TW201742629A - Method for improving active ingredient content, chemical component consistency of medicinal material, and/or therapeutic benefits of Uraria spp. plants, and products and applications thereof comprising a drying treatment conducted at a temperature of 35 to 45 DEG C - Google Patents

Abstract

An improved method for processing plants of Uraria spp., including performing a drying treatment of Uraria spp. before performing an optional extraction treatment for the harvested plants of Uraria spp. to improve the active ingredient contents of the Uraria spp. and its extracts, the chemical component consistency of this medicinal material, and/or the therapeutic benefits. The drying treatment is conducted at a temperature of 35 to 45 DEG C.

Description

Method for improving the content of active ingredients of genus Brassica, chemical composition of medicinal materials, and/or therapeutic benefit, products and applications thereof

The present invention relates to a processing improvement of the genus Urania spp., wherein the temperature is between 35 and 45 ° C before the desired extraction treatment of the harvested genus Brassica . The genus Brassica is subjected to a drying treatment to increase the active ingredient content of the genus Brassica or its extract, the chemical composition of the medicinal material, and/or the therapeutic benefit. The present invention also relates to a Brassica plant or an extract thereof obtained by the above-described processing improvement method

Brassica ( Uraria crinita (L.) Desv.ex DC.), Brassica chinensis ( Uraria lagopodioides (L.) Desv.ex DC.), Brassica napus (Uria picta (Jacq.) Desv.ex DC.), Uraria neglecta Prain and other plants of the genus Urania spp. are commonly used to spleen and stomach, cough and throat, remove hoarding, treat diarrhea, treat dysplasia in children, Detoxification, swelling, and treatment of bruises and medicinal materials. According to the literature, Urania spp. also has good anti-inflammatory, analgesic, antibacterial, anti-oxidant, nitric oxide scavenging, insect repellent and insecticidal effects. Wild Brassica plants have been found in areas such as India, Thailand, Indonesia, southern China, and Taiwan. Among them, the cultivation of the genus Brassica has been carried out in Taiwan through artificial cultivation in order to respond to the Chinese herbal medicine market. high demand.

It is known that improving the consistency of the quality of Chinese herbal medicines is one of the important means to improve the consistency of clinical efficacy. Previous studies have pointed out that the variation of the active ingredient content of Chinese herbal medicines will affect the quality and efficacy of the herbs, and it is generally believed that the changes in the content of active ingredients in the herbs are derived from many agricultural factors, including planting and harvesting. However, these studies ignore the effects of post-harvest processing methods on the quality of the medicinal materials.

In addition to the quality of Chinese herbal medicines, such as variety, growth period, growth environment, origin, climate, harvesting season, etc., primary processing is also an important factor affecting the quality of Chinese herbal medicines, among which different drying methods The influence of the quality of the medicinal materials has been neglected for a long time in the initial processing of the production area. When Chinese herbal medicines are harvested, the water content is high. If it is not processed in time, it is easy to be mildewed and deteriorated, which seriously affects the quality of the medicine, clinical efficacy and economic benefits of the farmers.

Taking the drying process of medicinal materials as an example, the traditional method relies on the drying method of sun drying or dry drying, the efficiency is low, and the conditions are not dried in time or the drying conditions are inconsistent, which causes a large deviation between the dry and dry drying methods, thus affecting the quality of the medicinal materials.

Or, in order to prevent the mildew and corruption of the medicinal materials, some farmers use sulfur fumigation to process, which easily causes the residual amount of sulfur dioxide to exceed the standard and endanger human health.

At present, the genus Brassica is processed directly by the farmers in the production area after harvesting. Because there is no good processing norm, the processing methods and conditions adopted by farmers everywhere are different, which leads to heterogeneity. Heterogeneousness, which seriously affects the biological activity and active ingredient content in the medicinal materials, causes the quality of the medicinal materials of the genus Brassica to be unstable, which is not conducive to the treatment and control of related diseases. See, for example, Production of salvianolic acid B in Roots of Salvia miltiorrhiza (Danshen) during the post-harvest drying process. Molecules , 2012. 17(3): p. 2388-407, the entire disclosure of which is incorporated herein by reference.

Therefore, in the application level of the Chinese herbal medicine of the genus Brassica, how to standardize the processing method of the Chinese medicinal materials of the genus Brassica, in order to improve the active ingredient content and therapeutic benefit of the genus Brassica To ensure the safety and effectiveness of medicinal materials is one of the major issues to be solved in this field.

The present invention provides a method for effectively improving the content of the active ingredient of the genus Brassica or its extract, the composition of the chemical composition of the medicinal material, and/or the therapeutic benefit, in view of the above research results, and in particular, the improvement thereof. Amino acid, flavonoids, triterpenoids, betaine, and salicylic acid. In addition, the Brassica plant or the extract thereof provided by the method of the present invention has excellent estrogen stimulating activity, NRF2 stimulating activity, and/or COX-2 inhibitory activity, and thus can be used for treating or preventing estrogen activity, A disease associated with NRF2 activity, and/or COX-2 activity.

It is an object of the present invention to provide a method for improving the content of an active ingredient of a plant of the genus Urania spp. or a part thereof or the aforementioned extract, the composition of the chemical composition of the medicinal material, and/or the therapeutic benefit thereof. The drying process is carried out at a temperature of 35 to 45 ° C before being subjected to a desired extraction treatment on the harvested genus of the genus Brassica. .

Another object of the present invention is to provide a Brassica plant or a part thereof or the aforementioned extract obtained by the above method.

A further object of the present invention is to provide a use of the above-obtained Brassica plant or a part thereof or the aforementioned extract for the manufacture of a medicament for stimulating estrogenic activity and stimulating nuclear factor E2 Related factor 2 (nuclear erythroid 2-related factor 2, NRF2) activity, and / or inhibition of cyclooxygenase-2 (COX-2) activity.

The detailed technical content and some of the specific embodiments of the present invention will be described in the following, and the present invention will be apparent to those of ordinary skill in the art.

Figure 1 shows the ¹ H-NMR spectrum of rabbit tail herbs processed by different methods; Figure 2 shows the results of principal component analysis (PCA) of the metabolites of rabbit tail herbs processed by different methods. Dot maps, including "drying group", "dry group", and "drying group"; Figure 3 shows ¹ H-NMR spectrum of rabbit tail herbs dried at 40 ° C, 55 ° C or 70 ° C, respectively Figure 4 shows the results of a principal component analysis (PCA) analysis of the metabolites of rabbit-tailed herbal materials dried at different temperatures (40 ° C, 55 ° C, 70 ° C); Figure 5 shows A plot of the results of a chemical composition consistency comparison of different processed samples of rabbit tail herbs by principal component analysis (PCA); Figure 6 shows the first batch and the second batch of rabbit tail herbs treated under different conditions The estrogen activity (%) bar graph of the material; Figure 7 shows the NRF2 stimulating activity (%) bar graph of the first batch and the second batch of rabbit tail herbs treated under different conditions; Figure 8 shows the COX-2 inhibitory activity (%) bar graph of the first batch and the second batch of rabbit tail herbs treated under different conditions; Figure 9 shows the The DPPH radical scavenging rate (%) bar graph of the first batch and the second batch of rabbit tail herbs treated under the same conditions; and the 10th graph shows the first batch and the second batch of rabbits treated under different conditions The TEAC value (μg/ml) of the tail herb material is bar graph.

The invention will be described in detail below with reference to the embodiments of the present invention. The present invention may be practiced in various different forms without departing from the spirit and scope of the invention. . In addition, the terms "a", "an" and "the" and "the" Refers to the reduction of a disease or symptom of a body, and may also mean reducing the cause of the disease or the lesion itself, and is not limited to completely eliminating the disease or the lesion; the term "prevention" refers to inhibiting or preventing the occurrence of a disease or symptom.

It is known that Urania spp. contains the main active ingredients such as amino acids, flavonoids, triterpenoids, betaine, and salicylic acid. It can be used to spleen and stomach, relieve cough and throat, and remove hoarding. , treatment of diarrhea, treatment of dysplasia in children, detoxification, swelling, and treatment of bruises, and anti-inflammatory, analgesic, antibacterial, anti-oxidation, deworming, insecticidal and other effects. However, the post-harvest processing methods used in the origin of the genus Brassica are different, which results in unstable quality of the genus Brassica, which is not conducive to the treatment and control of the disease. In view of this, if we can find a standardized method to improve the content of the active ingredients of the genus Brassica, and even improve the consistency of the chemical composition of the medicinal materials, it will effectively improve the application of the genus Brassica in the treatment of diseases and improve the rabbit grass. The therapeutic benefits of genus.

The inventor of the present invention found that the dried genus of the genus Brassica is dried at 35 to 45 ° C, compared to the dry treatment at 22 to 25 ° C, and dried at 22 to 28 ° C. Or drying at 55 ° C or 70 ° C, can significantly improve the content of amino acids, flavonoids, triterpenoids, betaine, and salicylic acid of the genus Brassica, and can Improve the consistency of the chemical composition of the herbs of the genus Brassica. The inventors of the present invention have also found that the Brassica plant provided by the method of the present invention has estrogen stimulating activity and nuclear factor E2 related factor 2 in addition to enhanced DPPH free radical scavenging ability and TEAC total antioxidant capacity ( Nuclear erythroid 2-related factor 2, NRF2) stimulating activity, and cyclooxygenase-2 (COX-2) inhibitory activity.

Accordingly, the present invention provides a method for enhancing the content of active ingredients, consistency of chemical composition of a medicinal material, and/or therapeutic benefit of a plant of the genus Urania spp. or a portion thereof or the aforementioned extract, which is included in Before subjecting the harvested genus of genus to a desired extraction process, the harvested genus of the genus Brassica is subjected to a drying process, wherein the drying process is performed at a temperature of 35 to 45 ° C. Go on.

By the above drying treatment at 35 to 45 ° C, the content of at least one of the following active ingredients in the herbaceous plant or part thereof or the aforementioned extract can be increased: amino acids, flavonoids, triterpenoids , betaine, and salicylic acid. Among them, the amino acid isocyanic acid (Isoleucine, Ile), threonine (Threonine, Thr), arginine (Arginine, Arg), aspartate (Aspatate, Asp), aspartame At least one of aphoraine (Asn), tyrosine (Tyrosine, Tyr), tryptophan (Trp) and Phenylalanine (Phe), the flavonoid apigenin glycosides And at least one of Spatholosineside A.

Since the method of the invention can increase the content of the active constituents such as amino acids, flavonoids, triterpenoids, betaines, and salicylic acid in the harvested Schnauzer or part thereof or the aforementioned extracts, Elevating the benefits of the genus Brassica or a part thereof or the aforementioned extract in one or more of the following, but not limited thereto: spleen and stomach, cough and throat, removal of hoarding, treatment of diarrhea, treatment of children Dysplasia, detoxification, swelling, treatment of bruises, anti-inflammatory, analgesic, antibacterial, anti-oxidant, deworming, and insecticidal.

The method of the present invention is applicable to any suitable plant of the genus Brassica, including, for example, Brassica chinensis ( Uraria crinita (L.) Desv.ex DC.), and Brassica chinensis ( Uraria lagopodioides (L.) Desv.ex DC .), Urania picta (Jacq. Desv.ex DC.), Uraria neglecta Prain, Uraria acaulis Schindl., Camptotheca acuminata ( Uraria acuminata Kurz), Uraria balansae Schindl., Uraria barbata Lace, Uraria campanulata (Benth. Gagnep.), Urania candida Backer ), Uraria clarkei Gagnep., Urania cochinchinensis Schindl., Uraria cordifolia Wall., Urania crinita (L.) DC., column Urania cylindracea Benth., Uraria fujianensis YCYang & PHHuang, Uraria gossweileri Baker f., Uraria kurzii Schindl., Shan State rabbit tail grass (Uraria lacei Craib), raccoon tail grass (Uraria lagopodoides (L.) DC. ), rabbit pulled Perth Grass (Uraria lagopus DC.), Beaver tail smith beans (Uraria longibracteata YCYang & PHHuang), petri Lagurus grass (Uraria pierrei Schindl.), Glass Lagurus Blue Grass (Uraria poilanei Phon), the Prussian rifampicin Li Uraria prunellifolia Baker, Uraria rotundata Craib, Uraria rufescens (DC. Schindl.), and Urania sinensis (Hemsl.) Franch). Preferably, the method of the present invention is used in Brassica chinensis ( Uraria crinita (L.) Desv.ex DC.), Urania lagopodioides (L.) Desv. ex DC., and Brassica napus. ( Uraria picta (Jacq.) Desv.ex DC.), and Urania neglecta Prain.

According to the method of the present invention, the use site of the genus Brassica is not particularly limited, and may be a whole plant of the genus Brassica or a different part of the genus Brassica, such as the root, stem, and leaf of the genus Brassica. Department, and/or flower department. In some embodiments of the present invention, the roots of the genus Brassica are used to prepare a Chinese medicinal material of the genus Brassica.

In the process of the invention, the drying temperature used is preferably about 40 °C. Further, the extract of the genus Brassica can be subjected to an extraction treatment after the drying treatment to provide a Brassica plant extract.

Accordingly, the present invention further provides a plant of the genus Brassica or a part thereof or the aforementioned extract obtained by the above method of the present invention. Among them, the extract is preferably obtained by extracting a Brassica plant or a part thereof using a polar solvent such as an alcohol, water, or a mixture of an alcohol and water. For example, the polar solvent may be a C1-C6 alcohol (eg, methanol, ethanol, ethylene glycol, propanol, isopropanol, propylene) Alcohol, butanol, isobutanol, butanediol, pentanol, isoamyl alcohol, hexanol, cyclohexanol, etc.), water, or a combination of the foregoing. More preferably, the polar solvent is selected from the group consisting of C1-C4 alcohols, water, and combinations of the foregoing. In some embodiments of the invention, ethanol is used as the extraction solvent.

As described above, the Brassica plant or a part thereof or the extract thereof according to the present invention contains an active ingredient such as an amino acid, a flavonoid, a triterpenoid, a betaine, and a salicylic acid, wherein the amino acid is Isoleucine (Ile), threonine (Thr), arginine (Arg), aspartate (Aspate, Asp), aspartic acid (Asparagine, Asn), cheese At least one of Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe), the flavonoids Apigenin glycosides and Spatholosineside A At least one of them. Further, the Brassica plant or a part thereof or the aforementioned extract system provided by the method of the present invention has various biological activities including estrogen stimulating activity, NRF2 stimulating activity, and COX-2 inhibitory activity.

It is known that increasing estrogen activity can achieve treatment or prevention of osteoporosis, Parkinson's disease, Alzheimer's disease, cardiovascular disease, obesity, stroke and the like. Therefore, the herbaceous stimulating activity of the genus Brassica or its extract can also be used for the treatment or prevention of diseases caused by estrogen activity as described above or caused by low estrogen. See, for example, Estrogen receptors and human disease. J Clin Invest. 2006 Mar; 116(3): 561-70, the entire disclosure of which is hereby incorporated by reference.

It is known that increasing the activity of the NRF2/ARE (nuclear erythroid 2-related factor 2/antioxidant responsive element) message transduction pathway can achieve treatment or prevention of Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke, heart Vascular diseases (stroke, cardiac hypertrophy, cardiomyocyte apoptosis, myocardial fibrosis, heart failure), liver damage, liver fibrosis, kidney damage, diabetes, etc. Therefore, the Brassica plant having an NRF2 stimulating activity or an extract thereof can also be used for treating or preventing a disease caused by the above-mentioned NRF2/ARE message transduction pathway activity or low activity due to the NRF2/ARE message transduction pathway. See, for example, Natural product-derived pharmacological modulators of Nrf2/ARE pathway for chronic diseases. Nat Prod Rep. 2014 Jan;31(1):109-39, and Targeting the Nrf2 pathway against cardiovascular disease. Expert Opin Ther Targets. 2009 Jul; 13(7): 785-94, the entire contents of which are hereby incorporated by reference.

Studies have shown that COX-2 does not perform in most normal body tissues and cells, but COX-2 activity can be detected in the body of various cancer patients. Therefore, the activation of COX-2 is closely related to the development of cancer and tumor. If it can inhibit the activity of COX-2, it can achieve anti-cancer and anti-tumor effects. See, for example, Chemoprevention in gastrointestinal physiology and disease. Anti-inflammatory approaches for colorectal cancer chemoprevention. Am J Physiol Gastrointest Liver Physiol. 2015 Jul 15;309(2): G59-70.doi:10.1152/ajpgi.00101.2014.Epub 2015 May 28, the entire disclosure of which is incorporated herein by reference. Therefore, a Brassica plant having an COX-2 inhibitory activity or an extract thereof can also be used for anticancer and antitumor.

Accordingly, the present invention also provides the use of the above-obtained Brassica plant or a part thereof or the aforementioned extract for the manufacture of a medicament for stimulating estrogenic activity, stimulating NRF2 activity and/or inhibiting COX-2 activity. In addition, the agent can also be used to treat or prevent one or more of the following, but not This is limited to: osteoporosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, obesity, stroke, cardiac hypertrophy, cardiomyocyte apoptosis, myocardial fibrosis, heart failure, liver damage, liver Fibrosis, kidney damage, diabetes, cancer, and cancer.

The agent produced by using the Brassica plant of the present invention or a part thereof or the aforementioned extract may be in any suitable form, and is not particularly limited, and is in a suitable dosage form depending on the intended use. For example, but not limited thereto, the drug can be administered to an individual in need thereof by oral or non-oral administration (for example, subcutaneous, intravenous, intramuscular, intraperitoneal, or nasal). Depending on the form of use and use, a suitable carrier may be employed to provide the agent.

In the case of a dosage form suitable for oral administration, the medicament provided by the present invention may contain any pharmaceutically acceptable carrier which does not adversely affect the desired benefits of the Brassica plant or part thereof or the aforementioned extract of the present invention. For example: solvent (water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof), oily solvents, diluents, stabilizers, absorption delaying agents, disintegrating agents, emulsifiers, antioxidants , binders, binders, tackifiers, dispersants, suspending agents, lubricants, moisture absorbents, solid carriers (such as starch, bentonite), and the like. The agent may be provided in a dosage form suitable for oral administration by any convenient method, for example, a tablet (for example, a sugar-coated tablet), a pill, a capsule, a granule, a powder, a flow extract, a solution, a syrup, a suspension. , emulsions, and tinctures.

As for an injection form or a drip form suitable for subcutaneous, intravenous, intramuscular, or intraperitoneal injection, one or more e.g. isotonic solutions, such as phosphate buffer or the like, may be contained in the medicament provided by the present invention. Intravenous infusion of citrate buffer, solubilizer, emulsifier, 5% sugar solution, and other carriers The medicament is provided in the form of a liquid, an emulsion intravenous infusion solution, a dry powder injection, a suspension injection, or a dry powder suspension injection. Alternatively, the medicament is prepared as a pre-injection solid, the pre-injection solid is provided in a dosage form, or emulsifiable, which is soluble in the other solution or suspension, and the injection is administered prior to administration to the individual in need thereof. The pre-solids are dissolved in other solutions or suspensions or emulsified to provide the desired injectables. Further, it is suitable for intranasal or transdermal administration of an external dosage form such as an emulsion, a cream, a gel (for example, a hydrogel), a paste (for example, a dispersion cream, an ointment), a spray, or a solution (for example, washing) Liquid, suspension).

Optionally, a suitable amount of an additive may be further included in the medicament provided by the present invention, for example, a flavoring agent, a toner, a coloring agent, etc., which can improve the mouthfeel and visual sensation of the medicament when taken, and can be improved. A buffer, a preservative, a preservative, an antibacterial agent, an antifungal agent, and the like for stability and storage of the agent. In addition, the agent may optionally contain one or more other active ingredients, or may be used in combination with the drug containing the one or more other active ingredients to further enhance the efficacy of the agent or increase the flexibility and formulation of the formulation, as long as the agent The other active ingredient does not adversely affect the desired benefits of the Brassica plant or part thereof or the aforementioned extract of the present invention.

The amount of the agent produced by using the Brassica plant of the present invention or a part thereof or the aforementioned extract may be adjusted depending on the age of the subject to be administered and the purpose of administration, and the frequency of use may be adjusted as needed. The agent may also contain other ingredients depending on the final form of the agent and the purpose of use. In principle, the type and amount of other ingredients added may not adversely affect the desired efficacy of the plant of the genus Brachnia or a part thereof or the aforementioned extract.

The invention is further illustrated by the following examples. Of which The examples are provided by way of illustration only and are not intended to limit the scope of the invention. The scope of the invention is shown in the appended claims.

Example [Preparation Example]

A. Harvesting and processing of Brassica

The genus Brassica used in the following examples was collected from the farmland of the same area in the same area of Nanjian, Nantou, Taiwan (Uria crinita (L.) Desv.ex DC.), and two batches were collected. The first batch was harvested in October and the second batch was harvested in November. The two batches of the rabbittail specimen (LMS-1001) were deposited in the Department of Chinese Pharmacy and Chinese Medicine Resources, China Medical University. .

After harvesting the first batch of the latter, the roots were taken and washed quickly with water, then divided into three groups, and then dried separately under the following conditions to provide rabbit tail herbs: (1) drying group: Outdoor (outdoor room temperature is about 22-28 ° C) exposure for 5-7 days; (2) dry group: in the dark (outdoor room temperature is about 22-25 ° C), air drying, lasting 12-14 days And (3) drying group: drying in an oven at 40 ° C for 2 days.

Similarly, after harvesting the second batch of the latter, the roots were taken and quickly washed with water, and then divided into three groups, but dried under the following conditions to provide rabbit tail herbs: (1) 40 °C drying group: drying in an oven at 40 ° C for 2 days; (2) drying at 55 ° C: drying in an oven at 55 ° C for 2 days; (3) Drying group at 70 ° C: drying in an oven at 70 ° C for 2 days.

B. Brassica medicinal materials and their extraction

B-1. Each group of rabbit-tailed herbal materials obtained from [Preparation Example] A. was ground to obtain a rabbit-tailed herbal material powder for subsequent experimental analysis.

B-2. The medicinal material powders of each of the above B-1 groups were treated as follows. Take 100 grams of the medicinal material powder, soak in 500 ml of 50% ethanol for 7 days; repeat the aforementioned ethanol extraction step for three times; combine the extracts obtained by three extractions, and concentrate under reduced pressure to remove the ethanol to obtain the rabbit tail. The grass extract was prepared in 50% ethanol to a concentration of 1 mg/ml for subsequent analysis.

C. ¹ H-NMR spectrum ( ¹ H-Nuclear Magnetic Resonance Spectroscopy, ¹ H-NMR analysis

C-1. Preparing the sample to be tested

Take the above-mentioned B-1. Each group of rabbit tail herbs powder, each group take 50 mg into a microcentrifuge tube (Eppendorf), and carry out the following sample preparation steps: (i) add to the microcentrifuge tube 0.75 ml of deuterated methanol (CH ₃ OH-d4) and 0.75 ml of phosphate buffer solution (containing 90 mM potassium dihydrogen phosphate (KH ₂ PO ₄ ) phosphate buffer solution, the pH was adjusted to 6.0 with 1 M NaOD, wherein Using 0.01% trisodium phosphate (TSP) as internal standard and D ₂ O as solvent); (ii) vortexing the microcentrifuge tube at room temperature for 1 minute; Iii) shaking at 298 K (ie, 25 ° C) with an ultrasonic oscillator for 30 minutes; (iv) at 298 K (ie, 25 ° C) for 10 minutes centrifugation (13,200 rpm); (v) 600 μl of the supernatant was transferred to a 5-mm NMR tube for testing (see, for example, NMR-based metabolomic analysis of plants. Nat Protoc. 2010. 5 (3): p.536-49 And as needed, the entire text of which is incorporated herein by reference.

C-2. Analysis steps and conditions:

The NMR tubes provided with the samples to be tested provided by C-1 above were placed in a Bruker 600 MHz NMR spectrometer (Bruker AVANCE 600 AV) with a cryoprobe (CryoProbe 5mm CPTXI (1H)) at 298 K (ie The spectra were collected at 25 ° C).

In order to obtain high-quality nuclear magnetic resonance spectroscopy, manual locking and manual shimming were used in the experiment, and the half-height width (FWHM) of the TSP was used to determine the magnetic field uniformity.

A standard one-pulse sequence with water saturation (zggppr) sequence was collected during the experiment, wherein the line width factor (LB) was set to 0.3 Hz, and the TSP half-height width of each sample was necessary. ≦1.8Hz is the pass standard to ensure the quality of the map. Experimental conditions: number of scans (NS) = 128, number of sweeps (DS) = 0, number of samples (TD) = 32K data points, spectral width = 20 ppm, relaxation time (RD) = 2.0 seconds, The sampling time is 1.36 seconds, and the 90 degree pulse length is about 10.5 μs, which is adjusted according to each sample.

In order to identify the metabolites of rabbit tail herbs, 2D-NMR spectra were collected, including COSY ( ¹ H- ¹ H correlation spectroscopy), TOCSY ( ¹ H- ¹ H total correlation spectroscopy), JRES (J-resolved spectroscopy), HSQC( ¹ H- ¹³ C heteronuclear single-quantum coherence), and HMBC (heteronuclear multiple bond correlation), the above steps and conditions can be found, for example, Combined NMR and LC-DAD-MS analysis reveals comprehensive metabonomic variations for three phenotypic cultivars of Salvia Miltiorrhiza Bunge. J Proteome Res. 2010. 9 (3): p.1565-78, and Combined NMR and LC-MS analysis reveals the metabonomic changes in Salvia miltiorrhiza Bunge induced by water depletion. J Proteome Res. 2010. 9 (3 ): p. 1460-75, the entire disclosure of which is hereby incorporated by reference.

C-3. Data Processing and Multivariate Statistical Analysis:

The ¹ H NMR free induction decay (FID) raw data is multiplied by a window function with a linewidth factor (LB) of 0.3 Hz, zero fill (were zero-fille) is 65536 points, and then the complex leaf transform (Fourier) Transform). Next, manual phase correction and manual baseline correction were performed, and the chemical shift was corrected by using TEST (δ=0.00 ppm) as an internal standard by Mestrenova (version 8.0.2, Mestrelab research SL). The corrected map is binned and normalized. The integral interval of each segment is 0.005ppm, and the integral range is δ 0.04-10.0ppm, which has a total of 1992 integration intervals. Thereafter, methanol (δ 3.300-3.400) and water peak signal (δ 4.500-5.000) are removed, and total area normalization is performed on the peak area to eliminate water content and sample volume between samples. The difference in concentration caused.

The integrated data matrix was imported into the multivariate analysis software (SIMCA-P version 13.0, Umea, Umetrics, Sweden) for analysis, and the Pareto (par) calibration method was used for multivariate statistical analysis. Perform principal component analysis (PCA). Wherein the fitting (Fitting) The quality of the model using the value R ² (R ² value) and a value Q ² (Q ² value) as the basis for judgment, R ² represents a value of the variable may be interpreted in a direction of total variable Total amount variation explained by the model, and Q ² value indicates the predictive of the model under cross validation.

For the above data processing and multivariate statistical analysis, see, for example, Gallic acid ameliorated impaired glucose and lipid homeostasis in high fat diet-induced NAFLD mice. PLoS One. 2014.9(2): p.e96969, and Gut microbiota composition modifies fecal J. Proteome Res. 2013.12(6): p. 2987-99, the entire contents of which are hereby incorporated by reference.

C-4. Metabolite structure identification:

Metabolites in the tested samples were identified by literature alignment, metabolite library pairs, standard alignments, and 2D NMR spectroscopy confirmations (including JRES, COSY, HSQC, TOCSY, and HMBC). The data obtained by the analysis are expressed as mean ± standard error (mean ± SE) (see Table 2, Table 3). As for the metabolite data, a one-way analysis of variance (ANOVA) was used, and a post hoc test was performed using Bonferroni analysis technique, with p < 0.05 as a statistically significant limit. .

D. Plasmid construction and cell transfection

D-1. Plasmid construction

pERE-Luc plasmid: The synthetic tetra-estrogen response element (ERE: 5'-GGTCACAGTGACCTA-3', SEQ ID NO: 1) was cloned into the pGL4 plasmid (Promega, Madison). , firefly luciferase gene in WI USA reported upstream of the gene, and the pERE-Luc plasmid was obtained.

pER-α plasmid: clone ER-α gene full-length cDNA to pcDNA Downstream of the CMV promoter in granules (Promega, Madison, WI USA), the pER-α plasmid was obtained.

NRF2-Luc plasmid: A four-repetitive antioxidant response element (ARE: 5'-GGTCACAGTGACC-3', SEQ ID NO: 2) was cloned into the pGL4 plasmid to obtain a NRF2-Luc plasmid.

D-2. Cell transfection

The pERE-Luc plasmid and the pER-α plasmid were simultaneously stably transfected into human embryonic kidney 293 cells (HEK-293 cell, purchased from the American Type Culture Collection (ATCC), American horses. A stable transfected pER-α-Luc HEK-293 cell line was established in Manassas (VA, USA). In addition, the NRF2-Luc plasmid was stably transfected into HEK-293 cells to establish stable transfection. pNRF2-Luc HEK-293 cell line.

D-3. Cell culture

The pER-α-Luc HEK-293 cell line and the pNRF2-Luc HEK-293 cell line were separately cultured in 10% fetal bovine serum (FBS) and 100 at 37 ° C under 5% CO ₂ . Micrograms per milliliter of Kanamycin in DMEM medium (Dulbecco's modified Eagle's medium, available from Life Technologies, USA, model: gibco 11965).

Among them, the pER-α-Luc HEK-293 cell strain was washed with PBS before inoculation, and phenol-free red containing 5% activated carbon/dextran-treated fetal bovine serum (charcoal dextran-treated FBS, cdFBS) was added. DMEM without phenol red, purchased from Life technologies, USA, model: gibco In 21063), the cells were starved and lasted for 2 days. Next, the pER-α-Luc HEK-293 cell strain was inoculated into a 96-well plate, and 40,000 cells were seeded per well.

The pNRF2-Luc HEK-293 cell line was seeded in a 96-well dish in DMEM medium containing 10% FBS, and 20,000 cells were seeded per well.

Example 1: Effect of different processing methods on the active ingredients of rabbit tail herbs

1-1. Metabolite analysis

In order to understand the effects of different processing methods on the harvested rabbit tail herbs, ¹ H-NMR metabolite analysis was performed on the "dried group", "dry group" and "drying group" rabbit tail herbs. In Figure 1 and Table 1. Among them, (A) is the drying group, the temperature is 22-28 ° C; (B) is the dry group, the temperature is 22-25 ° C; (C) is the drying group, the temperature is 40 ° C; "The Arabic numerals" The metabolite names represented are shown in Table 1.

As shown in Table 1, 33 metabolites were identified in rabbit tail herbs, which can be divided into primary and secondary metabolites, and their structures include amino acids, organic acids, sugars, nucleic acids, amines, and Triterpenoids, flavonoids, phenolic acids, and the like.

As shown in Figure 1, whether it is compared to the "dry group" or "drying group", the signals of the amino acid, lipid, and triterpenoids of the "drying group" rabbit tail herbs are significantly increased by 1 to At 3 ppm, the signals of flavonoids, phenolic acids, and aromatic amino acids were significantly up-regulated by 6 to 9 ppm, while the sugar signal was significantly down-regulated by 3 to 6 ppm. The above results show that the content of amino acids, lipids, triterpenoids, flavonoids, and phenolic acids in the rabbit-tailed herbal materials provided by drying at 40 ° C is high.

Table 1

1-2. Metabolite analysis

Principal component analysis (PCA) was used to analyze the data of the "drying group", "dry group" and "drying group" rabbit tail herb metabolites. The results showed In Fig. 2, the X-axis can clearly distinguish the drying group from the other groups, indicating that the chemical composition of the rabbit-tailed herbal material after drying treatment is significantly different from other groups.

As can be seen from Figure 2, the first main component (PC1) of the "drying group", "drying group", and "dry group" rabbit tail herbs can be clearly distinguished, and the first principal component (PC1) and The interpretation rate (R ² X) of the two principal components (PC2) is 0.759, and the prediction rate (Q ² ) is 0.675, indicating that the model is good. Among them, the composition of the "drying group" rabbit-tailed herbal medicines was significantly different from that of the "drying group" and "dry-drying group" group of rabbit-tailed herbal materials, while the "dry group" and "drying group" rabbit tails were grouped. The ingredients of herbal materials are not clearly grouped. The above results showed that there was a significant difference in the metabolite composition between the "drying group" rabbit tail herbs and the "dry group" and "drying group" groups.

1-3. Semi-quantitative screening of metabolites

It can be seen from the results of Example 1-2 that the metabolite composition of the "drying group" rabbit-tailed herbal material is significantly different from that of the "drying group" and the "dry group", so the integral value of the metabolite front is half. Quantitatively, to understand the differential metabolites between the "drying group" rabbit tail herbs and the "drying group" and "dry group" two groups of rabbit tail herbs, the results are shown in Table 2.

As can be seen from Table 2, compared to the "drying group" and "yin dry group" two groups, the "drying group" of the rabbit tail herb amino acid (isoleucine, lysine, threonine, alanine , arginine, aspartic acid, tyrosine, tryptophan, phenylalanine), organic acids (salicylic acid, formic acid), betaine, triterpenoids, and flavonoids (apigenin, red blood vine The amount of glycoside A) is significantly higher, while the amount of sugars (glucose, sucrose) and choline is significantly lower.

The above results show that drying is compared to drying or drying. Treatment can significantly improve the amino acid (isoleucine, valine, threonine, alanine, arginine, aspartic acid, tyrosine, tryptophan, phenylalanine) in rabbit tail herbs. The content of active ingredients such as organic acids (salicylic acid, formic acid), betaine, triterpenoids, and flavonoids (apigenin, erythrostatin A).

Example 2: Effect of different drying temperatures on the active ingredients of rabbit tail herbs

2-1. Metabolite analysis

¹ H-NMR metabolite analysis was performed on the "40 ° C drying group", "55 ° C drying group", and "70 ° C drying group" rabbit tail herbs, and the results are shown in Fig. 3. Among them, the stronger the signal, the higher the content.

As shown in Figure 3, compared to the "55 °C drying group" or "70 °C drying group", the "40 °C drying group" of the rabbit tail herbs are amino acids, triterpenoids, and flavonoids. The content increased significantly, while the sugar content decreased significantly.

2-2. Metabolite analysis

The metabolites of the "40 °C drying group", "55 °C drying group", and "70 °C drying group" rabbit tail herbs were analyzed by principal component analysis (PCA). The results are shown in Fig. 4.

As can be seen from Figure 4, the composition of the "40 °C drying group" rabbit tail herbs is significantly different from the "55 °C drying group" and "70 °C drying group" group of rabbit tail herbs. The composition of the 55 °C drying group and the "70 °C drying group" rabbit tail herbs were not significantly grouped. The above results showed that the metabolite composition of the "40 °C drying group" rabbit tail herbs and the "55 °C drying group" and "70 °C drying group" group of rabbit tail herbs were significantly different.

2-3. Semi-quantitative screening of metabolites

From the results of Example 2-2, the metabolite composition of the "40 ° C drying group" rabbit tail herbs and the "non-40 ° C drying group" (including "55 ° C drying group" and "70 ° C drying group" There is a significant difference, so the integral value of the metabolite front is semi-quantified to understand the "40 °C drying group" and the "non-40 °C drying group" (including "55 °C drying group" and "70 °C drying" The differential metabolites between the dry groups") are shown in Table 3.

As can be seen from Table 3, compared to the "non-40 °C drying group" (including "55 °C drying group" and "70 °C drying group"), "40 °C drying group" rabbit tail herbal material amino acid (isoleucine, sulphate, arginine, aspartic acid, aspartic acid, tyrosine, tryptophan, phenylalanine), salicylic acid, betaine, triterpenoids, and flavonoids The amount of the genus (apigenin, erythrostatin A) was significantly higher, while the amount of sucrose and choline was significantly lower.

The above results show that after the rabbit's tail herbs are harvested and dried at a temperature of 35 to 45 ° C (for example, 40 ° C), the amino acid in the rabbit tail material (isoleucine, sulphate, essence) can be significantly improved. Aminic acid, proline, aspartic acid, aspartic acid, tyrosine, tryptophan, phenylalanine, salicylic acid, betaine, triterpenoids, and flavonoids (apigenin, red) The content of active ingredients such as saponin A).

Example 3: Comparison of different batches of rabbit tail herbs

Combine the first batch of "drying group", "drying group", "drying group" rabbit tail herbs and the second batch of "40 °C drying group", "55 °C drying group", "70 °C ¹ H-NMR data of the dried group "rabbit tail herbs" and principal component analysis (PCA) were performed to visually reflect the influence of different factors on the metabolite composition of the rabbit tail herb material. The results are shown in Fig. 5. Among them, the X-axis (PC1 axis, main component 1) analyzes different processing factors (including 40 °C (showing drying at 40 °C) and "non-40 °C (not 40 °C) shown on the PC1 axis. Other methods of dry treatment))) The effect on the consistency of chemical composition of rabbit tail herbs; Y-axis (PC2 axis, principal component 2) is used to analyze different batches (including the first batch shown on the PC2 axis) And "second batch") have an effect on the consistency of chemical composition of rabbit tail herbs, including the "drying group (first batch)" and "dry group" (first batch) in the upper half of the figure. ), the results of the "drying group (first batch)", and the "40 °C drying group (second batch)" and "55 °C drying group (second batch) in the lower half of the figure) ", 70 °C drying group (second batch)" results.

As can be seen from Figure 5, "40 °C drying group" and "non-40 °C drying group" (including "55 °C drying group", "70 °C drying group", "dry group", "drying group" The metabolites of rabbit tail herbs were clearly distinguished on the PC1 axis, and the chemical composition of the "40 °C drying group" was consistent. The metabolites of rabbit tail herbs in different batches were also clearly distinguished on the PC2 axis. The foregoing results show that the homogeneity between the two batches of drying treatment groups is significantly higher than that of other groups, and the drying treatment at 35 to 45 ° C (for example, 40 ° C) has different effects than the different batches. The effect of the harvest is more significant, and the chemical composition of the rabbit tail herbs can be made closer to each other, thereby improving the consistency of the clinical efficacy of the rabbit tail herbs.

Example 4: Effect of different processing methods on the biological activity of rabbit tail herbs

This study used a cell screening platform to analyze the biological activity of rabbit tail herbs. The results showed that rabbit tail herbs have estrogenic stimulating activity, COX-2 inhibitory activity, NRF2 stimulating activity, DPPH free radical scavenging ability, and total antioxidant capacity of TEAC. . Next, the effects of different processing conditions on the aforementioned activities were further analyzed.

4-1. Determination of estrogen stimulating activity

The pER-α-Luc HEK-293 cell line was inoculated into a 96-well plate, and each group of the rabbit extract (1 mg) provided in [Preparation Example] B-2. was added to each well. /ml) and the positive control drug 17?-estradiol (17?-estradiol ("E2"), 100 micromolar (100 nM), cultured for 24 hours. Thereafter, the medium was removed, and 50 μl of cell lysate was added to each well (composition: 1% Triton X-100, 2 mM DTT (dithiothreitol), 2 mM molar CDTA) (trans-1,2-Diaminocyclohexane-N,N,N ' ,N ' -tetraacetic acid monohydrate), 10% glycerol and 25 mM Tris-HCl (pH 7.8), oscillated for 10 minutes . Next, 100 microliters of luciferase assay buffer (1.25 mM Tris-HCl (pH 7.8), 10 mM NaHCO _{3 was added to each well.} MgSO _{4 at a} concentration of 2.5 millimolar, EDTA at a concentration of 0.1 millimolar, DTT at a concentration of 10 millimolar, coenzyme-A lithium at a concentration of 60 micromolar, potassium chromiferin at a concentration of 225 micromolar (potassium luciferin) ), ATP of 250 micromolar concentration, after mixing uniformly, the absorbance at 560 nm wavelength was measured using a spectrophotometer, and the absorbance ( A ) was recorded. Finally, estrogen activity (%) was calculated in the following formula 1, and the results after the statistics are shown in Fig. 6.

Formula 1: Estrogen activity (%) = [( A _sample - A _control / A _E2 - A _control )] x 100.

In Formula 1, " A _sample " is the absorbance of the sample at a wavelength of 560 nm; " A _control " is the absorbance of the control group at a wavelength of 560 nm; " A _E2 " is the absorbance of the positive control group at a wavelength of 560 nm.

As can be seen from Figure 6, compared to the "drying group" or "dry group", "bake The estrogen stimulating activity of the dry group was significantly higher; the estrogen stimulating activity of the "40 °C drying group" was significantly higher than that of the "55 °C drying group" or "70 °C drying group". On the other hand, there was no significant difference in the estrogenic stimulating activity between the "drying group" of the first batch and the "40 °C drying group" of the second batch. The above results show that the drying process of the rabbit tail material after harvesting at a temperature of 35 to 45 ° C (for example, 40 ° C) can effectively enhance the estrogen stimulating activity of the rabbit tail herb or its extract.

4-2. Determination of NRF2 stimulating activity

The pNRF2-Luc HEK-293 cell line was inoculated into a 96-well plate, and each group of the extracts of the rabbit grass (the concentration of 1 mg/ml) provided in [Preparation Example] B-2. was added to the respective wells. And the positive control drug andrographolide (20 micromolar concentration) was cultured for 16 hours. Thereafter, the medium was removed, and 50 μl of the cell lysate was added to each well and shaken for 10 minutes. Next, 100 μl of luciferase activity assay buffer was added to each well, and after mixing uniformly, the absorbance at 560 nm was measured using a spectrophotometer, and the absorbance ( A ) was recorded. Finally, the NRF2 activity (%) was calculated by the following formula 2, and the results after the statistics are shown in Fig. 7.

Formula 2: NRF2 stimulating activity (%) = [( A _sample - A _control / A _{andrographolide} - A _control )] x 100.

In Formula 2, " A _sample " is the absorbance of the sample at a wavelength of 560 nm; " A _control " is the absorbance of the control group at a wavelength of 560 nm; " A _{andrographolide} " is the _absorbance of the positive control group at a wavelength of 560 nm.

As can be seen from Figure 7, the NRF2 stimulating activity of the "drying group" was significantly higher than that of the "drying group"; the NRF2 stimulating activity of the "40 °C drying group" compared to the "55 °C drying group" Significantly higher. On the other hand, the first batch of "drying group" and the second batch There was no significant difference in the NRF2 stimulating activity of the "40 °C drying group". The above results show that the drying process of the rabbit tail material after harvesting at a temperature of 35 to 45 ° C (for example, 40 ° C) can effectively enhance the NRF2 stimulating activity of the rabbit tail herb or its extract.

4-3. Determination of COX-2 inhibitory activity

The COX Inhibitor Screening Assay Kit (Model 560131, available from Cayman Co., Ltd.) was used to determine the COX-2 inhibition of each group of Laxtail extracts provided by [Preparation Example] B-2. Activity, the results are shown in Figure 8.

As can be seen from Figure 8, the COX-2 inhibitory activity of the "drying group" is significantly higher than that of the "dry group"; compared to the "55 °C drying group" or "70 °C drying group", "40" The COX-2 inhibitory activity of the °C drying group was significantly higher. On the other hand, there was no significant difference in the COX-2 inhibitory activity between the "drying group" of the first batch and the "40 °C drying group" of the second batch. The above results show that the drying treatment of the rabbit tail material after harvesting at a temperature of 35 to 45 ° C (for example, 40 ° C) can effectively enhance the COX-2 inhibitory activity of the rabbit tail herb or its extract.

4-4. Determination of DPPH free radical scavenging ability

Take [Preparation Example] B-2. Each group of rabbit fura extract (concentration is 1 mg / ml), each group is taken 0.1 ml, respectively, with 0.1 ml of DPPH free radical methanol solution (concentration: Mix at 0.2 mm of DPPH/liter) and allow to stand at room temperature for 30 minutes in the dark. Next, the absorbance at 517 nm wavelength was measured using a spectrophotometer, and the absorbance ( A ) was recorded. The lower the absorbance value, the stronger the ability of the sample to scavenge DPPH radicals. Finally, the scavenging effect (%) is calculated in the following formula 3, and the statistical results are shown in Fig. 9.

Equation 3: Clearance rate (%) = [1 - ( A _sample - A _blank ) / ( A _control - A _blank )] x 100.

In Formula 3, " A _sample " is the absorbance of the sample at a wavelength of 517 nm; " A _blank " is the absorbance of the blank group at a wavelength of 517 nm; " A _control " is the absorbance of the control group at a wavelength of 517 nm.

As can be seen from Figure 9, the DPPH radical scavenging rate of the "drying group" is significantly higher than that of the "drying group"; compared to the "55 °C drying group" or "70 °C drying group", The DPPH free radical scavenging rate of the 40 °C drying group was significantly higher. On the other hand, there was no significant difference in the DPPH radical scavenging rate between the first batch of "drying group" and the second batch of "40 °C drying group". The above results show that the drying process of the rabbit tail material after harvesting at a temperature of 35 to 45 ° C (for example, 40 ° C) can effectively improve the DPPH scavenging ability of the rabbit tail herb or its extract.

4-5. Determination of total antioxidant capacity of TEAC (Trolox equivalent antioxidant capacity)

It is known that ABTS (2,2'-Azino-bis-[3-ethylbenthiazoline sulfonic acid] reacts with peroxidase to produce ABTS ^{. + a} cationic radical, which exhibits a stable blue-green color and absorbs bees at a wavelength of 734 nm. If the test sample is added, the ABTS is removed ^{. +} The ability of cationic free radicals, the blue-green color will be lighter and the absorbance will be reduced, which can be used to evaluate the total antioxidant capacity, and the Troxox solution is used as a positive standard to make a standard curve. This is the TEAC total. Determination of antioxidant capacity. Among them, the higher the TEAC value, the stronger the ability to scavenge free radicals and the stronger the antioxidant capacity.

Take the [Preparation Example] B-2. Each group of rabbit fura extract (concentration of 1 mg / ml) or Trolox solution, and 0.125 ml of ABTS (1000 micromolar concentration), 0.125 ml of H ₂ O ₂ (500 micromolar concentration) and 0.125 ml of peroxidase solution (44 units/ml) were mixed and placed in the dark for one hour until ABTS ^{. +} Free radical cation formation. Next, the absorbance at a wavelength of 734 nm was measured using a spectrophotometer for 10 minutes, and the absorbance ( A ) was recorded and converted into a TEAC value (microgram/ml). The results are shown in Fig. 10. The foregoing experimental methods can be found, for example, in Evaluation of antioxidant activity and inhibitory effect on nitric oxide production of some common vegetables. J Agric Food Chem. 2006. 54(5): p.1680-6, Antioxidant activity and bioactive compounds of tea seed (Camellia oleifera Abel) .) Oil. J Agric Food Chem. 2006.54(3): p.779-84, and Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999.26(9-10): p.1231-7, The entire contents of these documents are hereby incorporated by reference.

As can be seen from Figure 10, the TEAC value of the "drying group" is significantly higher than that of the "drying group"; compared to "55 °C drying group" or "70 °C drying group", "40 °C drying" The TEAC values of the dry group are significantly higher. On the other hand, there is no significant difference in the TEAC values of the first batch of "drying group" and the second batch of "40 °C drying group". The above results show that the drying process of the rabbit tail herb material at a temperature of 35 to 45 ° C (for example, 40 ° C) after harvesting can effectively improve the total antioxidant capacity of the TEAC of the rabbit tail herb or its extract.

It can be seen from the above experimental results that the drying treatment of the genus Brassica is carried out by drying at 35 to 45 ° C, which can not only effectively increase the active ingredient content of the genus Brassica or its extract, but also enhance The chemical composition of the medicinal material is conducive to the application of the medicinal material in the treatment of diseases, and can effectively improve the estrogen stimulating activity, COX-2 inhibitory activity, NRF2 stimulating activity, DPPH free radical scavenging ability, and TEAC total of the genus Brassica. Antioxidant ability, effectively improve the therapeutic benefits of the genus Brassica or its extract.

<110> Chongren Medical Management College

<120> Method for improving the content of active ingredients of the genus Brassica, the chemical composition of the medicinal materials, and/or the therapeutic benefit thereof, and the products and applications thereof

<130> None

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> ERE primer

<400> 1

<210> 2

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> ARE primer

<400> 2

Claims (10)

A method for enhancing the content of an active ingredient of a herb of the genus Urania spp. or a part thereof or the aforementioned extract, the composition of the chemical composition of the medicinal material, and/or the therapeutic benefit, which is included in the rabbit harvested The genus Hemerocallis is subjected to a drying treatment prior to the desired extraction treatment, wherein the drying treatment is carried out at a temperature of 35 to 45 °C. The method of the requested item 1, wherein the Lagurus plants of the genus is selected from at least one of the following: grass Lagurus (. Uraria crinita (L.) Desv.ex DC), big leaf Lagurus grass (Uraria lagopodioides (L. ) Desv.ex DC.), Urania picta (Jacq. Desv.ex DC.), Uraria neglecta Prain, Uraria acaulis Schindl., Hi Uraria acuminata Kurz, Uraria balansae Schindl., Uraria barbata Lace, Uraria campanulata (Benth. Gagnep.), White-tailed rabbit tail Uraria candida Backer, Uraria clarkei Gagnep., Urania cochinchinensis Schindl., Uraria cordifolia Wall., Uraria crinita (L.) DC.), Urania cylindracea Benth., Uraria fujianensis YCYang & PHHuang, Uraria gossweileri Baker f., Uraria kurzii Schindl. ), Shan Lagurus grass (Uraria lacei Craib), raccoon tail grass (Uraria lagopodoides (L.) DC. ), pulled Perot Lagurus grass (Uraria lagopus DC.), Beaver tail smith beans (Uraria longibracteata YCYang & PHHuang), petri Lagurus grass (Uraria pierrei Schindl.), Glass Lagurus Blue Grass (Uraria poilanei Phon), the Prussian Li Uraria prunellifolia Baker, Uraria rotundata Craib, Uraria rufescens (DC. Schindl.), and Chinese Brassica ( Uraria sinensis (Hemsl) .) Franch). The method of claim 1, wherein the portion of the genus Brassica is root, stem, leaf, and/or flower. The method of claim 1, wherein the drying treatment is carried out at a temperature of about 40 °C. The method of any one of claims 1 to 4, wherein the active ingredient is selected from at least one of the group consisting of amino acids, flavonoids, triterpenoids, betaines, and salicylic acid. The method of claim 5, wherein the amino acid is Isoleucine (Ile), threonine (Threon), arginine (Arginine, Arg), aspartate (Aspate, Asp) At least one of Aspartaine (Asn), Tyrosine (Tyr), Tryptophan (Trp) and Phenylalanine (Phe), the flavonoid is apigenin ( At least one of Apigenin glycosides) and Spatholosineside A. The method of any one of claims 1 to 4, which enhances the benefit of one or more of the following plants of the genus Brassica or a part thereof or the aforementioned extracts: spleen and stomach, cough and throat, and removal of sputum Accumulate, treat diarrhea, treat dysplasia in children, detoxification, reduce swelling, treat bruises, anti-inflammatory, analgesic, antibacterial, anti-oxidant, deworming, and insecticidal. A Schnauzer or a part thereof, or an extract thereof, obtained by the method of any one of claims 1 to 7. Use of a species of Brassica or a part thereof as claimed in claim 8 or an extract thereof For the manufacture of a medicament for stimulating estrogen activity, stimulating nuclear factor 2 (NRF2) activity, and/or inhibiting cyclooxygenase-2 (Cycloxygenase- 2, COX-2) activity. The use of claim 9, wherein the agent is for treating or preventing one or more of the following: osteoporosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, obesity, stroke, Cardiac hypertrophy, cardiomyocyte apoptosis, myocardial fibrosis, heart failure, liver damage, liver fibrosis, kidney damage, diabetes, cancer, and tumors.