CN-121975031-A - Extraction method and application of orchid root polysaccharide

CN121975031ACN 121975031 ACN121975031 ACN 121975031ACN-121975031-A

Abstract

An extraction method of orchid root polysaccharide and application thereof can effectively extract orchid root polysaccharide GLHG a from orchid roots, and realize application in preparing medicaments for treating lung injury. The method comprises the steps of cutting orchid roots, reflux extracting, concentrating, centrifuging to obtain a precipitate, adding water for suspension after deproteinization treatment, adding sevage reagent, magnetically stirring, standing, layering, taking an upper solution, concentrating under reduced pressure, adding H 2 O 2 , regulating the pH of a reaction system by using a NaOH solution for decoloring, dialyzing by using a semipermeable membrane, freeze-drying to obtain powder, dissolving by using deionized water, centrifuging, taking supernatant, filtering, gradient eluting, detecting, drawing an elution curve, concentrating under reduced pressure, dialyzing, freeze-drying, selecting water component polysaccharide to prepare a polysaccharide solution, purifying and separating. The raw materials are rich, the preparation method is simple and easy to operate, provides technical support for developing novel medicaments for treating lung injury, and has practical popularization and application values.

Inventors

CHEN SUIQING
GAO ZHINING
GUO QIYU
XUE SHUJUAN
WANG YIFAN
ZHANG LIKE
WANG YANDONG

Assignees

河南中医药大学

Dates

Publication Date: 20260505
Application Date: 20260122

Claims (3)

1. The extraction method of the orchid root polysaccharide is characterized by comprising the following specific steps: S1, preparing concentrated solution: cutting orchid roots into segments with the thickness of 6-8 mm, sequentially adding 10 times of the weight volume of the orchid roots and 95% ethanol with the volume concentration of 95% into the segments, carrying out reflux extraction twice, 1.5 h each time, removing fat-soluble components, carrying out reflux extraction twice by using 10 times of the weight volume of ultrapure water with the weight volume of the orchid roots after airing, 2h each time, merging extracting solutions, concentrating under reduced pressure to obtain concentrated solution with the weight volume of 1.25 times of the weight volume of the raw medicinal materials, wherein the weight volume refers to that solid is calculated by kg, and liquid is calculated by L; s2, extracting crude polysaccharide powder of orchid roots: Adding absolute ethyl alcohol into the concentrated solution, regulating the volume concentration of the absolute ethyl alcohol to 80%, standing for 24 hours at 4 ℃, centrifuging for 10 min at 10000 r.min -1 , collecting precipitate, adopting a Sevag method for precipitation, taking chloroform-n-butanol mixed solution with the volume concentration ratio of 4:1 as a sevage reagent, deproteinizing, then adding water to suspend, adding the sevage reagent according to the volume ratio of 4:1, magnetic stirring for 30 min, pouring into a separating funnel for standing, removing lower protein after the solution is layered, repeating for 2-3 times, merging the upper solution, concentrating under reduced pressure, adding H 2 O 2 into the concentrated solution, adding NaOH solution, regulating the pH value at 9 with pH test paper for decolorization, dialyzing for 48H by using a 3500Da semipermeable membrane, and freeze-drying to obtain crude polysaccharide powder of the purified radix isatidis; s3, separating and purifying the crude polysaccharide of the orchid roots: Dissolving the crude orchid polysaccharide powder with deionized water, centrifuging 10min by 10000 r min -1 , taking supernatant, passing through a 0.22 mu m water system microporous filter membrane, establishing a 0-0.6 mol L -1 NaCl gradient elution system, using a phenol-sulfuric acid method for detection, drawing each concentration elution curve, concentrating orchid polysaccharide solutions with different gradients under reduced pressure, dialyzing 48 h, then freeze-drying to obtain each gradient polysaccharide powder, selecting water component polysaccharide, preparing polysaccharide solution of 30mg ml -1 , and separating by using a gel purification system to obtain orchid polysaccharide GLHG.
2. The extraction method of the orchid root polysaccharide according to claim 1, which is characterized by comprising the following specific steps: S1, preparing concentrated solution: Cutting 4.0 kg orchid root samples into segments of 6-8 mm, sequentially extracting with 40L and 32L of 95% ethanol under reflux twice, 1.5 h each time, removing fat-soluble components, extracting with 40L and 32L of ultrapure water under reflux twice, 2h each time after air drying, mixing the extracts, and concentrating under reduced pressure to obtain concentrated solution 5L; s2, extracting crude polysaccharide powder of orchid roots: Adding absolute ethyl alcohol into the concentrated solution, regulating the volume concentration of the absolute ethyl alcohol to 80%, standing for 24 hours at 4 ℃, centrifuging for 10min at 10000 r.min -1 , collecting precipitate, adopting a Sevag method for precipitation, taking chloroform-n-butanol mixed solution with the volume concentration ratio of 4:1 as a sevage reagent, deproteinizing, then adding water for suspension, adding the sevage reagent according to the volume ratio of 4:1, magnetic stirring for 30 min, pouring into a separating funnel for standing, removing lower protein after the solution is layered, repeating for 2-3 times, merging the upper solution, concentrating under reduced pressure, adding 30 ml H 2 O 2 into the concentrated solution, adding NaOH solution, regulating the PH value at 9 by using PH test paper for decolorization, dialyzing for 48 h by using a 3500Da semipermeable membrane, and freeze-drying to obtain crude polysaccharide powder of the purified radix isatidis 32.65 g; s3, separating and purifying the crude polysaccharide of the orchid roots: Weighing 500 mg orchid root crude polysaccharide powder, fixing the volume to 20 ml by deionized water, centrifuging 10min by 10000 r min -1 , taking supernatant, passing through a 0.22 mu m water system microporous filter membrane, establishing a NaCl gradient elution system of 0-0.6 mol L -1 , using a phenol-sulfuric acid method for detection, drawing each concentration elution curve, concentrating orchid root polysaccharide solutions with different gradients under reduced pressure, dialyzing 48 h, then freeze-drying to obtain each gradient polysaccharide powder, selecting water component polysaccharide, preparing polysaccharide solution of 30mg ml -1 , and separating by using a gel purification system to obtain orchid root polysaccharide GLHG.
3. Use of the orchid polysaccharide GLHG a prepared by the method according to any one of claims 1-2 in the preparation of a medicament for the treatment of lung injury.

Description

Extraction method and application of orchid root polysaccharide Technical Field The invention relates to the field of medicines, in particular to an extraction method and application of orchid root polysaccharide. Background Orchid (Orchidaceae) plants are widely distributed in China, wherein root systems of orchid (Cymbidium Sw.) terrestrial or epiphytic herbaceous plants (such as cymbidium, and the like) have important medicinal values. The orchid root is used for treating sequelae of tumor radiotherapy and chemotherapy (such as improving nasopharyngeal carcinoma symptom by combining herba Selaginellae), hemoptysis of pulmonary tuberculosis (fresh root juice), urinary tract infection, neurasthenia, etc. Modern researches show that the orchid root is rich in alkaloid, flavone and phenols and other active ingredients, has multiple pharmacological effects of antibiosis, anti-inflammation, antioxidation, immunoregulation and the like, and has obvious curative effects on respiratory system (cough relieving and phlegm eliminating), digestive system (gastroenteritis resisting) and hemorrhagic diseases. However, the current development rate of orchid medicinal herbs is less than 30%, only 5 orchid medicinal herbs are collected in 2025 edition of Chinese pharmacopoeia, and polysaccharide research is still blank. The invention systematically develops the separation and purification, structural characterization and acute lung injury resistance mechanism research of the orchid root polysaccharide for the first time, fills the research blank in the field, provides a theoretical basis for developing novel medicines for treating lung injury, has important significance for promoting the high-value utilization of orchid medicinal resources, but has not been reported publicly so far. Disclosure of Invention Aiming at the situation, the invention aims to overcome the defects of the prior art and provide an extraction method and application of the orchid root polysaccharide, which can effectively extract orchid root polysaccharide GLHG a from orchid roots and realize the application in preparing medicaments for treating lung injury. The invention solves the technical proposal that the extraction method of the orchid root polysaccharide comprises the following specific steps: s1, preparing concentrated solution Cutting orchid roots into segments with the thickness of 6-8 mm, sequentially adding 10-8 times of ethanol with the volume concentration of 95% into the orchid roots, carrying out reflux extraction twice, 1.5 h each time, removing fat-soluble components, carrying out reflux extraction twice with 10-8 times of ultrapure water with the volume of orchid roots after airing, carrying out reflux extraction twice, 2-h each time, merging extracting solutions, and concentrating under reduced pressure to obtain a concentrated solution with the volume of 1.25 times of the weight of the raw medicinal materials, wherein the weight volume is calculated by solid in kg, and the liquid is calculated by L; S2, extracting crude polysaccharide powder of orchid roots Adding absolute ethyl alcohol into the concentrated solution, regulating the volume concentration of the absolute ethyl alcohol to 80%, standing for 24 hours at 4 ℃, centrifuging for 10 min at 10000 r.min -1, collecting precipitate, adopting a Sevag method for precipitation, taking chloroform-n-butanol mixed solution with the volume concentration ratio of 4:1 as a sevage reagent, deproteinizing, then adding water to suspend, adding the sevage reagent according to the volume ratio of 4:1, magnetic stirring for 30 min, pouring into a separating funnel for standing, removing lower protein after the solution is layered, repeating for 2-3 times, merging the upper solution, concentrating under reduced pressure, adding H 2O2 into the concentrated solution, adding NaOH solution, regulating the pH value at 9 with pH test paper for decolorization, dialyzing for 48H by using a 3500Da semipermeable membrane, and freeze-drying to obtain crude polysaccharide powder of the purified radix isatidis; s3, separating and purifying crude polysaccharide of orchid roots Dissolving the crude orchid polysaccharide powder with deionized water, centrifuging 10min by 10000 r min -1, collecting supernatant, passing through a 0.22 μm water system microporous filter membrane, establishing a 0-0.6 mol L -1 NaCl gradient elution system, using phenol-sulfuric acid method to detect, drawing each concentration elution curve, concentrating orchid polysaccharide solutions with different gradients under reduced pressure, dialyzing 48 h, freeze-drying to obtain each gradient polysaccharide powder, selecting water component polysaccharide (elution curve is shown as figure 1), preparing polysaccharide solution of 30mg ml -1, and separating by using a gel purification system to obtain orchid polysaccharide GLHG. The application of the orchid root polysaccharide GLHG0 prepared by the method in preparing medicaments for