total extract of Datura stramonium leaf ethanol obtained in step 1, and the extraction is performed 6 times.

[0021] Furthermore, the cation exchange resin mentioned in step 3 is a 732 type cation exchange resin, and the flow rate through the resin column is 3 BV / h. Here, "BV" is an abbreviation for Bed Volume.

[0022] Furthermore, in step 3, the components adsorbed on the cation exchange resin are eluted and recovered. Specifically, the components are washed with water until the effluent is colorless, then eluted sequentially with 95% ethanol and 4N ammonia-ethanol solution. The ammonia-ethanol eluent is collected, the solvent is recovered, and the components are extracted three times with chloroform. The chloroform layer is then recovered to obtain the alkaloid component.

[0023] Furthermore, the macroporous adsorption resin mentioned in step 4 is HPD-BJQH type macroporous adsorption resin.

[0024] Furthermore, the specific conditions for gradient elution in step 4 are as follows: first, elute with 2 BV of water, then elute with 2 BV of 30% ethanol, and finally elute with 4 BV of 95% ethanol, with an elution flow rate of 1 BV·h⁻¹.

[0025] Furthermore, in step 4, the glycoside component is a water-eluted component, the flavonoid component is a 30% ethanol-eluted component, and the solanolactone component is a 95% ethanol-eluted component.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. This invention achieves the systematic separation of multiple active components in Datura stramonium leaves, significantly improving the comprehensive utilization rate of resources. It overcomes the shortcomings of existing technologies that often focus only on a single component (alkaloid). Through a combined process of "ethanol extraction → acid water extraction → cation exchange resin → gradient elution with macroporous adsorption resin," alkaloid components, glycoside components, flavonoid components, and solanolactone components are separated from Datura stramonium leaves in a single step. This ensures the effective recovery of active components of different polarities from Datura stramonium leaves, avoiding resource waste.

[0028] 2. This invention effectively solves the cross-contamination problem between components, resulting in high purity and good non-overlapping properties of the eluted components. First, the total ethanol extract of Datura stramonium leaves is extracted with an acidic aqueous solution (e.g., 0.1% hydrochloric acid) to convert alkaloids into salt forms. Then, the alkaloids are completely adsorbed and eluted separately using a cation exchange resin, ensuring that the effluent is free of alkaloids. Subsequently, the effluent is passed through a macroporous adsorption resin and eluted using an optimized water-ethanol gradient (water → 30% ethanol → 95% ethanol), collecting each elution fraction separately. Since there is no alkaloid interference in the effluent, and the gradient elution conditions are optimized for the polarity differences of glycosides (water elution), flavonoids (30% ethanol elution), and solanolactones (95% ethanol elution), the eluted components exhibit good non-overlapping properties. Experiments show that the purity of the target components in each fraction is significantly higher than that of existing methods, and no residues of other types of components were detected.

[0029] 3. The combined process of cation exchange resin and macroporous adsorption resin has been optimized to avoid interference from residual alkaloids in subsequent separations. Existing resin combined processes often overlook the trace amounts of alkaloids that may remain in the effluent after cation exchange resin treatment. These alkaloids, upon entering the macroporous adsorption resin, can co-adsorb or cross-elute with flavonoids and solanolactones, leading to cross-elution. In the cation exchange resin step, this invention ensures no alkaloid residue in the effluent by controlling the sample loading flow rate (e.g., 3 BV / h) and employing a thorough regeneration method involving water washing, 95% ethanol washing, and 4N ammonia-ethanol elution. This design fundamentally blocks the residual pathway of alkaloids to the components separated by the subsequent macroporous adsorption resin, guaranteeing the non-cross-linking of the components.

[0030] 4. For the first time, optimized gradient elution parameters are provided to address the polarity differences among the components of Datura stramonium leaf extract, resulting in clear separation. This invention, through systematic screening, determined the gradient elution procedure for macroporous adsorption resins (such as HPD-BJQH type): first, elute with 2 BV of water to obtain the glycoside component, then elute with 2 BV of 30% ethanol to obtain the flavonoid component, and finally elute with 4 BV of 95% ethanol to obtain the solanolactone component. This elution condition overcomes the component cross-linking defects caused by general gradient elution in existing technologies, resulting in excellent separation between the components.

[0031] 5. The process parameters are reasonable, the operation is simple, and it is suitable for industrial-scale promotion. This invention provides a wide and verified reasonable range for key parameters in each step (such as solvent volume ratio, extraction time, acid volume ratio, number of extractions, etc.). (For example, ethanol volume 6-12 times, immersion time 2-5 days; hydrochloric acid volume 3-8 times, extraction 3-8 times, etc.), providing operational flexibility for industrial production while ensuring separation effect. The entire process requires no special equipment, uses common solvents, is low in cost, has good repeatability, and is easy to scale up for production.

[0032] 6. The system validated the separation effect of each component, demonstrating strong process reliability. This invention not only focuses on the yield of each component but also uses methods such as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) to detect the cross-linking of each eluted component. The results show that there is no cross-linking between the components. This systematic validation method provides a reliable basis for process stability and standardization, which is beneficial for subsequent pharmacodynamic studies and quality control in drug development.

[0033] In summary, this invention provides a method for simultaneously separating and preparing multiple high-purity active components from Datura leaves. The components do not overlap with each other, significantly improving the comprehensive utilization value of Datura leaves, overcoming many shortcomings of existing technologies, and showing good prospects for scientific research and industrial applications. Attached Figure Description

[0034] Figure 1 Here is a process flow diagram of an embodiment of the present invention:

[0035] Figure 2 HPLC chromatograms of the fractions separated by water, 30% ethanol, 95% ethanol and chloroform extraction;

[0036] Figure 3 The peak diagrams of the fractionated components of datura leaves;

[0037] Figure 4 PCA analysis chromatograms of each separated component;

[0038] Figure 5 PLS-DA analysis chromatograms of each separated component;

[0039] Figure 6 HPLC chromatogram and UV characteristic absorption of the aqueous component;

[0040] Figure 7 HPLC chromatogram and UV characteristic absorption of the 30% ethanol component;

[0041] Figure 8 HPLC chromatogram and UV characteristic absorption of the 95% ethanol component;

[0042] Figure 9 HPLC chromatogram and UV characteristic absorption of the chloroform extraction layer;

[0043] Figure 10 This is a diagram of the water maze positioning, navigation, and swimming trajectory in Embodiment 4 of the present invention;

[0044] Figure 11 This is a graph showing the changes in the 5-day escape latency of rats in each group in Example 4 of the present invention;

[0045] Figure 12 This is a bar chart showing the number of times rats crossed the platform and the percentage of time spent in the target quadrant in each group in Example 4 of the present invention;

[0046] Figure 13 HE staining images of the CA1 region of the hippocampus of rats in each group in Example 4 of this invention (×200x).

[0047] Figure 14 The images show the immunohistochemical analysis (×400x) of P-Tau in the CA1 region of rats in Example 4 of this invention and the percentage of protein-positive areas.

[0048] Figure 15 The figure shows the effect of various components of Datura stramonium leaf on the serum levels of TNF-α, IL-1β, IL-6, GSH-PX, SOD, and MDA in AD rats in Example 4 of this invention. Detailed Implementation

[0049] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0050] Example 1

[0051] Reference Figure 1Two kg of dried Datura leaves were extracted using 8 times the volume of 95% ethanol for 3 days each time. The filtrates were combined and recovered under reduced pressure to obtain 260 g of Datura leaf extract (yield 13.0%). The total ethanol extract of Datura leaves was extracted 6 times with 5 times the volume of 0.1% hydrochloric acid solution and filtered. Hyoscyamine alkaloids in the filtrate were removed by column chromatography using a 732 cation exchange resin at a flow rate of 3 BV / h. The eluent was recovered and concentrated, and then adsorbed using HPD-BJQH macroporous adsorption resin at a flow rate of 1 BV·h with water (2 BV), 30% ethanol (2 BV), and 95% ethanol (4 BV), respectively. -1 The column chromatography eluent was collected and concentrated under reduced pressure. The water eluent contained glycosides (17.3%), the 30% ethanol eluent contained flavonoids (25.5%), and the 95% ethanol eluent contained solanolone (39.5%). The 732 cation exchange resin column was washed with pure water until the eluent was colorless. It was then eluted sequentially with 95% ethanol and 4N ammonia-ethanol solution. The ammonia-ethanol eluent was recovered, and the column was extracted three times with chloroform. The recovered chloroform layer yielded the alkaloid component (1.3%).

[0052] Example 2

[0053] Reference Figure 1 Two kg of dried Datura leaves were extracted with 6 times the volume of 95% ethanol for two days each time. The filtrates were combined, and the solvent was recovered under reduced pressure to obtain approximately 186 g of total ethanol extract of Datura leaves (yield 9.3%). The extract was then extracted three times with 0.1% hydrochloric acid solution at a volume of three times the concentrate, and filtered. Hyoscyamine alkaloids in the filtrate were removed by column chromatography using a 732 cation exchange resin at a flow rate of 3 BV / h, and the eluent was collected. After recovery and concentration, the eluent was then adsorbed sequentially with water (2 BV), 30% ethanol (2 BV), and 95% ethanol (4 BV) at a flow rate of 1 BV·h using HPD-BJQH macroporous adsorption resin. -1 Column chromatography was performed at a specific flow rate, and the eluents were collected and concentrated under reduced pressure. The water eluent contained glycosides (approximately 14.2%), the 30% ethanol eluent contained flavonoids (approximately 21.6%), and the 95% ethanol eluent contained solanolone (approximately 33.7%). The 732 cation exchange resin column was washed with water until the eluent was colorless, and then eluted sequentially with 95% ethanol and 4N ammonia-ethanol solution. The ammonia-ethanol eluent was collected, the solvent was recovered, and the column was extracted three times with chloroform. The chloroform layer was recovered to obtain the alkaloid component (approximately 1.0%).

[0054] Example 3

[0055] Reference Figure 1Two kg of dried Datura leaves were extracted with 12 times the volume of 95% ethanol for 5 days each time. The filtrates were combined, and the solvent was recovered under reduced pressure to obtain approximately 265 g of total ethanol extract of Datura leaves (yield 13.25%). The extract was then extracted eight times with 0.1% hydrochloric acid solution at a volume of 8 times the concentrate, and filtered. Hyoscyamine alkaloids in the filtrate were removed by column chromatography using a 732 cation exchange resin at a flow rate of 3 BV / h, and the eluent was collected. After recovery and concentration, the eluent was then adsorbed sequentially with water (2 BV), 30% ethanol (2 BV), and 95% ethanol (4 BV) at a flow rate of 1 BV·h using HPD-BJQH macroporous adsorption resin. -1 Column chromatography was performed at a specific flow rate, and the eluents were collected and concentrated under reduced pressure. The water eluent contained glycosides (approximately 18.8% yield), the 30% ethanol eluent contained flavonoids (approximately 27.4% yield), and the 95% ethanol eluent contained solanolone (approximately 42.1% yield). The 732 cation exchange resin column was washed with water until the eluent was colorless, and then eluted sequentially with 95% ethanol and 4N ammonia-ethanol solution. The ammonia-ethanol eluent was collected, the solvent was recovered, and the column was extracted three times with chloroform. The chloroform layer was recovered to obtain the alkaloid component (approximately 1.5% yield).

[0056] 1. Study on the non-overlapping chemical composition of each component of Datura leaf

[0057] 1.1 Study on the non-overlapping chemical composition of each resolved component under HPLC detection

[0058] 1.1.1 HPLC Chromatographic Conditions

[0059] Instrument: 2695-2998-2424 analytical HPLC-UV-ELSD Column: Waters SunFire TM C 18 Column (4.6 × 150 mm, 5 μm) Flow rate: 1.0 mL / min; Column temperature: 25 ℃; Injection volume: 10 μL Mobile phase: methanol-water, gradient elution conditions are shown in Table 1.

[0060] 1.1.2 HPLC verification results of the non-overlapping chemical composition of each resolved component

[0061] The non-overlapping chemical composition of each separated component was verified primarily using HPLC, with results (e.g.) Figure 2As shown in the figure, the elution times of the main compounds in the water, 30% ethanol and 95% ethanol fractions are significantly different. The chloroform layer has some overlap in retention time with the other three groups, but by comparing the UV absorption spectra, it can be determined that their chemical composition types are completely different, indicating that there is basically no overlap among the four components.

[0062] 1.2 Study on the non-overlapping chemical composition of each separated component detected by UHPLC-MS

[0063] 1.2.1 Chromatographic conditions

[0064] liquid phase conditions

[0065] Instrument: Vanquish UHPLC ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA)

[0066] Column: Waters ACQUITY CSH T3 column (2.1 × 100 mm, 1.7 µm)

[0067] Flow rate: 300 μL / min; Column temperature: 35 ℃;

[0068] Mobile phase: acetonitrile-water (1‰ formic acid in water, v / v), gradient elution conditions are shown in Table 2.

[0069] Mass spectrometry conditions

[0070] Mass spectrometry system: Thermo Orbitrap Fusion Lumos mass spectrometry system;

[0071] Ionization mode: H-ESI source; Spray voltage: 3.5 kV; Cone voltage: 40 V;

[0072] Ion transfer tube temperature: 320 ℃; capillary tube temperature: 325 ℃;

[0073] Data acquisition range: m / z 100-1500.

[0074]

[0075] 1.2.2 Verification results of the non-overlapping properties of each split component

[0076] The baseline peak diagrams of the fractions separated by water, 30% ethanol, 95% ethanol, and chloroform extraction are shown below. Figure 3The results show that the elution times of the main compounds in the water, 30% ethanol, and 95% ethanol fractions are significantly different. Although some minor peaks have similar elution times, comparison of MS data indicates that the types of chemical components are completely different. Significant differences in polarity exist among the components of the three fractions, indicating that there is essentially no overlap between them. Compared with the chloroform extract, the elution times of the three components show significant overlap, but comparison of MS data again indicates that the types of chemical components are completely different, suggesting that there is essentially no overlap between the four components.

[0077] 1.3 PCA and PLS-DA Analysis of Each Fraction of Datura Leaf

[0078] Principal component analysis was used to analyze the data collected by UPLC-Orbitrap-Fusion-Lumos. Two-dimensional data analysis was performed using an unsupervised statistical model combined with orthogonal partial least squares. Scoring plots were obtained through PCA and PLS-DA analysis. The overall trend of each split component of the datura leaf was analyzed, and the non-overlapping nature of each split component of the datura leaf was verified from the perspective of multivariate mathematical statistics.

[0079] 1.3.1 PCA Analysis of Each Component of Datura Leaf

[0080] After preprocessing each fractionated component using QI software, the data was imported into SIMCA-P software. PCA was then used to analyze the imported data, and the PCA score graph is shown below. Figure 4 As shown, R²X = 0.862, Q² = 0.769. The fractions extracted by water, 30% ethanol, 95% ethanol, and chloroform were clearly distributed in different regions, indicating that there are significant differences in the chemical composition of each fraction.

[0081] 1.3.2 PLS-DA Analysis of Each Fraction of Datura Leaf

[0082] PLS-DA analysis was performed on the data of the fractions separated by water, 30% ethanol, 95% ethanol and chloroform extraction. The results are as follows: Figure 5 As shown, R²X = 0.83, R²Y = 0.995, and Q² = 0.992. The results indicate that the resolved components are clearly distributed in different regions, suggesting significant differences in their chemical composition, and that the components do not overlap.

[0083] 2. Chemical characteristics of each resolved component detected by HPLC

[0084] 2.1 HPLC Chromatographic Conditions

[0085] 2.2 Chemical characteristics of each chemically resolved component detected by HPLC

[0086] The water-eluted components were analyzed by HPLC and the UV absorption spectra were detected. The results are as follows: Figure 6 As shown in the figure. The results show that the aqueous component exhibits the characteristic absorption peak of aromatic glycosides at around 250 nm, therefore the aqueous component is identified as a glycoside component.

[0087] The elution fraction with 30% ethanol was analyzed by HPLC and the UV absorption spectrum was detected. The results are as follows: Figure 7 As shown in the figure. The results show that the 30% ethanol fraction exhibits characteristic absorption peaks of flavonoids at around 250-260 nm and 320-330 nm, therefore the 30% ethanol fraction was identified as the flavonoid fraction.

[0088] The 95% ethanol eluent was analyzed by HPLC and the UV absorption spectrum was detected. The results are as follows: Figure 8 As shown in the figure. The results showed that the 95% ethanol fraction exhibited the characteristic absorption peak of solanolactone compounds at around 226 nm, therefore the 95% ethanol eluent fraction was identified as the solanolactone fraction.

[0089] The chloroform extract layer was analyzed by HPLC and the UV absorption spectrum was detected. The results are as follows: Figure 9 As shown in the figure. The results showed that the chloroform layer exhibited characteristic absorption peaks of hyoscyamine alkaloids at around 220-230 and 270-280 nm, therefore the chloroform extract layer was identified as the alkaloid component.

[0090] Chemical fractions of Datura stramonium leaves were separated using 732 cation exchange chromatography combined with HPD-BJQH macroporous resin column chromatography. The retention times of the separated fractions under the same gradient elution conditions were investigated. Multivariate statistical methods, including HPLC-UV-ELSD, UHPLC-Orbitrap-MS, principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (PLS-DA), were used to study the non-overlapping chemical components of each fraction. The chemical characteristics of each fraction were studied using HPLC-UV, and principal component characterization was performed based on the ultraviolet absorption characteristics of the compounds. Experimental results showed that:

[0091] (1) The chemical components of Datura leaves were separated by 732 cation exchange combined with HPD-BJQH macroporous resin column chromatography. Water elution yielded glycosides, 30% ethanol elution yielded flavonoids, 95% ethanol elution yielded solanacrolides, and chloroform extraction yielded alkaloids.

[0092] (2) Using HPLC-UV-ELSD and UHPLC-Orbitrap-MS techniques, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (PLS-DA) and other multivariate mathematical statistical methods were applied to study the non-overlapping chemical composition of each split component. The results showed that the non-overlapping chemical composition of each split component was good.

[0093] Example 4

[0094] Pharmacodynamic experiments of the active components obtained by the method of this invention in the treatment of Alzheimer's disease

[0095] This embodiment evaluates the pharmacodynamics of the total extract of Datura stramonium leaves, glycosides (water-eluted), flavonoids (30% ethanol-eluted), solanolone (95% ethanol-eluted), and alkaloids (chloroform extract) prepared by the method of Example 1 of this invention in the context of Alzheimer's disease (AD), in order to verify the significance of the bioactivity and non-overlapping properties of each component in practical applications.

[0096] 4.1 Experimental Materials

[0097] 4.1.1 Test Drug

[0098] The following test samples were prepared according to the method described in Example 1 of the present invention:

[0099] Total ethanol extract of Datura stramonium leaves (Total);

[0100] Glycoside fraction (water-eluted fraction, H2O);

[0101] Flavonoid fraction (30% ethanol elution fraction, 30%)

[0102] Solanacolin fraction (95% ethanol eluent, 95%);

[0103] Alkaloid components (chloroform extract layer, alkaloids).

[0104] Before administration, prepare a suspension of the required concentration using 0.5% sodium carboxymethyl cellulose (CMC-Na).

[0105] 4.1.2 Laboratory Animals

[0106] SPF-grade male SD rats, weighing 200±20 g, were provided by the Experimental Animal Center, license number: No. SCXK (Liaoning) 2020-0001. They were housed in a constant temperature (22±2℃), constant humidity (50%±10%), 12 h light / 12 h dark cycle environment with free access to food and water.

[0107] 4.1.3 Main Reagents and Instruments

[0108] Streptozotocin (STZ, Sigma); Donepezil hydrochloride (positive control drug, Eisai Pharmaceutical); Rat TNF-α, IL-1β, IL-6, SOD, GPX, MDA ELISA kit (Nanjing Jiancheng Bioengineering Institute); Brain stereotaxic instrument (Shenzhen Ruiwode Company); Water maze video analysis system (Shanghai Xinruan Information Technology Co., Ltd.)

[0109] 4.2 Experimental Methods

[0110] 4.2.1 Animal modeling, grouping, and drug administration

[0111] An animal model of atrial hyperplasia (AD) was established by injecting STZ into the CA1 region of the hippocampus in the lateral ventricle. Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg) and fixed on a stereotaxic apparatus with the anterior fontanelle as the zero point. The coordinates were: 1.5 mm posterior to the anterior fontanelle, 1.5 mm lateral to the left and right, and 3.5 mm subdural. On the first and third days after surgery, STZ (3 mg / kg, 18 μL each time) was slowly injected into the CA1 region of the bilateral hippocampus. The sham-operated group received an equal volume of saline.

[0112] One hundred and twenty SD rats were randomly divided into the following 12 groups, with eight rats in each group: blank group (CON), sham-operated group (SHAM), model group (MOD), positive control group (donepezil hydrochloride, 1.30 mg / kg), high-dose total extract group (Total-H, 200 mg / kg), low-dose total extract group (Total-L, 50 mg / kg), high-dose glycoside group (H2O-H, 200 mg / kg), low-dose glycoside group (H2O-L, 50 mg / kg), high-dose flavonoid group (30%H, 200 mg / kg), low-dose flavonoid group (30%L, 50 mg / kg), high-dose asholinone group (95%H, 200 mg / kg), and low-dose asholinone group (95%L, 50 mg / kg).

[0113] The day after model establishment, the drug was administered via gavage at a volume of 10 mL / kg once daily for 30 consecutive days. The control group, sham-operated group, and model group were given an equal volume of physiological saline.

[0114] 4.2.2 Behavioral Experiments

[0115] (1) Navigation and Positioning Experiment: Adaptation training was conducted on the second day after drug administration. The formal experiment began on the third day. Rats were placed in the water facing the pool wall, and the time it took for them to find the hidden platform (escape latency) was recorded, limited to 60 seconds. Training continued for 4 days, and testing was conducted on the fifth day. The swimming trajectory diagram is shown below. Figure 10 See changes in the incubation period to avoid Figure 11 .

[0116] (2) Spatial exploration experiment: 24 hours after the end of the positioning and navigation experiment, the platform was removed, and the rats were placed in the water from the original entry point. The number of times the rats crossed the original platform position and the percentage of time spent in the target quadrant were recorded within 60 seconds. The results are shown in […]. Figure 12 .

[0117] 4.2.3 Tissue Sampling and Processing

[0118] After the behavioral experiment, blood was collected from the abdominal aorta of rats, centrifuged (4℃, 3500 rpm, 15 min) after standing at room temperature, and the serum was aliquoted and stored at -80℃ for later use. Brain tissue was collected, some of which was fixed with 4% paraformaldehyde and paraffin sections were prepared; the rest was quick-frozen and stored at -80℃.

[0119] 4.2.4 Indicator Testing

[0120] (1) Pathological observation of brain tissue (HE staining): Paraffin sections were dewaxed to water, stained with hematoxylin and eosin, dehydrated and mounted, and the cell morphology of the CA1 region of the hippocampus was observed under a 200x light microscope. The results are shown in the figure. Figure 13 .

[0121] (2) Detection of Tau protein expression (immunohistochemistry): Paraffin sections were antigen-retrieved, blocked with serum, incubated overnight at 4°C with P-Tau primary antibody, incubated at room temperature with secondary antibody, developed with DAB, counterstained with hematoxylin, observed under a 400x light microscope, and the positive area ratio was calculated. The results are shown in […]. Figure 14 .

[0122] (3) Detection of serum inflammatory factors and oxidative stress indicators: The levels of TNF-α, IL-1β, IL-6, SOD, GPX, and MDA in serum were measured using an ELISA kit, following the instructions of the kit. The results are shown in the table below. Figure 15 .

[0123] 4.3 Experimental Results

[0124] 4.3.1 Observation of the general condition of rats

[0125] After modeling, the rats in the model group exhibited symptoms such as dry and yellowish fur, poor appetite, emaciation, reduced activity, and sluggish response to external stimuli. With increasing administration days, these symptoms improved to varying degrees in all treatment groups, with the most significant improvements observed in the high-dose total extract group, the high-dose flavonoid group, and the high-dose solanolone group.

[0126] 4.3.2 Results of the behavioral experiment

[0127] The results of the positioning and navigation experiment show that ( Figure 10 , Figure 11The escape latency of rats in the model group was significantly longer than that in the control group (P < 0.01), indicating impaired learning and memory abilities. Compared with the model group, the escape latency of rats in the positive drug group, the high-dose total extract group, the high-dose flavonoid group, the high-dose ashlanolone group, and the low-dose group was significantly shortened (P < 0.01 or P < 0.05), while there was no significant improvement in the glycoside group (P > 0.05).

[0128] The results of space exploration experiments show that ( Figure 12 The number of times rats crossed the platform and the percentage of time spent in the target quadrant were significantly reduced in the model group compared with the control group (P < 0.01). Compared with the model group, the above indicators were significantly increased in the positive drug group, the high / low dose group of total extract, the high dose group of flavonoids, and the high / low dose group of solanolone (P < 0.01 or P < 0.05), while there was no significant change in glycosides.

[0129] 4.3.3 Effects on brain tissue pathology

[0130] HE staining results ( Figure 13 The results showed that the pyramidal cells in the CA1 region of the hippocampus of rats in the blank group were neatly and densely arranged with regular morphology; the pyramidal cells in the model group were shrunken, with condensed and deeply stained nuclei and loose arrangement; the morphology of pyramidal cells in the CA1 region of the hippocampus of rats in the positive drug group, the high-dose flavonoid group, and the high / low-dose solanolone group were all improved to varying degrees, and the cells were arranged more neatly; the improvement of glycoside group was not obvious.

[0131] 4.3.4 Effects on Tau protein expression in brain tissue

[0132] P-Tau immunohistochemical results ( Figure 14 The results showed that, compared with the blank group, the positive area ratio of P-Tau protein in the CA1 region of the hippocampus of rats in the model group was significantly increased (P < 0.01); compared with the model group, the positive drug group, the high-dose flavonoid group, and the high / low-dose asholinone group had significantly decreased P-Tau protein positive area ratio (P < 0.01 or P < 0.05), while the glycoside group had no significant effect.

[0133] 4.3.5 Effects on serum inflammatory factors and oxidative stress markers

[0134] ELISA test results ( Figure 15 )show:

[0135] The serum levels of TNF-α, IL-1β, and IL-6 in the model group rats were significantly higher than those in the control group (P < 0.01), the MDA level was significantly higher, and the SOD and GPX activities were significantly lower (P < 0.01).

[0136] Compared with the model group, the positive control group, the total extract group, the high-dose flavonoid group, and the high / low-dose asholinone group significantly reduced the levels of TNF-α, IL-1β, IL-6, and MDA, while increasing the activities of SOD and GPX (P < 0.05 or P < 0.01).

[0137] The glycoside components had no significant effect on the above indicators (P > 0.05).

[0138] 4.4 Summary of Example 4

[0139] This embodiment uses an ICV-STZ-induced AD rat model to evaluate the pharmacodynamics of the active components of Datura stramonium leaves isolated by the method of this invention. Results show that:

[0140] (1) The total ethanol extract of Datura stramonium leaves, the flavonoid component (30% ethanol elution component), and the solanacone component (95% ethanol elution component) can significantly improve the learning and memory ability of AD model rats, reduce neuronal damage in the CA1 area of ​​the hippocampus, inhibit the excessive phosphorylation of Tau protein, reduce the level of serum inflammatory factors and regulate oxidative stress indicators.

[0141] (2) The glycoside component (water-eluting component) did not show significant improvement in the above efficacy indicators. The alkaloid component (chloroform extract layer) was not tested separately due to its low content, but it did not overlap with other components in chemical analysis.

[0142] (3) This embodiment further verifies that the active components obtained by the method of the present invention have clear chemical non-overlapping and differentiated biological activities, providing experimental basis for the comprehensive utilization of Datura leaf resources and the development of AD treatment drugs.

Claims

1. A method for preparing multiple active components from Datura leaves, characterized in that, Includes the following steps: Step 1: Take dried Datura leaves, extract them by soaking in ethanol, recover the solvent from the extract under reduced pressure, and concentrate it to a certain volume to obtain a concentrated extract of total ethanol extract of Datura leaves. Step 2: Extract the concentrated total extract of Datura stramonium leaves in ethanol obtained in Step 1 with an acidic aqueous solution, filter, and obtain the filtrate; Step 3: Pass the filtrate obtained in Step 2 through a cation exchange resin column, collect the effluent, and simultaneously elute and recover the components adsorbed on the cation exchange resin to obtain the alkaloid component. Step 4: After recovering and concentrating the effluent collected in Step 3, pass it through a macroporous adsorption resin column and elute it sequentially with water and ethanol aqueous solution. Collect each eluent and concentrate it under reduced pressure to obtain glycoside, flavonoid and solanolactone components.

2. The method according to claim 1, characterized in that, The ethanol mentioned in step 1 is 95% ethanol by volume. The amount of ethanol used during maceration and extraction is 6 to 12 times the weight of the datura leaves, and the maceration and extraction time is 2 to 5 days each time.

3. The method according to claim 2, characterized in that, The amount of ethanol used during maceration extraction was 8 times the weight of the datura leaves, and the maceration extraction time was 3 days each time.

4. The method according to claim 1, characterized in that, The acidic aqueous solution mentioned in step 2 is a 0.1% hydrochloric acid solution, the amount of which is 3 to 8 times the volume of the concentrated total extract of Datura stramonium leaf ethanol obtained in step 1, and the extraction is performed 3 to 8 times.

5. The method according to claim 4, characterized in that, The amount of the acidic aqueous solution used is 5 times the volume of the concentrated total extract of Datura stramonium leaf ethanol obtained in step 1, and the extraction is performed 6 times.

6. The method according to claim 1, characterized in that, The cation exchange resin mentioned in step 3 is type 732 cation exchange resin, and the flow rate through the resin column is 3 BV / h.

7. The method according to claim 1, characterized in that, Step 3 describes the elution and recovery of the components adsorbed on the cation exchange resin. Specifically, the components are washed with water until the effluent is colorless, then eluted sequentially with 95% ethanol and 4N ammonia-ethanol solution. The ammonia-ethanol eluent is collected, the solvent is recovered, and the components are extracted three times with chloroform. The chloroform layer is then recovered to obtain the alkaloid component.

8. The method according to claim 1, characterized in that, The macroporous adsorption resin mentioned in step 4 is HPD-BJQH type macroporous adsorption resin.

9. The method according to claim 1, characterized in that, The specific conditions for gradient elution in step 4 are as follows: first elute with 2 BV of water, then elute with 2 BV of 30% ethanol, and finally elute with 4 BV of 95% ethanol, with an elution flow rate of 1 BV·h⁻¹.

10. The method according to claim 1, characterized in that, In step 4, the glycoside component is a water-eluted component, the flavonoid component is a 30% ethanol-eluted component, and the solanolone component is a 95% ethanol-eluted component.

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