WO2023173268A1 - 一种黄蜀葵花黄酮类有效部位及其制备方法与应用 - Google Patents

一种黄蜀葵花黄酮类有效部位及其制备方法与应用 Download PDF

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WO2023173268A1
WO2023173268A1 PCT/CN2022/080818 CN2022080818W WO2023173268A1 WO 2023173268 A1 WO2023173268 A1 WO 2023173268A1 CN 2022080818 W CN2022080818 W CN 2022080818W WO 2023173268 A1 WO2023173268 A1 WO 2023173268A1
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quercetin
marshmallow
extract
extraction
ethanol
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French (fr)
Chinese (zh)
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唐海涛
王丹丹
葛海涛
王殿广
彭小兰
梁慧
王富江
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Jiangsu Suzhong Pharmaceutical Research Institute Co Ltd
Suzhong Pharmaceutical Group Co Ltd
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Jiangsu Suzhong Pharmaceutical Research Institute Co Ltd
Suzhong Pharmaceutical Group Co Ltd
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Priority to CN202280093354.8A priority Critical patent/CN118973595A/zh
Priority to EP22931308.5A priority patent/EP4494646A4/en
Priority to US18/847,227 priority patent/US20250195598A1/en
Priority to JP2024554801A priority patent/JP2025507192A/ja
Priority to PCT/CN2022/080818 priority patent/WO2023173268A1/zh
Priority to CN202380026355.5A priority patent/CN118891053A/zh
Priority to PCT/CN2023/081052 priority patent/WO2023174205A1/zh
Publication of WO2023173268A1 publication Critical patent/WO2023173268A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/42—Respiratory system, e.g. lungs, bronchi or lung cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/577—Malvaceae (Mallow family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the invention belongs to the field of traditional Chinese medicine, and specifically relates to an effective part of marshmallow flower flavonoids and its application.
  • Medicinal hollyhock flower is the dried corolla of the Malvaceae plant Abelmoschus manihot (L.) Medic. Its taste is sweet and cold, and it returns to the kidney and bladder meridian. It has the effects of clearing away dampness and heat, reducing swelling and detoxifying, and is used for carbuncle, swollen poison, water-fire scald. This medicine was first recorded in "Jiayou's Materia Medica”. "Jiayou's Materia Medica” records: marshmallow flowers are used to treat malignant sores that have pus that have not healed for a long time. "Compendium of Materia Medica” says: The flowers of hollyhock are sweet, cold, slippery, and non-toxic. They can be used to treat malignant sores that have pus and water that have not bled for a long time. They can be cured immediately after applying it for a long time. It is an important medicine for treating sores.
  • Flavonoids are one of the main components of marshmallow flowers, and they are also one of its pharmacologically active ingredients. Huangkui capsules have been on the market for many years, and their main active ingredient is marshmallow flower extract. Research has confirmed that the flavonoids in marshmallow flower extract not only have a significant therapeutic effect on ulcers, but also have a significant protective effect on damage to the heart, brain, tissue, etc. caused by ischemia. In addition, many literature reports have shown that the total flavonoids of marshmallow flowers It may have good effects in anti-inflammatory, antipyretic and analgesic, protecting heart and brain from ischemic damage, lowering blood sugar, and anti-viral.
  • patent CN201210082553.7 discloses a total flavonoid extract of marshmallow flowers, containing gossipin-3'-glucoside, quercetin-3'-glucoside and isoquercetin in a weight ratio of (11-16 ): (2.5-6): (4-6.5) flavonoids can be used to prepare and treat nephritis diseases.
  • Patent CN200610097615.6 discloses a total flavonoid extract of marshmallow flowers, with a total flavonoid content of 50-90% by weight.
  • the flavonoid components contained in it are: quercetin-3-acaiglycoside 1.0-5.0%, golden silk peach glycoside 8-24.0%, isoquercetin 7.0-20.0%, quercetin-3'-glucoside 5.0-15.0%, gossyrin-3'-glucoside 3.0-10.0%, myricetin 0.5-5.0% , cottonseed 0.5-5.0%, quercetin 2.0-8.0%, and several other flavonoid components.
  • the total flavonoid extract of marshmallow flowers can be used to prepare drugs for treating nephritis.
  • the flavonoid components are qualitatively and quantitatively clear, and the curative effect is reliable. .
  • bioflavonoids have various therapeutic effects, there are many types of flavonoids, which can currently be divided into seven different subtypes, and different flavonoid compositions will lead to different therapeutic effects.
  • bioflavonoids have been reported in the literature to be effective in preventing or treating diabetic retinopathy
  • compounds with five hydroxyl groups, such as quercetin have negative effects on ocular blood flow
  • flavonoids are dehydrogenated to flavanones
  • Significant improvements in ocular blood flow were obtained.
  • compounds that increase blood flow also lead to a significant increase in retinal functional recovery after ischemic injury. How to develop such compounds to effectively treat eye diseases such as macular degeneration, visual fatigue and cataracts and even improve the effect on diabetic retinopathy is an important research task.
  • CNV Choroidal neovascularization
  • AMD age-related macular degeneration
  • AMD central exudative chorioretinitis
  • macular degeneration caused by high myopia.
  • CNV secondary to AMD is the most common and has become One of the main causes of irreversible visual impairment in the elderly. Insufficient blood flow is also a factor affecting visual fatigue. Enhancing eye blood flow or acting on eye smooth muscles can help restore visual fatigue.
  • Glucose reabsorption in the proximal renal tubule is mediated by the sodium-glucose cotransporter (SGLT) 1 and SGLT2 transporters, of which approximately 90% of glucose reabsorption is mediated by SGLT2 and the remaining 10% is mediated by SGLT1.
  • SGLT2 inhibitors inhibit renal tubular glucose reabsorption by selectively binding to the SGLT2 receptor, thereby lowering the renal glucose threshold, increasing urinary glucose excretion, and achieving significant blood sugar lowering purposes.
  • SGLT2 inhibitors have a renal protective effect on non-diabetic chronic kidney disease (CKD).
  • SGLT2 inhibitors can reduce the expression of collagen and fibronectin by reducing TGF ⁇ -1, PAI1, STAT 1, MMP 7 and inhibiting the AGEs-RAGE axis, thereby reducing the accumulation of extracellular matrix and alleviating the progression of DN renal fibrosis.
  • SGLT2 inhibitors can also reduce uric acid (SUA) in patients with diabetes.
  • SGLT2 inhibitors empagliflozin
  • serum uric acid levels significantly reduced serum uric acid levels.
  • SGLT2 inhibitors can be beneficial to the prevention and treatment of heart failure by reducing plasma volume, reducing before and after load, improving myocardial energy metabolism, and improving myocardial remodeling.
  • marshmallow flower reported in the literature include ethanol reflux extraction, ultrasonic extraction, and room temperature soaking extraction.
  • the components of marshmallow flower extract are complex, the content of active ingredients varies greatly, the extraction transfer rate is low, the extraction energy consumption is high, and the amount of solvent is large. . Therefore, it is necessary to explore a marshmallow flower therapeutic drug and its extraction process that can jointly treat kidney disease, especially diabetic nephropathy, and simultaneously treat multiple diseases such as eye diseases.
  • the present invention provides an effective part of flavonoids from marshmallow flowers and its application.
  • the present invention prepares a high-purity effective part of marshmallow flower flavonoids through an extraction coupling resin method. It is found that it has better therapeutic activity for kidney diseases, such as diabetic nephropathy, contrast agent kidney injury or lupus erythematosus nephritis. It also has the ability to treat eye diseases. The role of disease.
  • the effective part or extract or composition of Althea flower flavonoids of the present invention has lower side effects and provides a better therapeutic drug for patients.
  • the present invention provides an effective part of flavonoids from marshmallow flowers, including flavonoid components in the following mass ratio: gossyrin-8-O- ⁇ -D-glucuronide: hypericin: isoquercetin.
  • the mass ratio of dermatin: myricetin: quercetin-3'-O-glucoside is: 10:3.0 ⁇ 20:4.0 ⁇ 18:1.9 ⁇ 6.0:6.0 ⁇ 20.
  • the mass ratio of gossyrin-8-O- ⁇ -D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is: 10 :4.0 ⁇ 17:4.50 ⁇ 16:1.9 ⁇ 5.5:7.0 ⁇ 16; preferably: 10:6.0 ⁇ 15:5.0 ⁇ 13:2.0 ⁇ 5.0:8.0 ⁇ 14; further, it contains quercetin, among which gossydertin -8-O- ⁇ -D-glucuronide: quercetin is 10:1.0 ⁇ 10.0, preferably 10:1.0 ⁇ 6.0, preferably 10:1.5 ⁇ 5.0; further, it contains rutin, wherein cotton Cortin-8-O- ⁇ -D-glucuronide:rutin is 10:0.05-0.6, preferably 10:0.1-0.4, further preferably 10:0.1-0.3; further, it contains quercetin- 3-O-Acaiglycoside, wherein Gossipin-8-O- ⁇ -D-glucuronide: Qu
  • the present invention provides an effective part of flavonoids from marshmallow flowers, including flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin:
  • the mass ratio of gossydisin-8-O- ⁇ -D-glucuronide:hyperin is: 0.8-1.2:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.1-1.2:0.4-3.1:0.10-0.9:0.4-3.1:0.5-3.0; further 0.8-1.2 : 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8 -1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 0.8-1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.09-1.6:0.2-2.0:0.05-1.2:0.5-3.0:0.25-3.6.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.12-0.7:0.8-1.6:0.2-0.5:0.85-1.7:1.0-1.4.
  • the mass ratio value of the isoquercetin is 1.2 or 1.0.
  • the effective part of the flavonoids of the marshmallow flower further contains rutin, and the mass ratio of isoquercitrin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05 , preferably 1:0.007-0.04, preferably 1:0.009-0.04, or preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, it contains quercetin-3-O-acaxiglucoside, The mass ratio of quercetin and quercetin-3-O-sophoroside is 10:0.1-2.5, 10:0.1-0.9, preferably 10:0.15-0.5.
  • the effective part of the flavonoids of the marshmallow flower contains quercetin, quercetin-3'-O-glucoside, myricetin, and gossyderm-8-O- ⁇ -D-glucuronide.
  • the total content of isoquercetin and hyperoside is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%; further, quercetin-3'-o-glucose
  • the glycoside content is 12.1-25%, further the quercetin-3'-o-glucoside content is 12.6-23% or 13-20%, 13-20%, or 14-25%; or further , the content of quercetin is higher than 0.7%, further higher than 1.0%, further 1.0%-10%, further 1.5%-5%; or further, cottonseed-8-O- ⁇ -D-glucuronide is 8.5-30%, further 12%-23%.
  • the effective part contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O- ⁇ -D-glucuronide, and isoquercetin.
  • the total content of rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82%, or 69-90%; further, quercetin
  • the content of quercetin-3'-o-glucoside is 12.1-25%, and further, the content of quercetin-3'-o-glucoside is 12.6-23% or 13-20%.
  • the present invention provides a plant extract containing flavonoid components, including the following mass content components: hyperoside 10-25%, isoquercitrin 8-19%, gossipin-8- O- ⁇ -D-glucuronide 5-30%, quercetin-3'-o-glucoside 12.1-25%; further hyperoside 12-23%, isoquercetin 10-17% , gossyrin-8-O- ⁇ -D-glucuronide 8.5-25%, quercetin-3'-o-glucuronide 12.6-23%; further hyperoside 13-22%, isosin Quercetin 11-17%, gossyrin-8-O- ⁇ -D-glucuronide 12-21%, quercetin-3'-o-glucuronide 13-20%.
  • the extract also contains quercetin 2-10%, preferably quercetin 2.5-9%, preferably quercetin 3-8.5%, further 1.5%-5%; further, it contains bayberry. 2-11% of myricetin, preferably 3-7% of myricetin, preferably 3-5% of myricetin, and further, 0.08-2.5% of quercetin-3-O-sophoraside, preferably 0.1-1.5%, further 0.1 -0.9%, and further 0.1-0.5%; further, the plant is a flower of marshmallow okra, which can be a whole flower or a corolla, preferably a marshmallow flower.
  • the present invention provides a marshmallow flower extract, containing flavonoid components, including the following mass content components: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossipin-8 -O- ⁇ -D-glucuronide 8.5-30%, myricetin 3.0-4.9% or 5.1-6.0%, quercetin-3'-o-glucuronide 14-25%, quercetin 1.6-4.9 %; furthermore, hyperoside 11-22%, isoquercetin 12.0-17%, gossyrin-8-O- ⁇ -D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1-9.0%, quercetin-3'-o-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%.
  • mass content components including the following mass content components: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossipin-8 -O- ⁇ -D-glucuronide 8.5-30%, myricetin 3.0-4.9%
  • the extract also contains rutin with a mass content of 0.01-1.0%, further 0.05-0.8%, further 0.09-0.8%, further 0.1-0.6%; further, it contains quercetin- 3-O-Asophoroside 0.08-2.5%, preferably 0.1-1.5%, further 0.1-0.9%, still further 0.1-0.5%.
  • the mass content of flavonoids in the marshmallow flower extract is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82% or 69-90%. .
  • the present invention provides a composition, including flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-
  • the mass ratio of 8-O- ⁇ -D-glucuronide:hyperoside is: 1:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6; further 1:0.1- 1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 1: 0.2-0.6: 1.0- 1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3 or 1.2
  • the composition also includes rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05, preferably It is 1:0.007-0.04, preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, the content of quercetin-3'-o-glucoside in the flavonoids is 12.1-25%, Furthermore, the content of quercetin-3'-o-glucoside is 12.6-23% or 13-20%. Furthermore, it contains 0.08-2.5% of quercetin-3-O-axiglucoside, preferably 0.1-1.5 %, further 0.1-0.9%, further 0.1-0.5%.
  • the composition contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O- ⁇ -D-glucuronide, and isoquercetin.
  • the total content of rutin, rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%.
  • the present invention provides a pharmaceutical composition, which contains the above-mentioned effective part, the above-mentioned marshmallow extract or the above-mentioned composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, bacteriostatic agents, diluents, and wetting agents.
  • the lubricant does not contain magnesium stearate.
  • the dosage forms of the pharmaceutical composition include injections, tablets, suppositories, ointments, gels, pills, tablets, granules, capsules or mixtures, suspensions, and dispersible tablets.
  • the present invention provides the use of the above-mentioned effective part, the above-mentioned marshmallow extract, the above-mentioned composition or the above-mentioned pharmaceutical composition in the preparation of kidney disease drugs.
  • the kidney disease is diabetic nephropathy or diabetic nephropathy accompanied by renal fibrosis. or nephritis.
  • the present invention provides the use of the above-mentioned effective part, the above-mentioned marshmallow extract, the above-mentioned composition or the above-mentioned pharmaceutical composition in the preparation of medicines for eye diseases.
  • the eye diseases are preferably macular degeneration, visual fatigue or cataracts. Diabetic retinopathy, age-related macular degeneration, central exudative chorioretinitis, and macular degeneration caused by high myopia.
  • the present invention provides the above-mentioned effective part, the above-mentioned marshmallow extract, the above-mentioned composition or the above-mentioned pharmaceutical composition in the preparation of lupus erythematosus nephritis, or renal damage caused by contrast agents, or pulmonary fibrosis, or heart failure, or reducing Application of uric acid or SGLT2 inhibitor drugs.
  • the present invention provides a method for preparing the above-mentioned effective parts or the above-mentioned Althaea hollyhock extract.
  • the method includes the following steps: (1) Extracting the marshmallow flowers or the medicinal parts of the hollyhock with ethanol to prepare an extract; (2) The extract is concentrated and extracted to obtain an extract; (3) the extract is freed of solvent and eluted with a macroporous resin to obtain the effective parts or extracts of flavonoids from marshmallow flowers; the preferred extraction method is percolation.
  • the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution
  • the extraction agent used for extraction in step (2) It is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is level 1-5
  • the macroporous resin model described in step (3) is D101, HPD100 or AB-8.
  • step (2) further includes subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
  • the elution process of the macroporous resin described in step (3) is: the diameter-to-height ratio of the macroporous resin is 1:4-1:9, the concentration of the loading solution is 0.10-0.30g crude drug/mL, and the loading solution The volume is 4-12BV, load the sample at a flow rate of 1-4BV/h, use 4-8BV pure water and 1-5BV 3-15% ethanol to remove impurities at a flow rate of 0.5-4BV/h, use 2-8BV 50-80 % ethanol at a flow rate of 1-5 BV/h.
  • the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution;
  • the water bath temperature described in step (2) is 50-25%. 70°C, water bath time is 30-90min;
  • the resin diameter-to-height ratio described in step (3) is 1:4-1:9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, and the volume of the sample solution is 4- 12BV, elute with 4-8BV pure water and 4-8BV 60-95% ethanol.
  • the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned marshmallow extract, and the preparation method includes the following steps: (1) extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) Adjust the pH value of the extract to 2.0-3.0, refrigerate, filter to collect the precipitate, and add water to dissolve; (3) The dissolved liquid is extracted to obtain an extract; (4) The solvent is removed from the extract and eluted with polyamide resin to obtain marshmallow.
  • Effective parts or extracts of flower flavonoids; the preferred extraction method is reflux.
  • the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution;
  • the pH regulator described in step (2) is hydrochloric acid , sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid;
  • the extraction agent used for extraction in step (3) is n-butanol, petroleum ether or ethyl acetate, and the extraction method is continuous countercurrent extraction, so
  • the material-to-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is level 1-5;
  • the diameter-to-height ratio of the resin in step (4) is 1:4-1:9, and the loading liquid
  • the concentration is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and 4-8BV pure water and 4-8BV 60-95% ethanol are used for elution.
  • the volume/weighed sample amount (after water discount) the content is not higher than 2.0%, further not higher than 0.9%, preferably 0.1%-0.7%.
  • the total content of various flavonoids tested was above 70%, further above 75%, and further between 80% and 90%.
  • the effective parts or extracts of flavonoids from Althea flowers described in the present invention have the effect of treating various nephritis, such as diabetic nephropathy, contrast agent kidney injury or lupus erythematosus nephritis, and also have the effect of treating eye diseases, especially with Various diseases related to inhibiting blood flow and inhibiting ocular blood vessels, such as age-related macular degeneration, central exudative chorioretinitis, and macular degeneration caused by high myopia.
  • the marshmallow flower flavonoids of the present invention are effective Parts or extracts also have therapeutic effects on pulmonary fibrosis.
  • the preparation method of the present invention has simple technology, short production cycle, mild processing conditions, low energy consumption and good separation effect of 7 kinds of flavonoids from marshmallow flowers.
  • the raw materials and reagents used come from a wide range of sources, are low in cost, and are easy to achieve industrialized large-scale production.
  • Figure 1 shows the results of pathological changes detected in rats after repeated administration of different drugs for three months.
  • A normal group
  • B Example 9.2 sample group
  • C Huangkui capsule group
  • D Example 2-A4 group.
  • Figure 2 shows the pathological results of the DKD group, HK group and HT group.
  • Figure 3 shows the SLC5A2mRNA expression detection results of DKD, HK and HT groups.
  • marshmallow flowers in the following examples are dried marshmallow corollas. Unless otherwise specified, they are from the same batch of marshmallow flowers.
  • the contents of seven components of the flavonoid effective parts of the marshmallow flowers prepared in each example were measured. These 7 ingredients refer to: hyperoside, rutin, isoquercetin, gossyrin-8-O- ⁇ -D-glucuronide, myricetin, quercetin-3'-o-glucose Glycosides, quercetin.
  • the detection method adopts the UPLC method reported in "Determination of 7 Components in Hollyhock Flowers by One Test and Multiple Evaluation Method" (Journal of Pharmaceutical Analysis, Issue 12, 2013, 2082-2087) to determine the content of the components.
  • the material-to-liquid ratio of extraction (that is, extraction agent:extraction liquid) refers to the volume ratio.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, and the concentration of the sample solution (aqueous solution) is 0.15g crude drug/mL.
  • the volume of the sample solution is 7BV, the sample is loaded at a flow rate of 2BV/h, 6BV pure water and 3BV 5% ethanol are used to remove impurities successively at a flow rate of 2BV/h, and 4BV 60% ethanol is used to elute at a flow rate of 2BV/h to obtain the elution liquid, and then recover the ethanol under reduced pressure at 50°C to obtain the marshmallow flower flavonoid extract.
  • the test content is based on the content measurement results and the content of the seven components is adjusted by the addition method so that the content of each component is as shown in the table below.
  • the total solid content of the effective parts of marshmallow flower flavonoids obtained was 2.23g, and the total of 7 components was 1769 mg, accounting for 79.32% of the total solid content.
  • Example 2-1 Crush 100g of marshmallow flowers to coarse powder, and extract by percolation with 18 times of 70% ethanol to obtain marshmallow flower extract; ethanol is removed from the marshmallow flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate.
  • the material-to-liquid ratio i.e., extraction agent:extraction liquid
  • the injection speed of the extraction is 80mL/min
  • the speed of the extraction machine is 40Hz
  • the extraction level of the extraction is 4
  • the extraction liquid is placed at 40 After recovering the solvent under reduced pressure at °C, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, and the volume of the sample solution is 6BV, load the sample at a flow rate of 2BV/h, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain the eluent, and then wash the After deliquidizing and recovering ethanol under reduced pressure at 60°C, the marshmallow flower flavonoids extract was obtained. The content was tested.
  • the content of the seven components was adjusted by the addition method so that the contents of each component were as shown in the table below, and the marshmallow flower flavonoids were obtained.
  • Total solids of similar effective parts 2.99.
  • the content of quercetin-3-O-acaiglycoside is 0.28%.
  • Example 2-A Preparation of effective parts of flavonoids from marshmallow flowers
  • the marshmallow flowers were enlarged 10 times, and two batches of flavonoid effective parts of the marshmallow flowers were prepared separately to obtain the samples with the content measurement data of the samples in Table 2-2.
  • Quercetin-3-O-Acacia glycoside content is 0.20-0.50%.
  • 2-A1 contains 0.24% based on peak area.
  • Embodiment 2-A3 take the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin 3-O-acaiglycoside, according to The formula was mixed according to the ratio of Example 2-A1 (rutin was replaced with quercetin 3-O-aphoroside, and the ratio was adjusted to 0.02) to prepare a total flavonoid composition.
  • Embodiment 2-A4 take the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-o-glucoside, quercetin, and quercetin 3-O-acaiglycoside, according to The formulation was mixed according to the ratio of Example 2-A1 (rutin was replaced with quercetin 3-O-acaxiglucoside, and the ratio was adjusted to 0.05) to prepare a total flavonoid composition.
  • the material-to-liquid ratio of the extraction i.e., extraction agent:extraction liquid
  • the injection speed of the extraction is 120mL/min
  • the speed of the extraction machine is 60Hz
  • the extraction level of the extraction is 4.
  • the extraction liquid in After recovering the solvent under reduced pressure at 50°C, dilute with water and centrifuge, take the supernatant and treat it with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, and the concentration of the sample solution is 0.15.
  • the sample solution volume is 7BV, load the sample at a flow rate of 2BV/h, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, and use 4BV 60% ethanol to elute at a flow rate of 2BV/h.
  • Quercetin-3'-O-glucoside is 17.37%
  • quercetin is 1.77%
  • the peak area containing quercetin-3-O-Acacia glycoside is converted to 0.43%.
  • the mass ratio between each component is shown in the following table 2-4:
  • Example 3.1 Crush 100g of marshmallow flowers to coarse powder, and extract by percolation with 18 times of 60% ethanol to obtain marshmallow flower extract; ethanol is removed from the marshmallow flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate.
  • the extracted material The liquid ratio (that is, extraction agent:extraction liquid) is 3:1, the injection speed of extraction is 100mL/min, the speed of extraction machine is 40Hz, the extraction level of extraction is 5, and the extraction liquid is heated at 40°C After recovering the solvent under reduced pressure, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:8, the concentration of the sample solution is 0.15g crude drug/mL, and the volume of the sample solution is 8BV.
  • Load the sample at a flow rate of 2BV/h use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 3BV/h to obtain the eluate, and then use the eluate in After recovering ethanol under reduced pressure at 60°C, the flavonoid extract of the marshmallow flower was obtained.
  • test content was based on the content measurement results and the content of the seven components was adjusted by the addition method so that the content of each component is as shown in the table below.
  • the total effective part of the flavonoids of the marshmallow flower was obtained.
  • Quercetin-3-O-Acacia glycoside content is 0.20-0.50%.
  • Example 3.2 According to the preparation method of Example 3.1, 500g of marshmallow flowers were used for repeated experiments, and the content data of 2 batches were obtained as shown in Table 4 below. Quercetin-3-O-Acacia glycoside content is 0.30-0.80%. Among them, A represents “each ingredient/total solids (%)" and B represents “each ingredient/isoquercitrin”.
  • Example 4-1 100g of marshmallow flowers were percolated and extracted with 18 times of 70% ethanol to obtain marshmallow flower extract; the ethanol was removed from the marshmallow flower extract, diluted with water, and continuously countercurrent extracted with n-butanol.
  • the extracted solid-liquid ratio i.e. extraction agent:extraction liquid
  • the injection speed of extraction is 120mL/min
  • the speed of extraction machine is 60Hz
  • the extraction level of extraction is 5, and the extraction liquid is decompressed at 50°C After recovering the solvent, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 7BV, and the sample solution is 2BV. /h flow rate to load the sample, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 2BV/h to obtain the eluate, and then use the eluate at 60°C After recovering ethanol under reduced pressure, obtain the marshmallow flower extract. Test the content. According to the content measurement results, adjust the content of the 7 components through the addition method so that the contents of each component are as shown in the table below, and obtain the total solid content of the effective parts of the flavonoids of the marshmallow flower. 2.05g.
  • the resin treatment process is: the resin diameter-to-height ratio is 1:7, and the sample solution concentration is 0.15-0.5g crude drug/ mL, the volume of the sample solution is 8BV. Elute with 5BV pure water and 5BV 80% ethanol to obtain the eluate. Concentrate and dry under reduced pressure at 60°C. Test the content.
  • Example 5-1 100g of marshmallow flowers were percolated and extracted with 15 times of 70% ethanol to obtain marshmallow flower extract; ethanol was removed from the marshmallow flower extract, diluted with water, and continuously countercurrent extracted with n-butanol.
  • the extracted material-to-liquid ratio i.e., extraction agent:extraction liquid
  • the extracted material-to-liquid ratio is 2.5:1
  • the injection speed of extraction is 80mL/min
  • the speed of extraction machine is 50Hz
  • the extraction level of extraction is 4
  • the extraction liquid is decompressed at 50°C After recovering the solvent, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, the concentration of the sample solution is 0.2g crude drug/mL, the volume of the sample solution is 6BV, and the sample solution is 2BV. /h flow rate to load the sample, use 1BV pure water and 4BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 3BV 60% ethanol to elute at a flow rate of 2BV/h to obtain the eluent, and reduce the eluate at 60°C. After pressure recovery of ethanol, the marshmallow flower extract was obtained. The content was tested. According to the content measurement results, the content of the seven components was adjusted by the addition method so that the content of each component was as shown in the table below. 2.86g of the total solid content of the flavonoids effective part of the marshmallow flower was obtained. .
  • Extract the marshmallow flowers with 15 times of 70% ethanol under reflux Remove the ethanol from the extract to 0.5-1.0g crude drug/ml, add 10% hydrochloric acid solution to adjust the pH to 2.0-3.0, refrigerate, filter, wash, and take the precipitate. Add water to dissolve, and use ethyl acetate for continuous countercurrent extraction.
  • the solid-to-liquid ratio of the extraction i.e., extraction agent: extraction liquid
  • the extraction level of the extraction is 4.
  • the extraction liquid is recovered under reduced pressure at 50°C to recover the solvent. Then, add water to dilute it, and process it with polyamide resin.
  • the resin treatment process is: the resin diameter-to-height ratio is 1:6, the concentration of the sample solution is 0.15-0.5g crude drug/mL, the volume of the sample solution is 6BV, and 4BV pure water is used. Elute with 4BV 70% ethanol to obtain the eluate, concentrate, and dry under reduced pressure at 60°C to obtain. The content of quercetin-3-O-acaxiglucoside is 0.47%.
  • the extracted material-to-liquid ratio (that is, the extraction agent : Extraction solution) is 2:1, the injection speed of the extraction is 100mL/min, the speed of the extraction machine is 40Hz, and the extraction level of the extraction is 4.
  • the extraction solution is D101 macroporous resin treatment
  • the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.3g crude drug/mL, the volume of the sample solution is 3BV, and the sample is loaded at a flow rate of 2BV/h , use 3BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 70% ethanol to elute at a flow rate of 2BV/h to obtain the eluent, test the content and adjust 7 types by adding 7 specific ingredients Component content, so that the content of each component is as shown in the table below.
  • the eluate is recovered under reduced pressure at 60°C to recover ethanol to obtain marshmallow flower extract.
  • the content is tested. According to the content measurement results, the content of the 7 components is adjusted by the addition method. , the addition amount does not exceed 10% of the total weight, so that the content of each component is as shown in the table below, and 3.16g of the total solid content of the effective parts of marshmallow flower flavonoids is obtained.
  • Example 7-1 Crush 500g of marshmallow flowers to coarse powder, and extract by percolation with 20 times of 70% ethanol to obtain marshmallow flower extract. After recovering the solvent under reduced pressure, it is treated with D101 macroporous resin.
  • the processing technology of macroporous resin It is: the diameter-to-height ratio of the macroporous resin is 1:4, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 6BV, the sample is loaded at a flow rate of 2BV/h, and 7BV pure water and 4BV 5% ethanol are used at a flow rate of 2BV/ h flow rate to remove impurities, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain an eluate, and recover the ethanol under reduced pressure at 60°C to obtain 16.12g of total solids of marshmallow flowers, and its components The content is shown in the table below.
  • macroporous resin The diameter-to-height ratio is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 6BV, the sample is loaded at a flow rate of 2BV/h, and 6BV pure water and 3BV 5% ethanol are used to remove impurities at a flow rate of 2BV/h. , use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain an eluate. The eluate is recovered under reduced pressure at 60°C to recover ethanol to obtain 3.56g of total solids of marshmallow flowers.
  • the extracted material-to-liquid ratio is ( That is, the extraction agent:extraction liquid) is 2.5:1, the injection speed of the extraction is 80mL/min, the speed of the extraction machine is 40Hz, the extraction level of the extraction is 4, and the extraction liquid is recovered under reduced pressure at 40°C. After using the solvent, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/ml, the volume of the sample solution is 6BV, and the sample solution is 2BV/ h flow rate to load the sample, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain the eluate, test the content, and then in the eluate Rutin and hyperin were added so that the contents of each component are as shown in the table below, and then ethanol was recovered under reduced pressure at 60°C to obtain 4.28g of total solids of marshmallow flowers.
  • the extracted material-to-liquid ratio is ( That is, the extraction agent:extracting liquid) is 2.5:1, and the extraction stage of the extraction is 4. After recovering the solvent under reduced pressure at 40°C, the extracting liquid is treated with D101 macroporous resin.
  • the processing process of macroporous resin is:
  • the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 3BV, and the sample is loaded at a flow rate of 1.5BV/h.
  • Example 2-B 30kg of the marshmallow flower mixture was crushed to coarse powder, and extracted by percolation with 18 times of 70% ethanol to obtain a marshmallow flower extract; the ethanol was removed from the marshmallow flower extract, diluted with water, and continuously counter-currently used with ethyl acetate.
  • the material-to-liquid ratio of the extraction that is, the extraction agent: the extraction liquid
  • the injection speed of the extraction is 80mL/min
  • the speed of the extraction machine is 40Hz
  • the extraction level of the extraction is 4
  • the extraction After the solvent is recovered under reduced pressure at 40°C, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, and the sample is loaded The liquid volume is 5BV, load the sample at a flow rate of 1.5BV/h, use 3BV pure water and 4BV 5% ethanol to remove impurities at a flow rate of 2BV/h, and use 5BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain the eluate.
  • the - eluent was recovered under reduced pressure at 65°C to ethanol to obtain the total solids of marshmallow flowers, vacuum dried, crushed, and the flavonoid content (%) of the 7 components was detected.
  • the results are as follows, among which quercetin-3-O-a Sophora glycoside Content 0.20-0.50%.
  • Preparation process Prepare the effective part according to any method of Examples 1-7, add water for injection, stir and dissolve, add to 100L, adjust the pH to 7, filter, fill, and sterilize to obtain eye drops.
  • the pH adjuster used is sodium hydroxide or/and hydrochloric acid.
  • STZ sodium citrate solution is administered to the mice.
  • the blood sugar level of the mice is measured in the tail vein, and the blood sugar level is greater than 16.7 mmol/L as the standard for establishing a diabetes model. If blood sugar is greater than 13.8mmol/L, urine protein and renal function abnormalities appear, it indicates that the modeling is successful.
  • Experimental groups normal group, model group, positive drug group (dapagliflozin tablets, 45.5 mg/kg), sample group prepared by the method of Example 2-1 (62.4 mg/kg) and sample group prepared by the method of Example 6 (62.4 mg/kg), and the sample group (62.4 mg/kg) was prepared by the method of Example 8, with a total of 6 groups, 15 animals in each group. After 4 weeks of administration, urine protein and urinary creatinine were detected, and daily food intake and mouse status were calculated. The test results are shown in Table 12-1 below.
  • Each group of Examples has a reducing effect on urinary protein, among which the sample group of Example 2-1 can significantly reduce urinary protein and urinary protein/urinary creatinine (ACR).
  • ACR urinary protein/urinary creatinine
  • mice Animal models: db/db mice, 16 weeks old, C57BL/6 mice, 16 weeks old.
  • mice C57BL/6 mice were divided into normal group, db/db mice were divided into model group, positive drug group (dapagliflozin tablets, 45.5mg/kg), sample group prepared by the method of Example 3-1 ( 62.4mg/kg) and the sample group (62.4mg/kg) prepared by the method of Example 9.1, and the sample group (62.4mg/kg) prepared by the method of Example 2-A3, a total of 6 groups, 15 animals in each group, after 4 weeks of administration Detect urine protein, urinary creatinine and daily food intake, and observe the status of the mice.
  • the test results are shown in Table 12-2 below.
  • ACR urinary protein/urinary creatinine
  • Example 13 Effects of flavonoids on ocular blood flow and choroidal neovascularization
  • mice rats, body weight 150-180g, SPF grade, half male and half male.
  • Drug grouping and dosage normal group, model group, positive control drug group (vertibofen 1.35 mg/kg), Example 2-1 sample group (69.9 mg/kg), Example 3.1 sample group (87.36 mg /kg), Example 7 sample group (87.36mg/kg), Example 9.1 sample group (87.36mg/kg), Example 9.2 sample group (87.36mg/kg, solid prepared according to Example 9.2)
  • a group of 15 animals were used to test the choroidal blood flow inhibition rate and choroidal vascular neogenesis area in the eyes 4 weeks after administration. The test results are shown in Table 13 below.
  • Example 2-1 Sample Group 80.67 ⁇ 13.78 # 23.24 ⁇ 1.33 # Example 3 Sample Group 83.76 ⁇ 14.68 # 22.89 ⁇ 1.34 # Example 7-2 Sample Group 61.98 ⁇ 16.21 # 28.78 ⁇ 1.45 # Example 9.1 Sample Set 84.19 ⁇ 15.92 # 24.57 ⁇ 1.39 # Example 9.2 Sample Set 85.27 ⁇ 11.34 # 23.79 ⁇ 1.41 #
  • the samples of each example have inhibitory effects on the choroidal blood flow inhibition rate and choroidal angiogenesis area.
  • the example 2 sample group, the example 3 sample group, the example 9.1 sample, and the example 9.2 sample can significantly reduce choroidal angiogenesis.
  • mice SD rats, body weight 200-250g, SPF grade, half male and half male.
  • the modeling success criteria are based on the diagnostic criteria for acute kidney injury recommended by the 2012 KDIGOAKI clinical guidelines (people who meet one of the following conditions can be diagnosed): 1 Serum creatinine rises by more than 26.5umol/L (0.3mg/dl) within 48 hours; 2 Serum creatinine increases by more than 50% of the baseline - confirmed or presumed to occur within 7 days; 3 Urine output decreases ⁇ 0.5ml/(kg*h) and lasts for more than 6 hours. Among them, in animal models, an increase in serum creatinine exceeding 50% of the baseline is often used as the diagnostic criterion for acute kidney injury, that is, the modeling success criterion.
  • Drug grouping and dosage normal group, model group, positive control drug group (irbesartan 50 mg/kg/d), Example 2-1 sample group (69.9 mg/kg), Example 3.1 sample group (87.36 mg/kg), Example 7 sample group (87.36mg/kg), Example 9.1 sample group (87.36mg/kg), Example 9.2 sample group (87.36mg/kg, solid prepared according to Example 9.2)
  • Example 9.2 sample group 87.36mg/kg, solid prepared according to Example 9.2
  • the serum creatinine and serum urea nitrogen of rats with contrast agent renal injury in the model group were significantly higher than those of each administration group;
  • Example 9.1 sample group, Example 2-A1 sample group, Example 3.1 sample, Example 8 sample and Example 6 sample Both serum creatinine and serum urea nitrogen were reduced to varying degrees.
  • mice Twenty-six MRL/lpr male mice (lupus erythematosus nephritis mice) were selected and one group was observed. The body weight was 18 to 22 g and they were 13 weeks old. They were purchased from Nanjing Junke Bioengineering Co., Ltd. and 24 male C57BL/6 mice were selected. Only as the normal group, body weight 18-22g, 13 weeks old, purchased from Wuhan Hualianke Biotechnology Co., Ltd.
  • Drug grouping and dosage normal group, MRL/lpr mouse model group, positive control drug group (dexamethasone 1 mg/kg), Example 2-B sample group (69.9 mg/kg), Example 3.1 sample group (87.36mg/kg), Example 7 sample group (87.36mg/kg), Example 9.1 sample group (87.36mg/kg), Example 8 sample group (87.36mg/kg, solid form prepared according to Example 9.2 15 animals per group, administered for 4 weeks.
  • Example 9.1 sample group, Example 2-B sample group, and Example 3.1 sample, Example 8 sample and Example 7 sample can all improve urinary protein, serum antinuclear antibody levels, and IgG and C3 deposition in renal tissue, and Example 2-B sample, Example 3.1 sample, and Implementation Example 5-2 sample has better effect.
  • mice rats, body weight 150-180g, SPF grade, half male and half male.
  • Drug grouping and dosage normal group, Huangkui capsule group (extract 8g/kg), Example 2-A1 sample group (5g/kg), Example 9.1 sample group (8g/kg), 30 animals in each group , once a day for 12 weeks, the dosage is calculated based on the extract.
  • Example 17 Effect of effective fractions of total flavonoids on idiopathic pulmonary fibrosis
  • mice 60 Kunming mice, half male and half female, weighing 18-22 grams, clean grade.
  • Animal grouping randomly divided into 6 groups, namely normal group, model group, control drug group (rosiglitazone 5 mg/kg), sample group prepared by the method of Example 2-A3 (70 mg/kg), sample prepared by the method of Example 9.2 group (70mg/kg), Huangkui capsule sample group (180mg/kg based on extract), 10 mice in each group.
  • mice were adaptively raised for 3 days, they were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01ml/g). The patients were placed in a supine fixed position, routine disinfection was performed, a midline cervical incision was made, and the trachea was exposed by blunt dissection. The model group, control drug group, and treatment group were punctured and slowly injected bleomycin (5 mg/kg) into the tracheal cartilage ring space, and the normal control group was injected Equal volume of normal saline was injected. Immediately after injecting the drug, the mice were rotated upright for 3-5 minutes to allow the drug solution to be evenly distributed in the lungs on both sides. The skin was then sutured and the sutures were disinfected. After they woke up, they were sent to a clean observation room for feeding.
  • bleomycin 5 mg/kg
  • Administration method will begin on the second day after modeling.
  • the administration group will be administered the above dosage once a day.
  • the normal group and model group will be administered the same volume of distilled water 10ml/kg in the same way for 28 consecutive days.
  • Staining of pathological sections of lung tissue After administration, the right lung was removed and fixed, embedded, sectioned, and stained with HE according to conventional pathological methods. The degree of alveolitis and pulmonary fibrosis is divided into four grades.
  • Sodium-glucose contransporter2 (SLC5A2, also known as SGLT2) is a gene encoding a sodium-glucose cotransporter and can reabsorb more than 90% of the glucose filtered by the glomerulus. SLC5A2 is only expressed at the brush border within the epithelial cell membrane of the proximal tubule of the kidney and is considered a marker of the proximal tubule. The main function is to recover filtered sodium and glucose. In the kidney tissue of patients with diabetes and diabetic nephropathy, inhibiting the expression of SLC5A2 protein can play a role in excreting sodium and reducing blood sugar, thus protecting the kidney tissue of patients with diabetes and diabetic nephropathy.
  • mice were divided into 3 groups when UACR>200mg/g, diabetic nephropathy group (DKD), Huangkui capsule group (DKD+HK, 0.84g/kg/d) and total flavonoids group (DKD+HT, Example 2-A2 sample, 0.0755g/kg/d), Huangkui capsule group and total flavonoids group were administered for four weeks (28d).
  • DKD diabetic nephropathy group
  • DKD+HK Huangkui capsule group
  • DKD+HT total flavonoids group
  • Example 2-A2 sample 0.0755g/kg/d
  • Huangkui capsule group and total flavonoids group were administered for four weeks (28d).
  • the Enzyme-linked immunosorbent assay (ELISA) method is used to detect trace albumin (UACR) in the urine of db/db mice
  • ELISA Enzyme-linked immunosorbent assay
  • RT-PCR Reverse Transcription Polymerase Chain Reaction
  • IHC ImmunoHistoChemistry
  • DKD body weight before grouping of the HK group (Huangkui capsule group) and HT group (total flavonoids group)
  • HT total flavonoids group
  • some glomeruli in the DKD group were lobulated, the basement membrane was significantly thickened, and the glomerular capillaries were significantly compressed.
  • the ratio of SLC5A2 IHC-P quantitative expression to the number of glomeruli was significantly lower, and compared with the IHC- P quantification results are consistent.
  • Huangkui capsules and total flavonoids can effectively reduce microalbuminuria in db/db mice.
  • Huangkui capsules and total flavonoids can inhibit the expression of SLC5A2 protein on the cell membrane of the proximal convoluted tubule, reduce the expression of SLC5A2 protein in the kidneys of DKD mice, and effectively inhibit the activity of SLC5A2 protein, thereby effectively reducing the reabsorption of glucose in the kidneys of DKD mice.

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PCT/CN2022/080818 2022-03-15 2022-03-15 一种黄蜀葵花黄酮类有效部位及其制备方法与应用 Ceased WO2023173268A1 (zh)

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EP22931308.5A EP4494646A4 (en) 2022-03-15 2022-03-15 EFFECTIVE PARTS OF FLAVONOIDS FROM FLOS ABELMOSCHUS MANIHOT, THEIR PREPARATION METHOD AND THEIR USE
US18/847,227 US20250195598A1 (en) 2022-03-15 2022-03-15 Flavonoid active fraction of an abelmoschi corolla, preparation method and application thereof
JP2024554801A JP2025507192A (ja) 2022-03-15 2022-03-15 アベルモスカスマニホット花のフラボノイド有効部位およびその調製方法並びに用途
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WO2025055910A1 (zh) * 2023-09-16 2025-03-20 苏中药业集团股份有限公司 一种药物组合物在制备骨质疏松药物中的应用

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