CN106146445B - The extracting method of monomer dehydrogenation matricarin ketone and its application in sagebruss artemisia anomalas - Google Patents

Abstract

The invention relates to a method for extracting dehydromatriolactone, a monomer compound in Artemisia genus Artemisia, and its application, and belongs to the technical field of natural plant extraction and application. The extract is a unique sesquiterpene compound in Artemisia plants of Compositae. The dehydromatriolide ketone of the Artemisia annua extract is obtained through the following steps: pulverize the traditional Chinese medicine Artemisia annua, heat and reflux with 70% ethanol for extraction, and concentrate the extract under reduced pressure to obtain a crude extract. The crude extract was subjected to silica gel column chromatography, the crude extract was subjected to extraction, silica gel column chromatography, gel column chromatography, and reverse-phase high performance liquid phase preparative chromatography to obtain dehydromatriolide ketone with a purity of 99%. The method for extracting dehydromatriolactone from the Artemisia annua extract of the present invention is simple, and the obtained extract has been proved by in vitro cell experiments that it has significant anti-clinical Streptococcus agalactiae, and can be used to prepare drugs for inhibiting Streptococcus agalactiae .

Description

Extraction method and application of monomer dehydromatriolactone in Artemisia genus Artemisia annua

technical field

The invention belongs to the technical field of natural plant extraction and application, and in particular relates to the extraction of the monomeric artemisia flavonoids in Artemisia genus Artemisia and its application in the preparation of medicines for inhibiting Streptococcus agalactiae and Staphylococcus aureus.

Background technique

Streptococcus agalactiae (Streptococcus. agalactiae, SAG), also known as Group B Streptococcus, is a common Gram-positive coccus, often deposited in the female reproductive tract, rectum and respiratory tract, and is an opportunistic pathogen. The carrier rate is as high as 15% to 35%. SAG is likely to cause genitourinary tract infection when immunity is low, antibacterial drugs are widely used or nosocomial infection (Zhang Zhuoran, Ni Yuxing. Clinical Microbiology and Microbiological Examination [M]. 3rd Edition. Beijing: People's Health Publishing House, 2003: 86 ; Zhao Mingze. Clinical characteristics and drug resistance analysis of Streptococcus agalactiae infection[J]. Zhejiang Clinical Medicine, 2010,12(9):1019-1020.), skin infection, endocarditis, postpartum infection, newborn Septicemia and neonatal meningitis, etc., are common pathogenic bacteria that cause neonatal bacteremia and meningitis (Wu Jianning, Lin Runhua, Lin Jian, etc. Analysis of drug resistance of 112 cases of Streptococcus agalactiae infection in pregnant women's genitourinary tract[ J]. Journal of Medical Research, 2008, 37(3): 88-89.).

In western developed countries, SAG is the most common pathogenic bacteria for neonatal infection. The colonization rate of SAG in pregnant women in the United States is as high as 40%-50%. The incidence rate was significantly reduced (Newborn C O I D C O F A, CJ B, CL B, et al.Policy statement—Recommendations for the prevention of perinatal group B streptococcal (GBS) disease.[J].Pediatrics,2011,128(3):611- 616. JR V. Prevention of Perinatal Group B Streptococcal Disease Revised Guidelines from CDC, 2010 [J]. MMWR Recommendations & Reports, 2010.). There are few research reports on SAG infection in my country. Some domestic hospital laboratories have tested positive for neonatal SAG infection in blood culture, and the number of neonatal pneumonia and upper respiratory tract infection caused by SAG infection is relatively large (Wu Jianning, Lin Runhua, Lin Jian et al. Drug resistance analysis of 112 cases of Streptococcus agalactiae infection in pregnant women's genitourinary tract [J]. Medical Research Journal, 2008,37(3):88-89). As clinicians and laboratory workers pay more attention to SAG infection, the reported cases of neonatal SAG infection in my country have gradually increased in recent years. According to statistics, the number of SAG infection cases increased significantly in 2012 (Dai Yiheng, Zeng Lijun, Gao Pingming. Clinical analysis of 16 cases of neonatal group B streptococcal sepsis [J]. Chinese Journal of Neonatology, 2012, 27 (1): 44-46. Huang Xiaoyi, Liu Zhiwei. Analysis of early-onset bloodstream infection in newborns in maternal and child health care hospital [J]. Chinese Journal of Hospital Infection, 2012,22(11):2329-2332.). Therefore, Chinese medical workers should check pregnant women for SAG colonization as soon as possible, and carry out preventive interventions for high-risk groups to reduce the incidence of early-onset SAG in newborns.

SAG is easy to infect perinatal newborns and mothers, and also affects non-pregnant adults. According to related reports, the diseases caused by SAG to non-pregnant adults with low immunity mainly include skin and soft tissue infection, bacteremia, pneumonia, osteomyelitis, and other rare diseases such as meningitis, streptococcal toxic shock syndrome, infectious Endocarditis, etc. (TH S, MM F, SP, et al.Increasing Burden of Invasive Group B Streptococcal Disease inNonpregnant Adults,1990-2007[J].Clinical Infectious Diseases,2009,49(1):85-92. ). Endocarditis caused by Streptococcus agalactiae infection has a high mortality rate, and it is difficult to control the development of the disease with simple antibiotic treatment (SD P, AC M, DA M, et al. Infective endocarditis caused by Streptococcus agalactiae.[J] . Int J Cardiol, 1989, (2): 179-183.). SAG is also the main pathogen of pyogenic arthritis in non-pregnant adults, and the mortality rate of septic arthritis caused by this bacteria is the highest (Nolla JM, G, Corbella X M P, et al.Group B streptococcus (Streptococcus agalactiae) pyogenic arthritis in nonpregnant adults .[J].Medicine, 2003,82(2):119-128.). SAG in Wuxi People's Hospital of my country mainly occurs in the elderly and patients with underlying diseases, and the infections caused by non-pregnant adults mainly include urinary tract infection, respiratory tract infection, reproductive tract infection and skin and soft tissue infection (Wang Yanyan, Ren Jing, Xu Yan et al. Clinical distribution characteristics and drug resistance analysis of Streptococcus agalactiae in non-pregnant adult patients [J]. Chinese Medical Science, 2013, (7): 87-88.). The cases of non-pregnant adults infected with SAG are increasing and the condition is serious, so it is urgent to control the occurrence of SAG infection.

With the extensive use of antibiotics, SAG has developed resistance to a variety of antibiotics. Relevant data report that SAG is resistant to quinolones and aminoglycosides and highly resistant to macrolides (Spanish][I.[Streptococcus agalactiae highly resistant to fluoroquinolones].[J].Enfermedades Infecciosas y Microbiología Clínica ,2006,24(9):562-563.; M G,T L,GG,et al.High-Level Aminoglycoside Resistance In The Beta-Hemolytic Group G Streptococcus Isolate Bm2721[J].Antimicrob Agents Chemother.,1999,43(12 )::3008–3010.), the resistance rate to tetracycline is >90%, and the resistance rate to erythromycin, clindamycin and levofloxacin is relatively high, and it is increasing year by year (Gao Jing, Liu Xiaoyan. Female genitourinary Drug resistance analysis of Streptococcus agalactiae[J]. Laboratory Medicine, 2015, (1). Drug resistance, penicillin is often used as the drug of choice for the treatment of SAG infection (Wu Tingting, Min Xiaochun, Wang Wei. Analysis of drug resistance of clinical patients infected with Streptococcus agalactiae isolates[J]. Chinese Journal of Hospital Infection, 2015, ( 1).2015-131516.). Severe allergic reactions to penicillin limit the use of drugs. Patients allergic to penicillin can be prevented and treated with erythromycin or clindamycin. The use of lindamycin is subject to certain restrictions. This poses a challenge for treating infections caused by SAG in penicillin-allergic patients. Finding highly effective and low-toxic antibacterial active ingredients from plants is an important direction for the development of antibiotic drugs. This study is based on the clinical application of Artemisia annua Chinese medicinal materials (Rong Yuanming, Ye Qili, He Shanming. Treating acute bacillary dysentery with Chinese medicine Liu Jinu 34 cases [J]. Shanghai Journal of Traditional Chinese Medicine, 1983, (1): 21. Deng Cunguo. Liu Jinu's treatment of early mastitis was effective [J]. Chinese Journal of Traditional Chinese Medicine, 2008, (9): 820-821.), on The antibacterial active ingredients of Artemisia annua (Tan Weifeng, Wang Jing, Xing Xin. Experimental study on the antibacterial activity of the extract of Artemisia annua in vitro[J]. Journal of Pharmaceutical Practice, 2010, 28(2):101-104.) were separated and purified, The dehydromatriolide ketone monomer compound with antibacterial activity is obtained. The compound has significant antibacterial effect on Streptococcus agalactiae, and provides an important material basis for the research, development and utilization of clinical antibiotic drugs. The invention can guide the rational development and application of the medicinal resources of Artemisia annua, a traditional Chinese medicinal material.

Artemisia annua, a traditional Chinese medicine, is rich in chemical constituents, which have been reported by natural product chemistry research (Wen Jing, Shi Haiming, Zan Ke et al. Research on the chemical constituents of Liu Jinu[J]. Chinese Herbal Medicine, 2010,41:870-873.) Artemisia annua The types of compounds contained in it are mainly flavonoids, coumarins and sesquiterpene lactones and other components. Among them, both flavonoids and coumarins have good antibacterial pharmacological activity (Chen Qiurong. Analysis of the pharmacological effects of flavonoids [J]. China Practical Medicine, 2012, 07(21): 254-255. Zheng Ling, Zhao Ting, Sun Lixin. Research progress on pharmacological activity and pharmacokinetics of coumarin compounds[J]. antibacterial activity. In the early days, Chinese drug researchers discovered artemisinin, a sesquiterpene lactone compound with significant anti-shigella activity from Artemisia annua, which made a breakthrough in the research of anti-dysentery drugs. Literature reports (Literature. Research on the Antibacterial Effect of Artemisinin [J]. Ginseng Research, 2009, 21(4): 38-39) Artemisinin also has obvious antibacterial activity against Escherichia coli and Staphylococcus aureus, the least effective Concentrations were 0.125mg/mL and 0.25mg/mL; the sesquiterpenoid mansonone in Hibiscus poplaris had certain antibacterial activity against Bacillus, and its minimum inhibitory concentration was 4.69ug/mL (Boonsri S, Karalai C, Pongglimanont C, et al. Cytotoxic and Antibacterial Sesquiterpenes from Thespesia populnea [J]. J. Nat. Prod., 2008, 71(7): 1173-1177.). At present, the antibacterial activity of sesquiterpene compounds in Artemisia annua has not been reported, so this report uses common clinical pathogenic bacteria to detect the antibacterial pharmacological activity of sesquiterpene lactone compound dehydromatriolactone in Artemisia annua.

Monomer dehydromatriolide ketone is an important sesquiterpene chemical component in Artemisia genus Artemisia, which has only been reported in Artemisia argyi and Artemisia argyi;

In the early stage, foreign scholar Seung-Ho Lee separated and purified the traditional Chinese medicine Artemisia yindi, and obtained the sesquiterpene compound dehydromatricarin (de-hydromatricarin), which mainly exists in Artemisia plants and is An important reference for the identification of Artemisia plants.

Dehydromatriolactone is a sesquiterpene compound, and there are few reports on its pharmacological activity. At present, only scholar Seung-Ho Lee has studied the anticancer activity of this compound. Scholar Seung-Ho Lee isolated dehydromatriolactone from Artemisia plants, and carried out recombinant mouse FPTase (farnesyl transfer) to it. enzyme) inhibition test. The test results show that dehydromatriolactone has a slight inhibitory effect on recombinant mouse FPTase (farnesyltransferase), with an IC ₅₀ of 300 μM, and has certain anticancer activity.

Foreign scholar Seung-Ho Lee took 1 kg of dried Artemisia annua, extracted twice with 2L of methanol, concentrated the extract to obtain methanol extract of Artemisia annua, and the extract was subjected to silica gel column chromatography and methanol-chloroform system gradient elution to obtain each group Fractions, carry out FPTase (farnesyltransferase) activity inhibition test to each fraction to obtain active fraction, this active component is 9:1-8:2 elution to obtain by methanol-chloroform system, this active fraction Adopt C18 reverse-phase silica gel column chromatography, methanol-water gradient elution obtains the fraction of 70% methanol elution and the fraction of 80% methanol elution, carry out Sephadex LH-20 gel column chromatography separation to this 2 groups of fractions, Methanol was eluted, and then the obtained fraction was separated by reverse-phase high-efficiency preparative chromatography to obtain a monomeric compound of dehydromatriolide ketone. (Lee S, Kang H, Song H, et al. Sesquiterpene Lactones, Inhibitors of Farnesyl Protein Transferase, Isolated from the Flower of Artemisia sylvatica[J]. Tetrahedron, 2000,56(27):4711–4715.)

In the above dehydromatriolide ketone extraction process, column chromatography is used to directly separate the crude extract. The polarity range of the separated compound is large, the separation is blind, the workload is large, and it takes a long time to obtain the target compound. Moreover, the activity tracking method is used to determine and isolate the target compound, which is complicated and cumbersome, which brings inconvenience to the extraction process.

Contents of the invention

The object of the present invention is to provide a method for extracting dehydromatriolide ketone monomer in Artemisia genus Artemisia and its application in the preparation of drugs for inhibiting Streptococcus agalactiae.

The extraction method of monomer dehydromatriolide ketone in Artemisia genus Artemisia annua is carried out according to the following steps:

A. Preparation of total crude extract

Grind the traditional Chinese medicine Artemisia annua (Herba Artemisiae Anomalae), take 30kg of Artemisia artemisiae powder, add 300L of extraction solvent 70% ethanol water (the volume ratio of ethanol and water is 7:3), heat at 70°C and reflux for 2 hours, extract twice , Concentrate the extract under reduced pressure to obtain 5.3kg of crude extract.

B. Separation and purification

Dissolve 5.3 kg of crude extract in 10 L of water and suspend it to obtain the extract suspension, which is extracted three times by adding petroleum ether (the volume ratio of the extract suspension to the petroleum ether added for each extraction is 1:2), discard Remove the sherwood oil extract three times to obtain the extract suspension after sherwood oil extraction; then add chloroform to the extract suspension after sherwood oil extraction and extract three times (the extract suspension after sherwood oil extraction The volume ratio of the extracted chloroform to each addition was 1:2), the three extracts were combined, and the solvent was recovered under reduced pressure to obtain 1.5 kg of chloroform part extract. Put the chloroform part extract on the silica gel column (1.5kg of the chloroform part extract, add 1.5kg of silica gel to dry mix the sample and put it on the column, pack the column with 7kg of silica gel, and use petroleum ether-dichloromethane-methanol-formic acid system 6:2:1 :0.1 mixed solution dissolved in silica gel packing), and petroleum ether-dichloromethane-methanol-formic acid system was used for gradient elution with about 60L of 6:2:1:0.1 and about 20L of 4:2:1:0.1, and the flow rate was 3 drops/second, sample volume 1L, each fraction is sampled by thin-layer chromatography, in the developer system of petroleum ether-dichloromethane-methanol-formic acid 4:2:1:0.1, Rf is 0.5 The fractions were combined, and the solvent was recovered under reduced pressure to obtain fraction Fr1.

Fraction Fr1 was prepared by reverse-phase high-performance liquid phase, and the preparation conditions were chromatographic column Kromasil C18 (250mm×21.2mmi.d.; 5 μm); the mobile phase ratio was acetonitrile:0.2% formic acid water=28:72, and the flow rate was 20ml/min. The column temperature is 25°C, and the detection wavelength is 250nm. According to the peak position displayed on the detector spectrum, the fraction "I" is received within 28.5min to 29.5min, and monomer dehydromatriolide ketone is obtained.

The purity of the obtained fractions was detected by high performance liquid chromatography, and the fraction "I" was measured as a single chromatographic peak with a purity higher than 99%. After the solvent was evaporated, the fraction "I" was analyzed by MS, ¹ HNMR and ¹³ CNMR , according to the obtained data for structure confirmation, fraction "I" is dehydromatriolide ketone.

The structural formula of dehydromatriolide ketone is the following formula I:

The compound is white powder, ESI-MS m/z: 345.1487[M+HCOO] ^— , combined with ¹ H-NMR and ¹³ C-NMR spectrogram data, the molecular formula is determined to be C ₁₇ H ₁₈ O ₅ . ¹ H-NMR (CDCl ₃ , 600MHz) δ: 6.19 (1H, t, J = 1.2HZ, H-3), 3.49 (1H, d, J = 10.2HZ, H-5), 3.70 (1H, t, J＝10.2HZ, H-6), 3.25(1H, dt, J＝3.6, 10.2HZ, H-7), 4.91(1H, dt, J＝1.8, 12.6HZ, H-8), 2.71, 2.46( IH,dd,J＝2.4,13.8HZ,H-9),6.23,5.64(2H,d,J＝3HZ,H-13),2.33(3H,s,H-14),2.44(3H,s, H-15), 2.15 (3H, s, H-17); ¹³ C-NMR (CDCl ₃ , 600MHz) δ: 133.5 (C-1), 194.8 (C-2), 135.9 (C-3), 169.1 (C-4), 51.5(C-5), 81.3(C-6), 54.9(C-7), 69.2(C-8), 44.3(C-9), 144.5(C-10), 135.9( C-11), 169.2(C-12), 121.7(C-13), 19.7(C-14), 21.2(C-15), 169.5(C-16), 20.9(C-17); NMR spectrum HMBC shows that C10 is related to H15, C16 is related to H17, C4 is related to H14, shown by COZY spectrum, H5 is related to H6, H6 is related to H7, H7 is related to H8, H7 is related to H5 and H9 is remotely related, and the structure of compound I was obtained through the above information. The structure data is basically consistent with the dehydromatricarin reported in the literature, so the structure of the compound was identified as (dehydromatricarin). (Lee S, Kang H, Song H, et al. Sesquiterpene Lactones, Inhibitors of Farnesyl Protein Transferase, Isolated from the Flower of Artemisia sylvatica[J]. Tetrahedron, 2000,56(27):4711–4715.)

The present invention has further found that the monomer dehydromatriolide ketone prepared by the above method has a better effect on Streptococcus agalactiae than the current preferred drug penicillin against Streptococcus agalactiae through antibacterial test results. The effect is better, and it also has certain antibacterial activity against other common clinical pathogenic bacteria.

Application of the monomer dehydromatriolactone obtained by the above preparation method in the preparation of drugs for inhibiting Streptococcus agalactiae.

Beneficial effect:

When a company prepares compounds on a large scale, it first crushes and extracts Chinese herbal medicines to prepare bold substances, then extracts the bold substances with different solvents to obtain polar parts, and determines the polar parts of the target compound by TLC thin layer chromatography, and then The polar part was further separated and purified. The target compound is determined by TLC thin-layer chromatography for each fraction obtained by column chromatography, and the method for determining the target compound is simpler, faster and easier to operate than the activity tracking method.

The monomer compound dehydromatriolide ketone prepared above has antibacterial activity, and has obvious activity to Gram-positive cocci, and weaker activity to Gram-negative bacilli. Antibacterial experiments in vitro show that dehydromatriolide ketone has antibacterial activity against gram-positive cocci. The bacteriostatic concentration of Streptococcus agalactiae (CICC10465) is 0.1mg/mL, and the bactericidal concentration is 0.3mg/mL; the bacteriostatic concentration of Staphylococcus aureus (CMCC26003) is 0.3mg/mL, and the bactericidal concentration is 1mg/mL, The antibacterial concentration for Staphylococcus aureus (ATCC6538) is 0.1mg/mL, and the bactericidal concentration is 1mg/mL; the antibacterial concentration for Gram-negative bacillus Escherichia coli (CMCC44102) is 1mg/mL, and the bactericidal concentration is 3mg /mL; the bacteriostatic concentration to Shigella dysenteriae (CMCC(B)51252) is 0.1mg/mL, and the bactericidal concentration is 3mg/mL, and the bacteriostatic effect diagram is shown in Figure 9-13.

Description of drawings

Figure 1 The MS spectrum of dehydromatricolide ketone negative ion mode scanning, the formate anion detection peak of dehydromatricolactone ketone is [M+HCOO] ^— =345.1487m/z

Fig. 2 The high performance liquid chromatogram of dehydromatriolide ketone in the normal phase column separation fraction Fr3 of Example 1.

Fig. 3 The high performance liquid chromatogram of dehydromatriolide ketone in fraction A obtained after fraction Fr3 in Example 1 was separated by gel column.

Fig. 4 is a schematic diagram of the position of the detector in the reversed-phase high-performance liquid phase preparative chromatography for dehydromatriolide ketone in fraction A in Example 1.

Fig. 5 is a high performance liquid chromatogram of purity detection of fraction "I" (dehydromatriolactone) in Example 1.

The high performance liquid chromatogram of dehydromatriolide ketone in normal phase column fraction Fr3 in Fig. 6 embodiment 2.

Fig. 7 is a schematic diagram of the position of the detector in the reversed-phase high-performance liquid phase preparative chromatography of the dehydromatriolide ketone in the fraction Fr3 in Example 2.

Fig. 8 is a chromatogram of high performance liquid chromatography for purity detection of fraction "I" (dehydromatriolactone) in Example 2.

Figure 9 The antibacterial effect of dehydromatriolide ketone on Streptococcus agalactiae

The antibacterial effect diagram of Fig. 10 dehydromatriolide ketone to Staphylococcus aureus (CMCC26003)

Figure 11 The antibacterial effect of dehydromatriolide ketone on Staphylococcus aureus (ATCC6538)

Figure 12 The antibacterial effect of dehydromatriolide ketone on Shigella dysenteriae

Figure 13 The antibacterial effect of dehydromatriolide ketone on Escherichia coli

Figure 14 The antibacterial effect of penicillin G on Streptococcus agalactiae

Detailed ways

The present invention is described in further detail below in conjunction with embodiment, but does not limit the present invention in any way.

Example 1

The method for extracting monomeric artemisia flavonoids in Artemisia plants is carried out according to the following steps:

A. Preparation of total crude extract

Grind 30 kg of Nanliu Jinu Herba Artemisiae Anomalae dried medicinal materials, heat to 70°C with 300 L of 70% ethanol water (the volume ratio of ethanol and water is 7:3) and extract twice under reflux, combine the extracts, recover the solvent under reduced pressure, and obtain crude Extract 5.3kg.

B. Separation and purification

Dissolve 5.3 kg of crude extract in 10 L of water and suspend it to obtain the extract suspension, which is extracted three times by adding petroleum ether (the volume ratio of the extract suspension to the petroleum ether extracted each time is 1:2), and the combined Three extractions, decompression recovery solvent to obtain petroleum ether part extract, discarding three times petroleum ether part extract, to obtain the extract suspension after petroleum ether extraction; then to the extract suspension after petroleum ether extraction Chloroform was added to extract three times (the volume of the extract suspension after petroleum ether extraction and the chloroform added each time were 1:2), the three extracts were combined, and the solvent was recovered under reduced pressure to obtain the chloroform part extract; Silica gel column chromatography on the paste (1.5kg of extract from the chloroform part, add 1.5kg of silica gel to dry mix the sample and put it on the column, fill the column with 7kg of silica gel, and use the solution of petroleum ether-dichloromethane-methanol-formic acid system 8:2:1:0.1 Dissolve silica gel for packing.), use petroleum ether-dichloromethane-methanol-formic acid system at 8:2:1:0.1, 6:2:1:0.1, 4:2:1:0.1, 2:2:1: 0.1, 1:1:1:0.1 for gradient elution, each gradient is about 60L, the flow rate is 3 drops/second, and the sampling volume of each fraction is 1L, and each fraction is sampled by thin layer chromatography, and combined For fractions with the same polarity, 7 partial eluents Fr1-Fr7 were obtained according to the polarity difference from small to large, and the Fr3 part was 6:2:1:0.1 and 4:2:1:0.1. The Rf value of some parts is 0.5 in the developing agent system of petroleum ether-dichloromethane-methanol-formic acid with a polarity of 4:2:1:0.1. Fr3 was detected by HPLC, and the peak time t=26.232 was the peak of the target compound. See 2 for the HPLC spectrum of Fr3.

Fraction Fr3 is separated by Sephadex G-20 gel column chromatography, eluted isocratically with 2 L of chloroform-methanol 1:1 solution, the flow rate is 6 seconds/drop, and the sampling volume is about 50 mL, and the fractions of the same polarity are combined to obtain Components Fr3a-Fr3q, each fraction was detected by HPLC, the peak time of 26.232min was the peak of the target compound dehydromatriolactone, and the sample fractions Fr3h-Fr3n containing the target peak were combined to obtain Fraction A. The HPLC detection spectrum of fraction A is shown in Figure 3, and the retention time is 25.228min, which is the peak of the target compound.

In the above preparation process, the HPLC chromatographic conditions are chromatographic column Kromasil C18 (250mm×4.6mm i.d.; 5 μm); the mobile phase ratio is acetonitrile (A): 0.2% formic acid water (B); gradient elution, 0 ~ 20min, 35% ～50%A, 20min～30min, 50%～60%A, 30min～45min, 60%～95%A, flow rate 1ml/min, column temperature 30℃, detection wavelength 250nm.

Fraction A was prepared by reverse-phase high-performance liquid phase, and the preparation conditions were chromatographic column Kromasil C18 (250mm×21.2mmi.d.; 5 μm); the mobile phase ratio was acetonitrile: 0.2% formic acid water=28:72, and the flow rate was 20ml/min. The column temperature is 25°C, and the detection wavelength is 250nm. According to the detector spectrogram, the fraction "I" is received within 28.5min to 29.5min, as shown in Figure 4 below (the peak at t=29min is within dehydromatricaria absorption peak of ester ketone).

The purity of the obtained fractions was detected by high performance liquid chromatography, and the fraction "I" was measured as a single chromatographic peak with a purity higher than 99%. After the solvent was evaporated, the fraction "I" was analyzed by MS, ¹ HNMR and ¹³ CNMR , according to the obtained data for structure confirmation, fraction "I" is dehydromatriolide ketone. The MS spectrum of dehydromatricolide is shown in Figure 1, and the HPLC detection spectrum of fraction "I" is shown in Figure 5 (the peak time t=8.696min is the absorption peak of dehydromatricolide).

The structural formula I of dehydromatriolactone is as follows:

The compound is white powder, ESI-MS m/z: 345.1487[M+HCOO] ^— , combined with ¹ H-NMR and ¹³ C-NMR spectrogram data, the molecular formula is determined to be C ₁₇ H ₁₈ O ₅ . ¹ H-NMR (CDCl ₃ , 600MHz) δ: 6.19 (1H, t, J = 1.2HZ, H-3), 3.49 (1H, d, J = 10.2HZ, H-5), 3.70 (1H, t, J＝10.2HZ, H-6), 3.25(1H, dt, J＝3.6, 10.2HZ, H-7), 4.91(1H, dt, J＝1.8, 12.6HZ, H-8), 2.71, 2.46( IH,dd,J＝2.4,13.8HZ,H-9),6.23,5.64(2H,d,J＝3HZ,H-13),2.33(3H,s,H-14),2.44(3H,s, H-15), 2.15 (3H, s, H-17); ¹³ C-NMR (CDCl ₃ , 600MHz) δ: 133.5 (C-1), 194.8 (C-2), 135.9 (C-3), 169.1 (C-4), 51.5(C-5), 81.3(C-6), 54.9(C-7), 69.2(C-8), 44.3(C-9), 144.5(C-10), 135.9( C-11), 169.2(C-12), 121.7(C-13), 19.7(C-14), 21.2(C-15), 169.5(C-16), 20.9(C-17); NMR spectrum HMBC shows that C10 is related to H15, C16 is related to H17, C4 is related to H14, shown by COZY spectrum, H5 is related to H6, H6 is related to H7, H7 is related to H8, H7 is related to H5 and H9 is remotely related, and the structure of compound I is obtained through the above information. The structure data is basically consistent with the dehydromatricarin reported in the literature ^[13] . Therefore, the structure of the compound is identified as (dehydromatricarin) . (Lee S, Kang H, Song H, et al. Sesquiterpene Lactones, Inhibitors of Farnesyl Protein Transferase, Isolated from the Flower of Artemisiasylvatica [J]. Tetrahedron, 2000, 56(27): 4711–4715.)

Example 2

A. Preparation of total crude extract

Take 30kg of dry medicinal materials from Herba Artemisiae Anomalae, grind them, heat them to 70°C with 300L of 70% ethanol water (the volume ratio of ethanol and water is 7:3) and extract twice, combine the extracts, and recover under reduced pressure solvent to obtain 5.3kg of crude extract.

B. Separation and purification

Dissolve 5.3 kg of crude extract in 10 L of water and suspend it to obtain the extract suspension, which is extracted three times by adding petroleum ether (the volume ratio of the extract suspension to the petroleum ether extracted each time is 1:2), and the combined Three extractions, decompression recovery solvent to obtain petroleum ether part extract, discarding three times petroleum ether part extract, to obtain the extract suspension after petroleum ether extraction; then to the extract suspension after petroleum ether extraction Chloroform was added to extract three times (the volume of the extract suspension after petroleum ether extraction and the chloroform added each time were 1:2), the three extracts were combined, and the solvent was recovered under reduced pressure to obtain the chloroform part extract; Silica gel column chromatography on the paste (1.5kg of extract from the chloroform part, add 1.5kg of silica gel to dry mix the sample and put it on the column, fill the column with 7kg of silica gel, and use the solution of petroleum ether-dichloromethane-methanol-formic acid system 8:2:1:0.1 Dissolve silica gel for packing.), use petroleum ether-dichloromethane-methanol-formic acid system at 8:2:1:0.1, 6:2:1:0.1, 4:2:1:0.1, 2:2:1: 0.1, 1:1:1:0.1 for gradient elution, each gradient is about 60L, the flow rate is 3 drops/second, and the sampling volume of each fraction is 1L, and each fraction is sampled by thin layer chromatography, and combined For fractions with the same polarity, 7 partial eluents Fr1-Fr7 were obtained according to the polarity difference from small to large, and the Fr3 part was 6:2:1:0.1 and 4:2:1:0.1. The Rf value of some parts is 0.5 in the developing agent system of petroleum ether-dichloromethane-methanol-formic acid with a polarity of 4:2:1:0.1. Fr3 is detected by HPLC, the chromatographic conditions are chromatographic column Kromasil C18 (250mm×4.6mmi.d.; 5μm), the mobile phase ratio is acetonitrile:0.2% formic acid water=43:57, the flow rate is 1ml/min, and the column temperature is 30°C. The wavelength is 250nm, and the peak time t=24.333 is the peak of the target compound. See 6 for the HPLC spectrum of Fr3.

Fraction Fr3 was prepared by reverse-phase high-performance liquid phase, and the preparation conditions were chromatographic column Kromasil C18 (250mm×21.2mmi.d.; 5 μm); the mobile phase ratio was acetonitrile: 0.2% formic acid water=28:72, and the flow rate was 20ml/min. The column temperature is 25°C, and the detection wavelength is 250nm. According to the detector spectrogram, the fraction "I" is received within 28.5min to 29.5min, as shown in Figure 7 below (the dehydromatricolactone at the peak time t=29min ketone absorption peak).

The purity of the obtained fractions was detected by high performance liquid chromatography, and the fraction "I" was measured as a single chromatographic peak with a purity higher than 99%. After the solvent was evaporated, the fraction "I" was analyzed by MS, ¹ HNMR and ¹³ CNMR , according to the obtained data for structure confirmation, fraction "I" is dehydromatriolide ketone. Fraction "I" is the HPLC spectrum for the purity detection of dehydromatricolide ketone, see Figure 8, and the MS spectrum of dehydromatricolide ketone is shown in Figure 1.

The structural formula I of dehydromatriolactone is as follows:

The compound is white powder, ESI-MS m/z: 345.1487[M+HCOO] ^— , combined with ¹ H-NMR and ¹³ C-NMR spectrogram data, the molecular formula is determined to be C ₁₇ H ₁₈ O ₅ . ¹ H-NMR (CDCl ₃ , 600MHz) δ: 6.19 (1H, t, J = 1.2HZ, H-3), 3.49 (1H, d, J = 10.2HZ, H-5), 3.70 (1H, t, J＝10.2HZ, H-6), 3.25(1H, dt, J＝3.6, 10.2HZ, H-7), 4.91(1H, dt, J＝1.8, 12.6HZ, H-8), 2.71, 2.46( IH,dd,J＝2.4,13.8HZ,H-9),6.23,5.64(2H,d,J＝3HZ,H-13),2.33(3H,s,H-14),2.44(3H,s, H-15), 2.15 (3H, s, H-17); ¹³ C-NMR (CDCl ₃ , 600MHz) δ: 133.5 (C-1), 194.8 (C-2), 135.9 (C-3), 169.1 (C-4), 51.5(C-5), 81.3(C-6), 54.9(C-7), 69.2(C-8), 44.3(C-9), 144.5(C-10), 135.9( C-11), 169.2(C-12), 121.7(C-13), 19.7(C-14), 21.2(C-15), 169.5(C-16), 20.9(C-17); NMR spectrum HMBC shows that C10 is related to H15, C16 is related to H17, C4 is related to H14, shown by COZY spectrum, H5 is related to H6, H6 is related to H7, H7 is related to H8, H7 is related to H5 and H9 is remotely related, and the structure of compound I is obtained through the above information. The structure data is basically consistent with the dehydromatricarin reported in the literature ^[13] . Therefore, the structure of the compound is identified as (dehydromatricarin) . (Lee S, Kang H, Song H, et al. Sesquiterpene Lactones, Inhibitors of Farnesyl Protein Transferase, Isolated from the Flower of Artemisiasylvatica [J]. Tetrahedron, 2000, 56(27): 4711–4715.)

Example 3

In Vitro Experiment of Dehydromatriolactone

1. Main test materials

Strains: Staphylococcus aureus (CMCC26003), Escherichia coli (CMCC44102) were purchased from Beijing Bei Nachuanglian Biotechnology Research Institute; Staphylococcus aureus (ATCC6538), Shigella dysenteriae [CMCC(B)51252] and milk Streptococcus (CICC10465) was purchased from Nanjing Biological Technology Co., Ltd.

Main reagents: Anhydrous penicillin G sodium salt was purchased from Shanghai Sangon Bioengineering Co., Ltd.; modified broth medium and blood agar medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.; 96-well plates and culture dishes were purchased from Guangzhou Jiete Biofiltration Products Ltd.

Main instruments: LDZX-50KB vertical pressure steam sterilizer, Shanghai Shen'an Medical Equipment Factory; SW-CJ-ID single-person purification workbench, Suzhou Purification; ELX800 microplate reader, Gene Co., Ltd.; HZQ-X100 constant temperature oscillation Incubator, Huamei Biochemical Instruments; DHP-022 Electric Heating Constant Temperature Incubator, Shanghai Shenxian Constant Temperature Equipment Factory.

2. Antibacterial experiment of dehydromatriolactone

Drug preparation: the sample to be screened was dissolved in ethanol solvent with a volume fraction of 50% at 10 mg/ml, and the positive drug (penicillin G sodium salt) was dissolved in distilled water at 100 mg/ml.

Activation and cultivation of strains:

a. Weigh 18g of modified broth culture based on 300ml of distilled water, and sterilize at 121°C for 20min. Under sterile conditions, dispense into 15ml centrifuge tubes, 10ml per tube.

b. Take 1ml of the culture medium into the lyophilized powder of the strain, pipette and mix well, transfer to a 15ml centrifuge tube, and incubate at 37°C, 150rpm for 24h.

c. Take 100 ul of the bacterial suspension and spread it on the surface of the blood agar medium, and incubate it in a 37°C electric constant temperature incubator for 24 hours. Single clones were picked and cultured in broth medium for 24 hours.

3. Determination of MBC and MIC:

a. Take the 96-well plate of bacterial species cultured for 24 hours, 100ul per well; set up no drug-dosing wells, drug-dosing wells and positive control drug wells. The dosing concentration starts from 3mg/ml, and the culture medium is used for 3-fold gradient dilution, and there are 6 gradients in total. Positive control drug wells start from 10mg/ml, 3-fold serial dilution, a total of 6 gradients.

b. Measure the OD value at 490nm on the microtiter plate: measure 6 time points of 0h, 2h, 4h, 8h and 24h after adding the drug, and calculate the MIC.

c. Take the bacterium solution with antibacterial effect in the microplate plate, spread it evenly on the plate medium, continue to cultivate in the biochemical incubator for 24 hours, and the minimum concentration without colony formation is MBC.

3. Test results and analysis

In this study, penicillin was used as the positive control drug, and the antibacterial activity of dehydromatriolactone against Escherichia coli, Staphylococcus aureus, Shigella dysenteriae and Streptococcus agalactiae were investigated respectively. The results show that the inhibitory concentration of dehydromatriolide ketone to Streptococcus agalactiae (CICC10465) is 0.1mg/mL, and the bactericidal concentration is 0.3mg/mL; the inhibitory concentration to Staphylococcus aureus (CMCC26003) is 0.3mg /mL, the bactericidal concentration is 1mg/mL, the antibacterial concentration to Staphylococcus aureus (ATCC6538) is 0.1mg/mL, and the bactericidal concentration is 1mg/mL; The concentration is 1mg/mL, and the bactericidal concentration is 3mg/mL; the bacteriostatic concentration for Shigella dysenteriae (CMCC(B)51252) is 0.1mg/mL, and the bactericidal concentration is 3mg/mL. The inhibitory concentration of penicillin to Streptococcus agalactiae (CICC10465) is 3 mg/mL, and the bactericidal concentration is 3 mg/mL. See Figure 9-13 for the effect of dehydromatriolactone on various clinical pathogenic bacteria, and Figure 14 for the antibacterial effect of penicillin on Streptococcus agalactiae.

The above activity data shows that the order of antibacterial activity of dehydromatriolide ketone to detection of various clinical pathogenic bacteria is: Streptococcus agalactiae (CICC10465)=Shigella dysenteriae (CMCC (B)51252)=golden yellow Staphylococcus (ATCC6538) > Staphylococcus aureus (CMCC26003) > Escherichia coli (CMCC44102); the order of the bactericidal activity of dehydromatriolactone to various clinical pathogenic bacteria is: Streptococcus agalactiae (CICC10465) > Staphylococcus aureus (ATCC6538) = Staphylococcus aureus (CMCC26003) > Shigella dysenteriae (CMCC (B) 51252) = E. The order of the antibacterial activity of each detected pathogenic bacteria was: Streptococcus agalactiae (CICC10465) > Staphylococcus aureus (ATCC6538) > Shigella dysenteriae (CMCC (B) 51252) > Staphylococcus aureus (CMCC26003) > large intestine Bacillus (CMCC44102), indicating that dehydromatriolactone has the most significant antibacterial effect on Streptococcus agalactiae, and the above antibacterial effect diagrams are shown in Figure 9-13.

According to the in vitro antibacterial experiment that monomer dehydromatriolide ketone is carried out according to example 3 scheme, activity data shows that dehydromatriolide ketone is 0.1mg/mL to the bacteriostatic concentration of Streptococcus agalactiae (CICC10465), its positive The antibacterial concentration of contrast drug penicillin to Streptococcus agalactiae (CICC10465) is 3mg/mL, and the antibacterial activity of dehydromatricolide ketone to Streptococcus agalactiae (CICC10465) is 30 times of penicillin; The bactericidal concentration of ester ketone to Streptococcus agalactiae (CICC10465) is 0.3mg/mL, and the bactericidal concentration of penicillin to Streptococcus agalactiae (CICC10465) is 3mg/mL, that is, dehydromatricolide ketone is to Streptococcus agalactiae ( CICC10465) has 10 times the bactericidal activity of penicillin. The above comparative data show that dehydromatriolide ketone has significant anti-Streptococcus agalactiae activity, can effectively inhibit and kill Streptococcus agalactiae, and its antibacterial activity is significantly higher than that of the positive drug penicillin. The antibacterial effect of penicillin is shown in Figure 14.

my country's attention to SAG infection and research on prevention and treatment are late, and a large number of non-standard use of antibiotics for a long time has led to SAG's resistance to a variety of antibiotics. At present, SAG is less resistant to penicillin, and it is of great significance to control SAG resistance to seek antibiotic alternatives from natural products to prevent them from developing resistance to current antibiotics. The present invention finds that dehydromatriolide ketone has significant antibacterial activity against SAG, and is more than 10 times stronger than the antibacterial activity of penicillin, the current preferred anti-SAG infection drug. Infection drugs provide effective material resources. Dehydromatriolide ketone also shows obvious antibacterial activity to Staphylococcus aureus, and has certain antibacterial effect on Gram-negative bacilli Shigella dysenteriae and Escherichia coli, therefore, dehydromatriolide ketone can Research and development as a broad-spectrum antibacterial drug. The present invention investigates the in vitro antibacterial activity of dehydromatriolide ketone, and provides scientific basis for further studying the in vivo pharmacokinetics and bioavailability of the compound.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments, and that described in the above-mentioned embodiments and the description only illustrates the principles of the present invention, and the present invention also has various aspects without departing from the spirit and scope of the present invention. Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

1. The method for extracting monomer dehydromatriolide ketone in Artemisia genus Artemisia annua is carried out according to the following steps: A. Preparation of total crude extract Grind the traditional Chinese medicine Artemisia annua, take 10-50kg of Artemisia annua powder, add 100--500L of 70% ethanol aqueous solution, heat at 70°C and reflux for 2 hours to extract twice, and concentrate the extract under reduced pressure to obtain the total crude extract; B. Separation and purification Dissolve the total crude extract obtained in step A with water and suspend it to obtain an extract suspension, add petroleum ether for extraction three times, discard the petroleum ether extract for three times, and obtain the extract suspension extracted by petroleum ether Then add chloroform to the extract suspension after petroleum ether extraction and extract three times, combine the three extracts, and reclaim the solvent under reduced pressure to obtain the chloroform part extract; put the chloroform part extract on the silica gel chromatography column, and use petroleum ether- Dichloromethane-methanol-formic acid system 6:2:1:0.1 60L and 4:2:1:0.120L were used for gradient elution, the flow rate was 3 drops/second, and the sampling volume was 1L. Fractions were sampled, and in a developer system with a ratio of petroleum ether-dichloromethane-methanol-formic acid of 4:2:1:0.1, the fractions with an Rf value of 0.5 were combined, and the solvent was recovered under reduced pressure to obtain fraction Fr1; Fraction Fr1 was prepared by reverse-phase high-performance liquid phase. The preparation conditions were chromatographic column Kromasil C18 250mm×21.2mmi.d.; 25°C, detection wavelength 250nm, according to the peak position displayed on the detector spectrum, fraction "I" was received within the time period of 28.5min to 29.5min, and the monomer dehydromatriolide ketone was obtained. 2. the method for extracting the monomeric compound dehydromatriolide ketone in Artemisia genus Artemisia Artemisiae according to claim 1, is characterized in that: After fraction Fr1 is obtained in step B, fraction Fr1 is separated by Sephadex G-20 gel column chromatography, eluted isocratically with 2 L of chloroform-methanol 1:1 solution, the flow rate is 6 seconds/drop, and the sampling volume is 50 mL, to obtain Components Fr1a-Fr1q, each fraction was detected by HPLC, and the sample fractions Fr1h-Fr1n containing the target peak were combined to obtain Fraction A; Fraction A was prepared by reversed-phase high-performance liquid phase. The preparation conditions were chromatographic column Kromasil C18 250mm×21.2mm i.d.; 5μm; the mobile phase ratio was acetonitrile: 0.2% formic acid water = 28:72, the flow rate was 20ml/min, and the column temperature was 25°C , the detection wavelength is 250nm, according to the peak position displayed on the detector spectrum, the time period is 28.5min to 29.5min to receive the fraction "I", and obtain the monomer dehydromatriolide ketone. 3. the method for extracting the monomeric compound dehydromatriolactone in Artemisia genus Artemisia annua according to claim 1, characterized in that: In step A, crush the traditional Chinese medicine Artemisia annua, take 30 kg of Artemisia annua powder, add 300 L of 70% ethanol aqueous solution, heat at 70° C. for reflux extraction for 2 hours, extract twice, and concentrate the extract under reduced pressure to obtain the total crude extract. 4. the method for extracting monomer dehydromatriolide ketone in Artemisia genus Artemisiae according to claim 3, is characterized in that: in step B, add 10L water to dissolve the total crude extract that step A makes . 5. the method for extracting monomer dehydromatriolide ketone in Artemisia genus Artemisiae according to claim 1, characterized in that: the extract suspension in the petroleum ether extraction described in step B and each addition The volume ratio of the extracted petroleum ether is 1:2; in the chloroform extraction, the volume ratio of the extract suspension after petroleum ether extraction and the extracted chloroform added each time is 1:2. 6. the method for extracting monomer dehydromatriolide ketone in Artemisia genus Artemisiae according to claim 1, is characterized in that: get 1.5kg of chloroform part extractum in step B, add 1.5kg silica gel dry method to mix Put the sample on the column, pack 7kg of silica gel into the column, and dissolve the silica gel into the column with a solution of petroleum ether-dichloromethane-methanol-formic acid system 6:2:1:0.1.