WO2023231582A1 - Extrait de saponine cucurbitane de picrorhizae rhizoma et son utilisation dans la préparation d'un médicament pour traiter la constipation - Google Patents

Extrait de saponine cucurbitane de picrorhizae rhizoma et son utilisation dans la préparation d'un médicament pour traiter la constipation Download PDF

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WO2023231582A1
WO2023231582A1 PCT/CN2023/087473 CN2023087473W WO2023231582A1 WO 2023231582 A1 WO2023231582 A1 WO 2023231582A1 CN 2023087473 W CN2023087473 W CN 2023087473W WO 2023231582 A1 WO2023231582 A1 WO 2023231582A1
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cucurbitane
compound
methyl
norlanoster
extract
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PCT/CN2023/087473
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Chinese (zh)
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吉海杰
郝淑兰
王晞星
仝立国
王若瑜
王晓玲
李仲云
王淑敏
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山西省中医药研究院（山西省中医院）
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J17/005—Glycosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of research and development of new traditional Chinese medicines. Specifically, it relates to a cucurbitane-type saponin extract of Huahuanglian and its application in preparing medicines for treating constipation.
  • Coptidis Coptidis as a "hu medicine”, has been introduced since the Tang Dynasty and has been gradually absorbed by the theoretical system of traditional Chinese medicine. It is an important medicine for clearing deficiency and heat in clinical TCM. It is cold in nature and bitter in taste. It belongs to the liver, stomach and large intestine meridians. It has the functions of clearing away heat, cooling blood and dryness. It has the effect of removing dampness and eliminating rickets, and is used for infantile malnutrition, bone steaming hot flashes, damp-heat diarrhea, jaundice, red urine, hemorrhoids, swelling and pain, etc.
  • Coptidis Coptidis contains compounds such as iridoids, cucurbitane-type triterpenes, phenethyl glycosides and phenolic glycosides, as well as phenolic acids such as vanillic acid, cinnamic acid and ferulic acid, 2020 edition of "Chinese Pharmacopoeia” It is stipulated that the total content of Coptidis Coptidis I and Coptidis II shall not be less than 9.0%. Modern pharmacology shows that Coptidis Coptidis has hepatoprotective, choleretic, blood lipid-regulating, anti-asthmatic, anti-inflammatory and hypoglycemic effects. Generally, studies believe that it is related to the iridoids, phenylethyl glycosides and phenolic glycosides it contains.
  • Coptidis Coptidis is a commonly used medicine in pediatrics. During long-term clinical practice, some scholars have discovered that Coptidis Coptidis decoction-free has a purgative effect that has not been recorded in traditional herbal books ("The Purgative Effect of Coptidis Coptidis Decoction-Free", Shanxi Traditional Chinese Medicine, 2016 Volume 32, Issue 2, 2017), animal studies further confirmed that the decoction of Coptidis chinensis also has a purgative effect ("Study on the new purgative effect of Coptidis chinensis and its material basis based on spectrum-effect relationship analysis", Shanxi College of Traditional Chinese Medicine, 2017 Master Dissertation), through spectral effect research and analysis, it was determined that vanillic acid is one of the laxative active substances of Coptidis Coptidis ("New Uses of Vanillic Acid from Coptidis Coptidis Extract", CN106983737B).
  • Vanillic acid is a phenolic acid compound produced by secondary metabolism of plants. It is used in the food industry as a spice, food additive, preservative, etc. It is widely found in rice, strawberry, sugar cane, mango, wheat and other plants. Pharmacological activity studies have confirmed that vanillic acid has antioxidant, anti-inflammatory, anti-apoptotic and anti-platelet aggregation effects. This suggests that in addition to vanillic acid, the purgative active ingredients in Coptidis Coptidis require further confirmation.
  • Chronic constipation is a common clinical digestive system disease or symptom, characterized by reduced defecation frequency, dry and hard stool, and/or difficulty in defecation, which seriously affects the patient's quality of life.
  • Diseases related to chronic constipation in the Rome IV diagnostic system promulgated in 2016 include functional constipation, opioid-induced constipation, constipation-predominant irritable bowel syndrome and functional defecation disorders.
  • laxatives are used to improve symptoms of different types of chronic constipation.
  • volumetric laxatives such as methylcellulose, which can play a cathartic effect by increasing the water content and volume of feces
  • osmotic laxatives such as lactulose form hypertonicity in the intestines. state, absorb water, and stimulate intestinal peristalsis
  • stimulating laxatives such as plant-based laxatives containing anthraquinone act on the intestinal nervous system and enhance intestinal motility
  • (4) lubricating laxatives such as glycerin can lubricate the intestinal wall and soften it. Shit.
  • laxatives such as methylcellulose, which can play a cathartic effect by increasing the water content and volume of feces
  • osmotic laxatives such as lactulose form hypertonicity in the intestines. state, absorb water, and stimulate intestinal peristalsis
  • stimulating laxatives such as plant-based laxatives containing anthraquinone act on the intestinal nervous system and enhance intestinal mot
  • Short-term use can significantly improve defecation, but long-term use has unsatisfactory effects or may cause cancer risks.
  • long-term use of anthraquinone-containing laxatives can aggravate constipation and induce black bowel disease, while long-term use of phenolphthalein may cause cancer risks.
  • 2021 The State Food and Drug Administration has stopped the production, sale and use of phenolphthalein tablets and phenolphthalein lozenges in my country.
  • linaclotide (trade name: Lingzeshu)
  • CFTR cystic fibrosis transmembrane conductance regulator
  • ASBT bile acid transporter
  • the present invention divides the Coptidis Coptidis extract into different elution components based on polarity differences after being adsorbed by macroporous resin. Based on activity guidance, animal models are used to track the purgative active components, and then combined with chemical Techniques such as color identification reaction, spectroscopy, chromatography, mass spectrometry and nuclear magnetic resonance confirmed that cucurbitane saponins are the effective parts of Phytophthora chinensis for diarrhea. Further pharmacological experiments confirmed that the cucurbitane type saponin extract of Phytophthora chinensis has the effect of treating constipation.
  • the invention provides a cucurbitane type tetracyclic triterpenoid saponin, white powder, with a molecular formula of C 36 H 58 O 10 , a molecular weight of 650, and a chemical name of 2 ⁇ -glucosyloxy-3,16,20-trihydroxy-9.
  • -Methyl-19-norlanoster-5,25-diene-22-one compound IX
  • Huahuanglian cucurbitacin A the structural formula is as follows .
  • the invention also provides a Cucurbitane-type saponin extract of Huahuanglian.
  • the Cucurbitane-type saponin extract contains 2 ⁇ -glucosoxy-3,16,20,25-tetrahydroxy-9-methyl-19- Norlanosterol is 10 kinds of cucurbitane-type tetracyclic triterpenoid saponins with a core structure, specifically: Compound I: 25-acetoxy-2 ⁇ -glucoseoxy-3,16-dihydroxy-9-methyl- 19-norlanoster-5-en-22-one, compound II: 2 ⁇ -glucosoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanoster-5-ene -22-one, compound III: 2 ⁇ -glucosoxy-3,16,20,25-tetrahydroxy-9-methyl-19-norlanoster-5,23-(E)-diene-22- Ketone, compound IV: 2 ⁇ -glucoseoxy-3,16,20,22-te
  • the method for preparing the Huahuanglian-cucurbitane saponin extract has the following steps:
  • the traditional Chinese medicine Coptis chinensis is decoctioned in water or extracted with organic solvent reflux.
  • the water extract is directly adsorbed by macroporous adsorption resin.
  • the organic solvent extract first recovers the organic solvent, adds water to disperse and then adsorbs by macroporous adsorption resin;
  • Picrorrhiza scrophulariiflora is the rhizome of the Scrophulariaceae plant Picrorrhiza kurroa Royle ex Benth or Picrorhiza scrophulariiflora Pennell.
  • the macroporous adsorption resin is any one of AB-8, D4020, D101, D860021 and HP20;
  • the column chromatography is any one of silica gel, alumina or ODS column chromatography;
  • the organic solvent is methanol or ethanol.
  • the present invention determines the cucurbitane-type saponin extract of Coptis chinensis with compound I to compound X as the main components and its preparation process.
  • the sum of the content of 10 compounds is >50%; it does not contain iridoids.
  • This preparation process can obtain a Cucurbitane-type saponin extract with a total content of the above-mentioned Cucurbitane-type triterpene saponins between 50% and 100%.
  • the present invention establishes a HPLC content determination method for Compound I to Compound X.
  • Another object of the present invention is to provide the use of Huahuanglian cucurbitane saponin extract in the preparation of medicines for treating constipation.
  • Pharmacological experiments show that the cucurbitane-type saponin extract of Huahuanglian can promote defecation in normal mice and constipation model mice, and significantly increase the moisture content of feces, and has a clear laxative effect.
  • Figure 1 HPLC spectrum of the ethanol extract of Coptidis Coptidis and each eluate; in the figure: A is 275nm, B is 230nm, and C is 200nm;
  • Figure 2 is the infrared spectrum of compound IX;
  • Figure 3 is the high-resolution mass spectrum of compound IX;
  • Figure 4 is the 1 H-NMR spectrum of compound IX;
  • Figure 5 is the 13 C-NMR spectrum of compound IX;
  • Figure 6 is the key 1 H, 1 H-COSY and HMBC signals of compound IX;
  • Figure 7 is cucurbitane The base peak intensity chromatogram of the saponin extract; in the figure: A: negative ion mode B: positive ion mode;
  • Figure 8 is the liquid chromatogram of the cucurbitane-type saponin extract of pheasanthus;
  • Figure 9 is the tetracyclic tricyclic chromatogram of the cucurbitane-type saponin extract Ultraviolet ab
  • Example 1 Take 2 kilograms of Vietnamese Rhizoma Coptidis, add water to decoct and extract 3 times, add 20 liters of water each time, decoct for 1 hour, filter, combine the extracts, adsorb with D101 macroporous adsorption resin, and use water in turn , elute with 30% ethanol by volume, and collect the 60% ethanol eluate until 25-acetoxy-2 ⁇ -glucosoxy-3,16,20-trihydroxy-9-methyl-cannot be detected by thin layer chromatography. 19-Norlanoster-5,23-(E)-dien-22-one (V).
  • Example 2 Take 2 kilograms of Vietnamese Rhizoma Coptidis, add absolute ethanol to heat and extract 3 times, 10 liters each time, reflux for 1 hour, filter, combine the extracts, concentrate under reduced pressure until there is no ethanol smell, add water to dilute, and pass
  • the AB-8 macroporous adsorption resin column is adsorbed, and is eluted sequentially with water and 20% ethanol in volume concentration. Collect the 50% ethanol eluate until 25-acetoxy-2 ⁇ -glucosoxy-3 cannot be detected by thin layer chromatography. 16,20-trihydroxy-9-methyl-19-norlanoster-5,23-(E)-diene-22-one (V).
  • Example 3 Take 2 kilograms of Vietnamese Rhizoma Coptidis, add 95% ethanol to heat and extract 3 times, 10 liters each time, reflux for 1 hour, filter, combine the extracts, concentrate under reduced pressure until there is no ethanol smell, add water to dilute, and pass HP-20 macroporous adsorption resin column adsorption, sequentially elute with water and volume concentration 30% ethanol, collect 60% ethanol eluate until thin layer chromatography cannot detect 25-acetoxy-2 ⁇ -glucosoxy-3. 16,20-trihydroxy-9-methyl-19-norlanoster-5,23-(E)-diene-22-one (V).
  • the eluate was decompressed to recover ethanol, and 157 grams of the crude extract of cucurbitane-type saponins from Coptidis rhizome was obtained.
  • the crude extract of cucurbitane saponin was added to ethyl acetate for purification, and the ethyl acetate was recovered under reduced pressure to obtain 31 grams of cucurbitane saponin extract of Coptidis rhizome. After HPLC detection, it was found that cucurbitane type tetracyclic triterpene saponin I- The total content of X is 62%.
  • Example 4 Take 2 kilograms of Indian Coptidis rhizome, add water to decoct and extract 3 times, add 20 liters of water each time, decoct for 1 hour, filter, combine the extracts, adsorb with D4020 macroporous adsorption resin, and use water in turn , elute with 30% ethanol by volume, and collect the 60% ethanol eluate until 25-acetoxy-2 ⁇ -glucosoxy-3,16,20-trihydroxy-9-methyl-cannot be detected by thin layer chromatography. 19-Norlanoster-5,23-(E)-dien-22-one (V).
  • the total content of cucurbitane-type tetracyclic triterpenoid saponins I-X is 77%, among which 25-acetoxy-2 ⁇ -glucosoxy-3,16,20-trihydroxy-9-methyl-19-de
  • the content of metlanoster-5,23-(E)-diene-22-one (V) was 51%, and no iridoid glycosides were detected by liquid mass spectrometry.
  • Example 5 Take 2 kilograms of Indian Coptidis rhizome, add absolute ethanol and heat and extract 3 times, 10 liters each time, reflux time is 1 hour, filter, combine the extracts, concentrate under reduced pressure until there is no ethanol smell, add water to dilute, and pass D860021 macroporous adsorption resin column adsorption, sequentially elute with water and volume concentration 30% ethanol, collect 60% ethanol eluate until thin layer chromatography cannot detect 25-acetoxy-2 ⁇ -glucosoxy-3,16, 20-trihydroxy-9-methyl-19-norlanoster-5,23-(E)-diene-22-one (V).
  • the eluate was decompressed to recover ethanol, and 161 grams of the crude extract of cucurbitane-type saponins from Coptidis rhizome was obtained.
  • the crude extract of cucurbitane saponin was added to ethyl acetate for purification, and the ethyl acetate was recovered under reduced pressure to obtain 34 grams of cucurbitane saponin extract of Coptidis rhizome. After HPLC detection, it was found that cucurbitane type tetracyclic triterpene saponin I- The total content of X is 52%.
  • Coptidis Coptidis slices were purchased from the herbal medicine shop of Shanxi Provincial Hospital of Traditional Chinese Medicine (batch number: 21010129); D101 macroporous adsorption resin was purchased from Tianjin Bochong Resin Technology Co., Ltd.; ethanol was purchased from Beijing Dimautei Science and Technology Development Center.
  • mice SPF grade KM mice, half male and half male, 120 in total, weighing 20 ⁇ 2g, production certificate number: SCXK (Beijing) 2014-0013, purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Preparation of macroporous resin eluate Weigh 500 g D101 macroporous adsorption resin and soak it in saturated salt solution for 18 to 20 hours, wash with distilled water until colorless, then soak in hydrochloric acid with a mass concentration of 5% for 2 to 4 hours, and drain it Then wash with distilled water until neutral, and put it into a 5 cm diameter glass column for later use.
  • the eluent was evaporated to dryness in a water bath to obtain 42.31 g, 6.63 g, 11.82 g, 9.14 g, 4.53 g, 3.21 g, 0.83 g, 0.42 g, 0.33 g and 0.24 g respectively.
  • Preparation of the test drug Weigh 4 g of the ethanol extract of Coptidis Coptidis, add 10 ml of distilled water, and prepare a 0.1g/ml suspension; also weigh each elution component equivalent to 4 g of the extract and prepare in the same way. into a suspension.
  • Mouse diarrhea experiment Grouping and administration: 120 mice were adaptively fed for 3 days and randomly divided into a blank group, a Coptis chinensis ethanol extract group, a water-eluted group, a 10% ethanol-eluted group, and a 20% ethanol-eluted group. Detachment group, 30% ethanol elution group, 40% ethanol elution group, 50% ethanol elution group, 60% ethanol elution group, 70% ethanol elution group, 80% ethanol elution group and 90% ethanol elution group Groups of 10 animals each, half male and half female. Except for the blank group, which was given distilled water by gavage, the other groups were given corresponding drugs with a gavage volume of 20 ml/kg.
  • test solution Precisely weigh an appropriate amount of each eluate of Coptidis Coptidis ethanol extract and macroporous resin, place it in a 50 ml volumetric flask, add methanol, and sonicate for 30 minutes to make a 10 mg/ml solution, and pass 0.22 ⁇ m microporous filter membrane, ready for use.
  • High performance liquid chromatography (HPLC-UV) analysis conditions Chromatographic column: Shim-pack C18-ODS column (250 mm 1.0 ml ⁇ min -1 ; injection volume: 10 ⁇ l; column temperature: 25 °C; detection wavelength: 200 nm, 230 nm and 275 nm.
  • Mobile phase gradient elution conditions are: 0-2 min, 15%B; 2-32 min, 15-25%B; 32-70min, 25%-50%B; 70-90min, 50-80%B;
  • Chromatographic column Diamonsil C18-ODS column (150 mm -1 ; Injection volume: 1000 ⁇ l; Detection wavelength: 200 nm.
  • UPLC-ESI-QTOF/MS analysis conditions Chromatographic conditions: ACQUITY UPLC HSS T3 C18 column (2.1 ⁇ 100 mm, 1.8 ⁇ m), mobile phase: 0.1% formic acid acetonitrile solution (A) - 0.1% formic acid aqueous solution (B) , gradient elution (0 to 1.2 min, 15% A; 1.2 to 6 min, 15% ⁇ 25% A; 6 to 36 min, 25% ⁇ 55% A), flow rate 0.2 mL/min, column temperature 40°C, The injection volume is 2 ⁇ L.
  • Mass spectrometry conditions XEVO G2-XS ESI ion source, positive/negative ion mode, cone voltage 40 V, ion source temperature 100 °C, desolvation temperature 400 °C, cone gas flow 50 L/h, desolvation gas flow 700 L/ h. Scanning range m/z 100 ⁇ 1500, calibration solution: leucine-enkephalin, [M+H] 556.2771, [MH] 554.2615. Masslynx 4.1 working software was used to collect mass spectrometry data in MS E Continuum mode, with a scan rate of 0.2/s and a collision energy of 20V to 35V.
  • HPLC-UV spectrum As shown in Figure 1, the maximum absorption wavelength of iridoid glycosides, phenolic glycosides and phenolic acids is around 280 nm, while the maximum absorption wavelength of cucurbitane-type tetracyclic triterpenes is around 230 nm. Or end absorption, the present invention uses high-performance liquid chromatography gradient elution combined with multi-wavelength analysis of chemical substances in each component of Coptidis Coptidis. The sample concentration is 10 mg/ml, and 10 ⁇ l is injected.
  • compound VII is a cucurbitane-type glucoside.
  • the planar structure of compound VII was further determined through 1 H, 1 H-COSY and HSQC, and it was highly similar to the structure of compound VI. The only difference was that the double bond position of the 17-position side chain was ⁇ 25 .
  • H 3 -27 is related to C-24/C-25/C-26
  • H 2 -26 is related to C-24/C-27
  • H 2 -24 is related to C- 22/C-23/C-25/C-26/C-27 related.
  • the structure of compound IX is determined to be 2 ⁇ - d -glucosoxy-3,16,20-trihydroxy-9-methyl-19-norlanoster-5,25-diene-22-one. It is a new cucurbitane-type glycoside compound that has not been reported before, named Cucurbita saponin A.
  • the structural formula is: .
  • the a and b signal peak assignments may be interchanged.
  • Coptidis bark can be extracted by decoction or refluxing with organic solvents.
  • organic solvents such as methanol and ethanol reflux extraction. Because ethanol is safe and cheap, ethanol reflux extraction is preferred.
  • the macroporous adsorption resin adsorption method can be used to separate cucurbitane-type triterpene saponins from polysaccharides, iridoids, phenolic glycosides and other impurities. Initial separation.
  • the water decoction liquid can be directly adsorbed by the macroporous adsorption resin, while the organic solvent extraction liquid recovers the solvent to a certain volume, adds water to disperse it, and then adsorbs it through the macroporous adsorption resin.
  • the macroporous adsorption resin described in the above preparation process can be selected from one of AB-8, D4020, D101, 860021, HP20 or other manufacturers' adsorption resins with the same or similar functions.
  • the organic solvent used for impurity removal and elution is generally a mixed solution of organic solvents such as methanol and ethanol and water, and an aqueous ethanol solution is preferred.
  • organic solvents such as methanol and ethanol and water
  • aqueous ethanol solution is preferred.
  • the so-called low concentration means that the saponin compounds are not eluted as the upper limit, which is generally not higher than 30%.
  • Silica gel, alumina or ODS can be used for the column chromatography.
  • the total content of cucurbitane saponins can be measured using spectrophotometry. However, this method is not accurate on the one hand, and cannot comprehensively detect the specific saponin components on the other hand. Therefore, a liquid mass spectrometry method for the cucurbitane saponin extract of Huahuanglian was established. Analysis and HPLC content determination methods.
  • Drugs and reagents Huahuanglian cucurbitane-type saponin extract, prepared in this laboratory; cucurbitane-type tetracyclic triterpenoid saponin compounds I-X, separated and purified in this experiment, and the purity reached 99.2% through liquid phase analysis; mass spectrometry grade Acetonitrile was purchased from Beijing Dimaoutai Science and Technology Development Center.
  • Preparation of standard material solution Precisely weigh 5 mg of compounds I-X each, place it in a volumetric flask, add an appropriate amount of methanol, sonicate for 30 minutes, let cool, adjust the volume to 50 ml, and pass through a 0.22 ⁇ m microporous filter for later use.
  • Mass spectrometry conditions XEVO G2-XS ESI ion source, positive/negative ion mode, cone voltage 40 V, ion source temperature 100 °C, desolvation temperature 400 °C, cone gas flow 50 L/h, desolvation gas flow 700 L/ h. Scanning range m/z 100 ⁇ 1500, calibration solution: leucine-enkephalin, [M+H] 556.2771, [MH] 554.2615. Masslynx 4.1 working software was used to collect mass spectrometry data in MS E Continuum mode, with a scan rate of 0.2/s and a collision energy of 20V to 35V.
  • High performance liquid chromatography conditions Chromatographic column: Shim -pack C18-ODS column (250 mm ; Injection volume: 10 ⁇ l; Column temperature: 20 °C; Detection wavelength: 200 nm. Gradient elution: 0 ⁇ 15 min, 15% ⁇ 25% B; 15 ⁇ 40 min, 25% ⁇ 30% B; 40 ⁇ 50 min, 30% ⁇ 30% B; 50 ⁇ 65 min, 30% ⁇ 40% B.
  • LC-MS analysis results The Cucurbitane-type saponin extract of Pangasius was analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and the positive and negative ion mode base peak intensity chromatograms were obtained respectively, as shown in Figure 7, refer to Comparison of peaks of standard substances showed that cucurbitane-type triterpenoid saponins I-X were detected, while iridoid glycosides such as corynoside I and glycoside II were not detected.
  • Liquid chromatography analysis results Precisely pipette 10 ⁇ l each of the mixed reference solution and test solution into the liquid chromatograph, as shown in Figure 8. Vanillic acid and cinnamic acid can be seen, peak 1 is compound I, and peak 2 is compound II. , Peak 3 is compound III, peak 4 is compound IV, peak 5 is compound V, peak 6 is compound VI, peak 7 is compound VII, peak 8 is compound VIII, peak 9 is compound IX, and peak 10 is compound X. Record the area of each peak and calculate that the sum of the contents of compounds I-X reaches 68.1%. Liquid chromatography also did not detect iridoid glycosides such as cooperside I and glycoside II.
  • Coptidis cucurbitane-type saponin extract prepared in our laboratory; vanillic acid and cinnamic acid were purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; Linaclotide capsules (Lingzeshu), Almac Pharma Services Limited, batch number W054121, specification: 290 ⁇ g/capsule.
  • mice SPF grade KM mice, half male and half female, body weight 20 ⁇ 2g, production certificate number: SCXK (Beijing) 2014-0013, purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • Preparation of the test drug Weigh 100 mg each of vanillic acid and cinnamic acid, add 5 ml of distilled water, ultrasonic for 10 min, and prepare a 20 mg/ml suspension; weigh an appropriate amount of cucurbitane saponin extract and follow the same method. Prepare 5 mg/ml, 10 mg/ml and 20 mg/ml suspensions; take the contents of the linaclotide capsule and grind it with distilled water to prepare a 5 ⁇ g/ml suspension. Place at 4°C and set aside.
  • Preparation of carbon powder suspension measure 80 ml of distilled water, add 10 g of gum arabic, boil, add 5 g of activated carbon after dissolving; boil 3 times, cool and dilute to 100 ml, place at 4°C for later use.
  • mice After adaptive feeding for 3 days, mice were randomly divided into blank group, linaclotide group (50 ⁇ g/kg), vanillic acid group (200 mg/kg), and cinnamic acid group (200 mg /kg), Huahuangliancucurbitane saponin extract low-dose group (50 mg/kg), medium-dose group (100 mg/kg) and high-dose group (200 mg/kg), 10 animals in each group, half male and half female . On the day of the test, mice in each group were given carbon powder suspension (10 ml/kg) by gavage.
  • mice were raised in a single cage and observed continuously for 12 hours. The time when each mouse first defecated black stool was recorded. The feces was collected and weighed to record the defecation volume. At the same time, the feces moisture content was calculated after the feces was completely dry.
  • mice After adaptive feeding, mice were continuously deprived of water and fasting for 72 hours to prepare a water loss constipation model. The next day, the model mice were randomly divided into model control group, linaclotide group (50 ⁇ g/kg), vanillic acid group (200 mg/kg), cinnamic acid group (200 mg/kg), and pestine group. saponin extract low-dose group (50 mg/kg), medium-dose group (100 mg/kg) and high-dose group (200 mg/kg), and mice that were not deprived of water were set as a blank group, with 10 mice in each group. Half and half were male and female.
  • mice in each group were given carbon powder suspension (10 ml/kg) by gavage. After 30 minutes, except the blank group which was given distilled water by gavage, the other groups were given corresponding drugs with a gavage volume of 10 ml/kg. The mice were raised in a single cage and observed continuously for 12 hours. The time when each mouse first defecated black feces was recorded. The feces were collected and weighed to record the defecation volume. At the same time, the feces moisture content was calculated after the feces was completely dry.
  • mice were used to evaluate the purgative effect of the cucurbitane-type saponin extract of pheasanthus chinensis.
  • Linaclotide was used as a positive control, and at the same time Vanillic acid and cinnamic acid control groups were established. The mice in each group were continuously observed for 12 hours after intragastric administration of water or test substance liquid. As shown in Table 3, the time to the first black stool of mice in the linaclotide group was significantly shorter than that of the blank group (P ⁇ 0.05).
  • the number of defecation particles There was a significant increase in fecal moisture content (P ⁇ 0.05, P ⁇ 0.05); compared with the blank group, there were no significant changes in the time to the first black stool, the number of defecation pellets and the fecal moisture content of the mice in the vanillic acid and cinnamic acid groups; Compared with the blank group, the three dosage groups of alkane saponin extracts showed a dose-dependent decrease in the time to first black stool, and increased the defecation volume and feces moisture content. The feces volume and feces moisture content in the medium-dose group (100 mg/kg) were lower than those of the blank group. There was a significant increase in both groups (P ⁇ 0.05, P ⁇ 0.05).
  • the amount and fecal moisture content were significantly reduced compared with the blank group (P ⁇ 0.01, P ⁇ 0.001); while the three indicators of mice in the linaclotide, vanillic acid and cinnamic acid control groups had no significant improvement compared with the model group; Coptis chinensis Compared with the model group, the three dose groups of cucurbitane saponin extract reduced the time to first black stool in a dose-dependent manner, and increased the defecation volume and fecal moisture content. The middle-dose group (100 mg/kg) had a lower defecation volume and fecal moisture content. There was a significant increase in the model group (P ⁇ 0.05, P ⁇ 0.05).
  • 100 mg/kg of Cucurbitane-type saponin extract of Panax chinensis can promote defecation in normal mice, reduce the time to first black stool, increase defecation volume, and increase the moisture content of feces; it was further confirmed by using dehydration constipation model mice that 100 mg/kg kg Huahuanglian cucurbitane saponin extract also reduced the time to first black stool, and increased the defecation volume and fecal moisture content, while vanillic acid (200 mg/kg) and cinnamic acid 200 mg/kg) reduced the defecation of the two model mice. All had no obvious effect.
  • Drugs and reagents Transpheniantris cucurbitane saponin extract (CTTS) and compounds I-X, prepared in our laboratory; Linaclotide capsules (Lingzeshu), Almac Pharma Services Limited, batch number W054121, specification 290 ⁇ g/capsule .
  • CTTS Transpheniantris cucurbitane saponin extract
  • Linaclotide capsules Linaclotide capsules (Lingzeshu), Almac Pharma Services Limited, batch number W054121, specification 290 ⁇ g/capsule .
  • mice SPF grade KM mice, half male and half female, body weight 20 ⁇ 2g, production certificate number: SCXK (Beijing) 2014-0013, purchased from Beijing Huafukang Biotechnology Co., Ltd.
  • test drugs Weigh appropriate amounts of cucurbitane-type tetracyclic triterpene saponins I-X compounds respectively, add 5 ml of distilled water, ultrasonic for 10 minutes, and prepare 5 mg/ml and 10 mg/ml suspensions respectively; weigh appropriate amounts.
  • the cucurbitane saponin extract of Coptidis Coptidis was prepared into a 10 mg/ml suspension in the same way; take the contents of the linaclotide capsule, grind it with distilled water, and prepare a 5 ⁇ g/ml suspension. Place at 4°C and set aside.
  • mice After adaptive feeding, the mice were continuously deprived of water and food for 72 hours to prepare a constipation model due to water loss and constipation. The next day, the model mice were randomly divided into a model control group, a linaclotide group (50 ⁇ g/kg), a cucurbitane saponin extract group (100 mg/kg), and a compound I-X low-dose group. (50 mg/kg) and high dose (100 mg/kg).
  • mice that were not deprived of water were set as a blank group, with 10 mice in each group, half male and half. Except for the blank group, which was given distilled water by gavage, the other groups were given corresponding Drug, intragastric volume 10 ml/kg. The mice were raised in a single cage and observed continuously for 12 h. The feces of each mouse were collected and weighed, and the feces moisture content was calculated after it was completely dry.
  • mice in each group were continuously observed for 12 hours after intragastric administration of water or test substances.
  • the defecation volume and fecal moisture content of the mice in the model group were significantly reduced compared with the blank group (P ⁇ 0.001, P ⁇ 0.001 ); however, there was no significant improvement in the mice in the linaclotide 50 ⁇ g/kg treatment group compared with the model group; the 100 mg/kg cucurbitane saponin extract treatment group showed a significant increase (P ⁇ 0.001, P ⁇ 0.001).
  • mice in the high-dose group 100 mg/kg
  • mice in the high-dose group 100 mg/kg
  • mice in the high-dose group 100 mg/kg
  • mice in the high-dose group 100 mg/kg
  • mice in the high-dose group 100 mg/kg
  • cucurbitane-type tetracyclic triterpenoid saponins I-X of Huahuanglian were significantly increased compared with the model group.

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Abstract

L'invention concerne un extrait de saponine cucurbitane de picrorhizae rhizoma et son utilisation dans la préparation d'un médicament pour traiter la constipation. Sur la base d'un guidage d'activité, la saponine de cucurbitane se révèle être une partie de picrorhizae rhizoma efficace pour son effet purgatif, et contient 10 saponines triterpénoïdes tétracycliques de cucurbitane ayant 2β-glucopyranosyloxy-3,16,20,25-tétrahydroxy-9-méthyl-19-norlanosta en tant que structure de noyau parent, la somme des teneurs des 10 saponines triterpénoïdes tétracycliques de cucurbitane étant supérieure à 50 %, et la saponine de cucurbitane ne contenant pas d'ingrédient iridoïde. Le composé 2β-glucopyranosyloxy-3,16,20-trihydroxy-9-méthyl-19-norlanosta-5,25-diène-22-one est une saponine triterpénoïde tétracyclique de cucurbitane nouvellement découverte. La recherche pharmacologique montre que l'extrait de saponine cucurbitane de picrorhizae rhizoma peut favoriser la défécation de souris modèles normales et constipées, et augmenter la teneur en eau des selles. L'extrait peut être utilisé pour préparer un médicament destiné au traitement de la constipation.
PCT/CN2023/087473 2022-05-29 2023-04-11 Extrait de saponine cucurbitane de picrorhizae rhizoma et son utilisation dans la préparation d'un médicament pour traiter la constipation WO2023231582A1 (fr)

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