CN107964031B - Triterpenoid extracted and separated from rhizoma alismatis, and method and application thereof - Google Patents
Triterpenoid extracted and separated from rhizoma alismatis, and method and application thereof Download PDFInfo
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Abstract
The invention discloses a triterpenoid extracted and separated from rhizoma alismatis, a method and application thereof, wherein the structural formula of the triterpenoid is shown as (I). The compound is obtained by extracting and separating dried tubers of the rhizoma alismatis serving as a raw material through the process steps of ethanol reflux extraction, ethyl acetate extraction, silica gel column chromatographic separation, C18 reversed-phase chromatographic packing high-pressure preparation separation and the like, is a novel medicinal compound with pharmacological activity, has a good inhibition effect on staphylococcus aureus, can be used for preparing antibacterial drugs, and provides a reliable basis for preparing novel antibacterial drugs.
Description
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to a triterpenoid extracted and separated from rhizoma alismatis and having pharmacological activity, and a method and application thereof.
Background
The Alismatis rhizoma is Alismatis rhizoma of AlismataceaeAlisma orientalis （Sam.）Juzep.The dried tuber of (1). Alisma orientale has been used for more than 3000 years in China. Poetry, three thousand years ago, in which the third passage of "fenyue gao" was written: 'Bi fen Yi Qu', a Chinese character of Bian Zi, a son thereof, Mei Yu. "Annotation by Lu Jiang" Shi Shu ": as for its fruit, its guo Ye, its herb, jin Xie Ye. Its leaves are as big as plantain and its taste is similar. Xuzhou and Guangling people. "modern scholars congratulate on the book Shi Jing Yinju of Zhao Shu et al will explain its guo wire' to be modern Chinese" I adopt Alisma orientale into basket. "Shi Jing Yinshi (Ling Jing Ying) of Chenzi Ming is translated into" Shang He of ze Xie. The notes and translations of the ancient and modern scholars are that the herba Ixeritis Denticulatae is rhizoma Alismatis. It is indicated that women collected folium sennae before three thousand years. In the process of eating, the medicine is found to have medical effect and is used as a medicine by people.
The earliest Chinese herbal work is Shen nong Ben Cao Jing, which firstly collects alisma orientale as a medicine and is listed as the top grade. Later generations of the herbal literature are also filed with alisma orientale, such as Ming Yi Bie Lu of the times of the North of Wei jin, Ben Cao Jing Ji Zhu, Tang Ben Cao of the Tang Dynasty, Ben Cao Tu Jing of the Song Dynasty, Standby Ben Cao, Ben Cao Yan Yi (derivative of the materia Medica), Tang Ben Cao of the first generation, Ben Cao Hui Jing essence (essence of the materia Medica), Ben Cao compendium of the Ming Dynasty, Ben Cao Yao semi-Yao (supplement of the materia Medica), Ben Mong St (true materia Medica of the Qing Dynasty, Ben Shu (sparse syndrome of the Ben), De Canon Ben. They are all enriched, corrected and perfected on the basis of the Shen nong Ben Cao Jing, and the alisma becomes a common traditional Chinese medicine.
The alisma orientale has the effects of promoting diuresis and excreting dampness, and the modern medical research shows that the alisma orientale can reduce the content of serum total cholesterol and triacylglycerol and slow down the formation of atherosclerosis; alismatis rhizoma and its preparation are also used for treating Meniere's disease, dyslipidemia, spermatorrhea, fatty liver and diabetes.
Modern pharmaceutical research proves that the rhizoma alismatis contains various active ingredients such as alisol A, B, C, alisol A monoacetate, alisol B monoacetate, alisol C monoacetate, epi-alisol A, alisol oxide, 16 β -methoxy alisol B monoacetate, 16 β -hydroxy alisol B monoacetate, sitosterol-3-O-stearoyl- β -D-glucopyranoside and the like, and also contains choline, polysaccharide, potassium, calcium, magnesium and the like, but at present, further research on rhizoma alismatis medicaments mostly focuses on the aspects of extraction, separation, purification and the like of various known active ingredients, and relevant patent documents are as follows:
CN101724009A provides an extraction and purification process of triterpenoids in Alisma orientale, and the invention screens out the best resin for adsorbing the triterpenoids in Alisma orientale, namely HPD-100 type macroporous adsorption resin, by comparing the adsorption and desorption characteristics of 5 macroporous adsorption resins. The invention discloses a result that the 80% ethanol solution can basically and completely elute the triterpenoid of the alisma rhizome and the 23-acetyl alisol B, and the concentration of the elution solution in the process of the invention is 80% ethanol, so that the process of the invention can basically and completely elute the triterpenoid of the alisma rhizome and the 23-acetyl alisol B. The process comprehensively considers two indexes of the content of the triterpenoid of the alisma rhizome and the content of the 23-acetyl alisol B, so that the extraction and purification process is more reasonable and scientific.
CN102372759A provides a purification method of alisol A with little pollution and low cost, and the process method comprises the following steps: pulverizing Alismatis rhizoma, extracting with 70-90% ethanol under reflux, decolorizing the extractive solution with active carbon, recovering ethanol, adsorbing the concentrated solution with macroporous resin at 60-80 deg.C, gradient eluting with water and alcohol, concentrating to obtain precipitate, washing, crystallizing, separating the crystal with high speed countercurrent chromatography, and selecting heptane-dichloromethane-acetonitrile solvent system to obtain the final product. The alisol A produced by the method has high purity and high yield.
CN102464694A discloses a method for preparing 23-acetyl alisol B by high-efficiency separation. Extracting total sterol extract from Alismatis rhizoma with supercritical CO2 fluid, and further separating and purifying by high speed countercurrent chromatography; and finally refining by adopting a recrystallization method to obtain the high-purity 23-acetyl alisol B. The method has simple steps, high efficiency, easy repetition, and applicability to industrial production, and the purity of the separated 23-acetyl alisol B can reach above 99%, and can be used as quality control reference substance and raw material medicine of Alismatis rhizoma.
CN104497092A provides a method for extracting 23-acetyl alisol C from alisma orientale, which mainly solves the problems of complex operation, low product content and low recovery rate in the prior art. The method for extracting 23-acetyl alisol C from alisma orientale comprises the following steps: 1] extracting; 2] membrane separation; 3, removing impurities; 4] crystallizing. The method for extracting 23-acetyl alisol C from Alismatis rhizoma adopts alkali water wetting, and can remove part of large polar impurity molecules; simple operation, high product content and high recovery rate, and is suitable for industrial production.
Because the compounds derived from plants have great potential for finding new drugs, the inventor discovers and separates a new medicinal compound when researching the active ingredients of the alisma orientale, the compound is a triterpenoid and has certain antibacterial activity, in particular to staphylococcus aureusS.aureusHas good inhibiting effect. Therefore, the chemical active ingredients contained in the alisma orientale are still necessary to be further developed and discussed.
Disclosure of Invention
One of the purposes of the invention is to provide a triterpenoid extracted and separated from alisma orientale, and to obtain a new medicinal compound with pharmacological activity by extracting and separating alisma orientale medicinal materials, so as to further explore the new medicinal effect of alisma orientale and provide a good reference value for preparing novel antibacterial drugs.
The invention also aims to provide a method for extracting and separating triterpenoids from rhizoma alismatis, which takes dried tubers of the rhizoma alismatis as raw materials and obtains a novel medicinal compound with pharmacological activity through the steps of ethanol reflux extraction, ethyl acetate extraction, silica gel column chromatography separation, high-pressure preparation separation of C18 reversed-phase chromatographic packing and the like.
The invention also aims to provide the application of the triterpenoid extracted and separated from the rhizoma alismatis in the preparation of the antibacterial drug, and provide a reliable basis for preparing the novel antibacterial drug.
The technical scheme adopted by the invention is as follows:
a triterpenoid extracted and separated from rhizoma alismatis has a structural formula shown as (I):
the triterpenoid is obtained by extracting and separating from dried tubers of the rhizoma alismatis, and has the chemical name:
(1S,3R) -1- ((R) -3,3-dimethyloxiran-2-yl) -3- ((5R,8S,9S,10S,11S,14R) -11-hydroxy-4,4,8,10,14-pentamethyl-3-oxo-2,3,4,5,6,7,8,9,10,11,12,14-dodecahydro-1H-cyclopenta [ a ] phenoanthren-17-yl) butyl acetate with the self-name: dehydro-23-acetyl alisol B.
The triterpenoid is white powder, and the color of the 10% sulfuric acid ethanol color developing agent is purple red.
The 10% sulfuric acid ethanol is a general color developing agent, and the preparation method comprises the following steps: pouring concentrated sulfuric acid into absolute ethyl alcohol according to the volume ratio of 1:9, and uniformly mixing to obtain the 10% sulfuric acid ethyl alcohol color developing agent.
The molecular weight of the triterpenoid is 512, and the molecular formula is C32H48O5。
A method for extracting and separating triterpenes from rhizoma Alismatis comprises the following steps:
A. crushing rhizoma alismatis dried tubers, heating and refluxing the crushed rhizoma alismatis dried tubers by using 95wt% ethanol, concentrating the crushed rhizoma alismatis dried tubers under reduced pressure until no ethanol exists, and adding water into the concentrated extract for dispersing to obtain a water dispersion;
B. extracting the water dispersion with ethyl acetate, and recovering the solvent to obtain an ethyl acetate extract and a water phase part;
C. dissolving the ethyl acetate extract with methanol completely, drying with silica gel as carrier, wet-loading, separating with silica gel column chromatography, collecting target substance chromatographic solution, concentrating under reduced pressure, drying, dissolving with ethyl acetate, filtering, adding petroleum ether, standing for crystallization to obtain crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, separating the filtrate with C18 reversed phase chromatography packing under high pressure, collecting corresponding chromatographic peak, concentrating the collected liquid to dry to obtain white powder product, i.e. triterpenoid with structural formula shown in (I).
In the step A, the dosage of the ethanol is 8-10 times of the weight of the crushed dried rhizoma alismatis tubers.
In the step A, the reflux extraction is carried out for 3-5 times, and each time lasts for 2 hours.
In the step A, water is added into the concentrated extracting solution according to the volume ratio of 1: 5-1: 10 for dispersing treatment.
In the step B, the water dispersion is extracted 3-5 times by using ethyl acetate with the same volume.
In step C, the silica gel column chromatography separation: a: petroleum ether B: ethyl acetate, A: B =5: 1-2: 1 (V/V) as a mobile phase.
In step D, the C18 reverse phase chromatography packing high pressure preparative liquid phase separation: a: methanol B: water, A: B =85:15V/V is a mobile phase; the detection wavelength is 210 nm.
The white powder product obtained by the invention is purple red in color development of a 10% sulfuric acid ethanol color developing agent, namely the compound is a triterpenoid compound.
On the basis, the further analysis results are as follows:
electrospray ionization mass spectrum ESI-MS of triterpenoid with the structural formula shown in (I) shows that: positive ion 535.12[ M + Na ]]+，1047.31[2M+Na]+(ii) a Anion 511.20[ M-H ]]-That is, the molecular weight of the compound is 512 and the molecular formula is C32H48O5。1H-NMR and13the C-NMR data are shown in Table 1 below.
TABLE 1 NMR data for triterpenoids of formula (I)
By passing1H、13C-NMR and DEPT135 ℃ and nuclear magnetism two-dimensional analysis technical means such as HSQC, HMBC, H-HCOSY, NOESY and the like determine that the compound is as follows: dehydro-23-acetyl alisol B (triterpenoid) has a structural formula shown in (I).
More intensive research shows that the triterpenoid has certain antibacterial activity and has the effect of resisting staphylococcus aureusS.aureusHas good inhibition effect, and can be used for preparing antibacterial drugs.
The invention has the advantages and beneficial effects that:
1. the triterpenoid compound with the structural formula shown in the formula (I) is obtained by extracting and separating from dried tubers of rhizoma alismatis, has a definite structure and definite pharmacological activity, has certain antibacterial activity, and has a certain inhibiting effect on staphylococcus aureus, escherichia coli, bacillus subtilis and the like.
2. The modern pharmacological action research shows that: the triterpene compounds have wide physiological activity, and have hemolytic, anticancer, antiinflammatory, antibacterial, antiviral, cholesterol reducing, and molluscicidal activities. The triterpenoid extracted and separated from the alisma rhizome is a new medicinal compound, and antibacterial pharmacological tests show that the triterpenoid has an inhibiting effect on drug-resistant staphylococcus aureus equivalent to that of broad-spectrum antibiotic kanamycin sulfate. Therefore, the new medicinal effect of the rhizoma alismatis is further developed, and the method has a good reference value for preparing a novel antibacterial medicament.
3. According to the invention, dried tubers of alisma orientale are used as raw materials, and the triterpenoid compound is obtained by adopting the modes of ethanol reflux extraction, ethyl acetate extraction, silica gel column chromatographic separation, C18 reversed-phase chromatographic packing high-pressure preparation separation and the like, and the triterpenoid compound is easy to extract and separate, easy to obtain, simple and controllable in method, short in time consumption of the whole production process, and suitable for industrial production.
4. The invention not only reports the structure of the dehydro-23-acetyl alisol B for the first time, but also determines a new compound with relative configuration according to relevant data such as nuclear magnetic two-dimensional data and the like, and pharmacological research shows that the compound has good inhibitory effect on staphylococcus aureus, and can be used as a potential compound for development and utilization in the aspect of developing novel antibacterial drugs.
Detailed Description
The present invention will be described in further detail with reference to examples. It should be noted, however, that the following examples are not to be construed as limiting the scope of the present invention, and that many insubstantial modifications and variations of the invention can be made by those skilled in the art without departing from the spirit and scope of the invention as set forth herein.
Example 1
A triterpene compound extracted from Alismatis rhizoma is white powder, is 10% sulphuric acid ethanol developer with purple red color, molecular weight of 512, and molecular formula of C32H48O5Having the formula (I):
the triterpenoid is obtained by extracting and separating from dried tubers of alisma orientale, and the specific process steps are as follows:
A. crushing rhizoma alismatis dried tubers, heating and refluxing the crushed rhizoma alismatis dried tubers by using 95wt% ethanol, concentrating the crushed rhizoma alismatis dried tubers under reduced pressure until no ethanol exists, and adding water into the concentrated extract for dispersing to obtain a water dispersion;
B. extracting the water dispersion with ethyl acetate, and recovering the solvent to obtain an ethyl acetate extract and a water phase part;
C. dissolving the ethyl acetate extract with methanol completely, drying with silica gel as carrier, wet-loading, separating with silica gel column chromatography, collecting target substance chromatographic solution, concentrating under reduced pressure, drying, dissolving with ethyl acetate, filtering, adding petroleum ether, standing for crystallization to obtain crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, separating the filtrate with C18 reversed phase chromatography packing under high pressure, collecting corresponding chromatographic peak, concentrating the collected liquid to dry to obtain white powder product, i.e. triterpenoid with structural formula shown in (I).
Example 2
A method for extracting and separating triterpenes from Alismatis rhizoma comprises the following steps:
A. crushing rhizoma alismatis dried tubers, heating and refluxing for 3 times by using ethanol with the concentration of 95wt% which is 8 times of the weight of the rhizoma alismatis, extracting for 2 hours each time, concentrating under reduced pressure until no alcohol exists, and adding water into the concentrated extracting solution according to the volume ratio of 1:5 to perform dispersion treatment to obtain water dispersion;
B. extracting the water dispersion with equal volume of ethyl acetate for 3 times, and recovering solvent to obtain ethyl acetate extract and water phase part;
C. dissolving the ethyl acetate extract with methanol completely, drying with silica gel as carrier, wet loading, separating with silica gel column chromatography (petroleum ether B: ethyl acetate, A: B =5: 1V/V as mobile phase), collecting target substance chromatographic solution, concentrating under reduced pressure, dissolving with ethyl acetate, filtering, adding petroleum ether, standing for crystallization to obtain crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: methanol B: water, A: B =85:15V/V as mobile phase; detection wavelength 210 nm), collecting corresponding chromatographic peak, concentrating the collected liquid to dry to obtain white powder product, namely the triterpenoid compound with the structural formula shown in (I).
Example 3
A method for extracting and separating triterpenes from Alismatis rhizoma comprises the following steps:
A. crushing rhizoma alismatis dried tubers, heating and refluxing and extracting for 5 times by using ethanol with the concentration of 95wt% which is 10 times of the weight of the rhizoma alismatis, extracting for 2 hours each time, concentrating under reduced pressure until no alcohol exists, and adding water into the concentrated extracting solution according to the volume ratio of 1:8 to perform dispersion treatment to obtain water dispersion;
B. extracting the water dispersion with equal volume of ethyl acetate for 5 times, and recovering solvent to obtain ethyl acetate extract and water phase part;
C. dissolving the ethyl acetate extract with methanol completely, drying with silica gel as carrier, wet loading, separating with silica gel column chromatography (petroleum ether B: ethyl acetate, A: B =2: 1V/V as mobile phase), collecting target substance chromatographic solution, concentrating under reduced pressure, dissolving with ethyl acetate, filtering, adding petroleum ether, standing for crystallization to obtain crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: methanol B: water, A: B =85:15V/V as mobile phase; detection wavelength 210 nm), collecting corresponding chromatographic peak, concentrating the collected liquid to dry to obtain white powder product, namely the triterpenoid compound with the structural formula shown in (I).
Example 4
A method for extracting and separating triterpenes from Alismatis rhizoma comprises the following steps:
A. crushing rhizoma alismatis dried tubers, heating and refluxing for 4 times by using ethanol with the concentration of 95wt% which is 9 times of the weight of the rhizoma alismatis, extracting for 2 hours each time, concentrating under reduced pressure until no alcohol exists, and adding water into the concentrated extracting solution according to the volume ratio of 1:10 to perform dispersion treatment to obtain a water dispersion;
B. extracting the water dispersion with equal volume of ethyl acetate for 4 times, and recovering solvent to obtain ethyl acetate extract and water phase part;
C. dissolving the ethyl acetate extract with methanol completely, drying with silica gel as carrier, wet loading, separating with silica gel column chromatography (petroleum ether B: ethyl acetate, A: B =3: 1V/V as mobile phase), collecting target substance chromatographic solution, concentrating under reduced pressure, dissolving with ethyl acetate, filtering, adding petroleum ether, standing for crystallization to obtain crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, separating the filtrate with C18 reversed phase chromatography packing under high pressure (A: methanol B: water, A: B =85:15V/V as mobile phase; detection wavelength 210 nm), collecting corresponding chromatographic peak, concentrating the collected liquid to dry to obtain white powder product, namely the triterpenoid compound with the structural formula shown in (I).
EXAMPLE 5 isolation of the Compound
A. Taking 10Kg of rhizoma alismatis dried tubers, crushing, heating and refluxing for 3 times by using 8 times of 95% ethanol with weight concentration, extracting for 2 hours each time, concentrating under reduced pressure until no ethanol exists to obtain 3.5Kg of concentrated extract, and adding water into the obtained concentrated extract according to the volume ratio of 1:8 for dispersion treatment;
B. sequentially extracting the aqueous dispersion with equal volume of ethyl acetate for 4 times, and recovering the solvent to obtain 1000g of ethyl acetate extract and a water phase part;
C. the ethyl acetate extract was completely dissolved in 800ml of methanol, and then loaded on silica gel as a carrier, dried, and subjected to wet loading and silica gel column chromatography, a: petroleum ether B: ethyl acetate, wherein A: B =2: 1V/V is a mobile phase, a target substance chromatographic solution is collected, is dried by decompression and concentration, is dissolved by 60ml of ethyl acetate, is filtered, is added with 300ml of petroleum ether, and is placed for crystallization to obtain 3g of a crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, and separating the filtrate by using C18 reversed phase chromatography packing under high pressure, A: methanol B: water, A: B =85:15V/V is a mobile phase; detecting wavelength at 210nm, collecting corresponding chromatographic peak, and concentrating the collected liquid to dryness to obtain 2.3g of triterpenes white powder compound with structural formula shown in (I).
The whole production process takes about 5 days;
the product purity was checked again by reversed-phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase component, and found to be 99.15%.
EXAMPLE 6 isolation of the Compound
A. Taking 10Kg of dried rhizoma alismatis tubers, crushing, heating and refluxing for extraction for 5 times by using 10 times of 95% ethanol in weight concentration, extracting for 2 hours each time, concentrating under reduced pressure until no ethanol exists to obtain 4Kg of concentrated extracting solution, and adding water into the obtained concentrated extracting solution according to the volume ratio of 1:10 for dispersion treatment;
B. sequentially extracting the aqueous dispersion with equal volume of ethyl acetate for 3 times, and recovering the solvent to obtain 1200g of ethyl acetate extract and a water phase part;
C. the ethyl acetate extract was completely dissolved in 1000ml of methanol, and then loaded on silica gel as a carrier, dried, and subjected to wet loading and silica gel column chromatography, a: petroleum ether B: ethyl acetate, wherein A: B =5: 1V/V is a mobile phase, a target substance chromatographic solution is collected, is dried after being concentrated under reduced pressure, is dissolved by 80ml of ethyl acetate after being concentrated under reduced pressure, is filtered, is added with 400ml of petroleum ether, and is placed for crystallization to obtain 4g of a crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, and separating the filtrate by using C18 reversed phase chromatography packing under high pressure, A: methanol B: water, A: B =85:15V/V is a mobile phase; detecting the wavelength of 210nm, collecting corresponding chromatographic peaks, and concentrating the collected liquid to dryness to obtain 3g of triterpenes white powder compound with the structural formula shown in the formula (I).
The whole production process takes about 7 days;
the product purity was checked again by reversed-phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase component, and found to be 99.45%.
EXAMPLE 7 isolation of the Compound
A. Taking 10Kg of dried rhizoma alismatis tubers, crushing, heating and refluxing for extraction for 4 times by using 95% ethanol with 9 times of weight concentration, extracting for 2 hours each time, concentrating under reduced pressure until no ethanol exists to obtain 3.2Kg of concentrated extracting solution, and adding water into the obtained concentrated extracting solution according to the volume ratio of 1:5 for dispersion treatment;
B. sequentially extracting the aqueous dispersion with equal volume of ethyl acetate for 4 times, and recovering the solvent to obtain 1100g of ethyl acetate extract and a water phase part;
C. the ethyl acetate extract was completely dissolved in 900ml of methanol, and then, silica gel was used as a carrier, and after drying, wet loading and silica gel column chromatography, a: petroleum ether B: ethyl acetate, wherein A: B =3: 1V/V is a mobile phase, a target substance chromatographic solution is collected, is dried after being concentrated under reduced pressure, is dissolved by 70ml of ethyl acetate after being concentrated under reduced pressure, is filtered, is added with 350ml of petroleum ether, and is placed for crystallization to obtain 3.2g of a crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, and separating the filtrate by using C18 reversed phase chromatography packing under high pressure, A: methanol B: water, A: B =85:15V/V is a mobile phase; detecting the wavelength of 210nm, and collecting corresponding chromatographic peaks to obtain 2.4g of triterpenes white powder compound with the structural formula shown in (I).
The whole production process takes about 6 days.
The product purity was checked again by reversed-phase analytical liquid chromatography (RP-HPLC) by replacing the mobile phase component, and found to be 99.25%.
EXAMPLE 8 test of antibacterial Activity of Compounds
First, experimental material
1.1 medicine
The triterpenes white powder compound with the structural formula shown in the formula (I) is obtained by extraction and separation in the above examples 5-7.
1.2 human pathogenic bacteria
Staphylococcus aureus (1)Staphylococcus aureus) ATCC 51650 was provided by the drug test in the hainan province.
1.3 Medium
Beef extract peptone agar medium (NA): 5g of beef extract, 7g of NaCl and 15g of peptone, wherein the volume is up to 1000ml, and the pH value is 7.4-7.6.
Second, Experimental methods
Measuring the antibacterial activity of the compound by a filter paper sheet agar diffusion method;S.aureusNA medium was used.
2.1 mixingS.aureusPreparing bacterial suspension (105-107 cfu/ml) with a certain concentration, uniformly coating 100 mu l of the bacterial suspension on a test sterile plate by using a cotton swab, and preparing the bacterial-containing plate.
2.2 the compounds to be tested were formulated as lmg/50. mu.l solution, 20. mu.l each was dropped onto a sterile filter paper sheet (phi =6 mm), and the filter paper sheet was attached to a bacteria-containing plate after the solvent had evaporated, repeating 3 times per treatment.
2.3 Positive control solution is kanamycin sulfate 0.08mg/ml (10/. mu.l), cultured at 37 ℃, observed after 24h, and the diameter of the inhibition zone is measured and recorded.
Third, experimental results
Example 5-7 extraction and separation to obtain white triterpenes with structural formula shown in (I)Powdered compound, kanamycin sulfate, methanol (negative control), against Staphylococcus aureusS.aureusThe results of the antibacterial activity are shown in Table 2.
TABLE 2 antimicrobial Activity results
| Numbering | Name (R) | Concentration (mg/. mu.l) | Diameter of antibacterial ring of staphylococcus aureus (mm) |
| 1 | Example 5 | 1mg/50μl | 11 |
| 2 | Example 6 | 1mg/50μl | 13 |
| 3 | Example 7 | 1mg/50μl | 12 |
| 4 | Kanamycin sulfate | 1mg/50μl | 31～33 |
| 5 | Methanol | 1mg/50μl | 0 |
The experimental result shows that the triterpenoid compounds can treat staphylococcus aureusS.aureusHas certain inhibiting effect, and can be used for preparing antibacterial agent.
Claims (6)
1. A method for extracting and separating triterpenoids from rhizoma alismatis is characterized in that: the method comprises the following process steps:
A. crushing rhizoma alismatis dried tubers, heating and refluxing the crushed rhizoma alismatis dried tubers by using 95wt% ethanol, concentrating the crushed rhizoma alismatis dried tubers under reduced pressure until no ethanol exists, and adding water into the concentrated extract for dispersing to obtain a water dispersion;
B. extracting the water dispersion with ethyl acetate, and recovering the solvent to obtain an ethyl acetate extract and a water phase part;
C. dissolving the ethyl acetate extract with methanol completely, drying with silica gel as carrier, wet-loading, separating with silica gel column chromatography, collecting target substance chromatographic solution, concentrating under reduced pressure, drying, dissolving with ethyl acetate, filtering, adding petroleum ether, standing for crystallization to obtain crystalline solid;
D. dissolving the crystalline solid with methanol, filtering, separating the filtrate with C18 reversed phase chromatography filler under high pressure, collecting corresponding chromatographic peak, concentrating the collected liquid to dry to obtain white powder product, i.e. triterpenoid compound with structural formula shown in (I);
（Ⅰ）。
2. the method for extracting and separating triterpenoids from alisma orientale as claimed in claim 1, wherein the triterpenoids are selected from the group consisting of: in the step A, the dosage of the ethanol is 8-10 times of the weight of the crushed dried rhizoma alismatis tubers; the reflux extraction is carried out for 3-5 times, and each time lasts for 2 hours.
3. The method for extracting and separating triterpenoids from alisma orientale as claimed in claim 1, wherein the triterpenoids are selected from the group consisting of: in the step A, water is added into the concentrated extracting solution according to the volume ratio of 1: 5-1: 10 for dispersing treatment.
4. The method for extracting and separating triterpenoids from alisma orientale as claimed in claim 1, wherein the triterpenoids are selected from the group consisting of: in the step B, the water dispersion is extracted 3-5 times by using ethyl acetate with the same volume.
5. The method for extracting and separating triterpenoids from alisma orientale as claimed in claim 1, wherein the triterpenoids are selected from the group consisting of: in step C, the silica gel column chromatography separation: a: petroleum ether B: ethyl acetate, wherein the volume ratio of A to B =5: 1-2: 1 is a mobile phase.
6. The method for extracting and separating triterpenoids from alisma orientale as claimed in claim 1, wherein the triterpenoids are selected from the group consisting of: in step D, the C18 reverse phase chromatography packing high pressure preparative liquid phase separation: a: methanol B: water, A: B =85:15V/V is a mobile phase; the detection wavelength is 210 nm.
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| CN113105522B (en) * | 2021-04-06 | 2022-04-01 | 神威药业集团有限公司 | Oriental water plantain triterpenes compound, preparation method, structure characterization method and application thereof |
| CN114874098B (en) * | 2022-06-15 | 2023-10-24 | 扬州大学 | Compound extracted and separated from glaucocalyxa, and preparation method and application thereof |
| CN115350197B (en) * | 2022-07-05 | 2023-06-13 | 华南农业大学 | Application of alisol A-24-acetate in improving sensitivity of MRSA to beta-lactam antibiotics |
| CN115569140A (en) * | 2022-09-29 | 2023-01-06 | 华南农业大学 | Application of alisol A-24-acetate in preparation of MRSA (methicillin resistant Staphylococcus aureus) virulence inhibitor |
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