Cistanche deserticola polysaccharide, preparation method and application thereof in slow transit constipation

Abstract

The present application relates to a kind of Cistanche polysaccharide and preparation method and its application in slow transit constipation, desert Cistanche medicinal material is collected in gansu province huili desert Cistanche planting base, after treating desert Cistanche dried medicinal material, after water extraction alcohol precipitation, dialysis treatment, Cistanche crude polysaccharide is prepared.The prepared Cistanche crude polysaccharide CDPs has protective effect on the colon intermuscular nerve of STC model mouse induced by loperamide (laxative), and can be used to prepare the drug for treating or preventing slow transit constipation, Cistanche crude polysaccharide CDPs is natural compound with biological activity, it has no side effect and low drug dependence.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, and in particular relates to a Cistanche deserticola polysaccharide, its preparation method, and its application in slow transit constipation. Background Technology

[0002] Functional constipation (FC) is a common functional bowel disorder, affecting 14% of the global population. In recent years, due to changes in dietary structure, faster pace of life, and complex social and psychological factors, the prevalence of FC has continued to increase. Studies have shown that FC increases the risk of cardiovascular events and mental disorders, having a serious negative impact on the physical and mental health and quality of life of patients of all ages. Slow transit constipation (STC) is a common type of FC, characterized by prolonged colonic transit time due to colonic motility dysfunction. Currently, research on the etiology and pathogenesis of STC is still in the exploratory stage. Treatment mainly focuses on long-term medication to relieve symptoms, failing to address the underlying cause, and severe cases often require surgical intervention. Therefore, exploring the pathogenesis of STC and finding safer and more effective treatment strategies remains a hot topic and a challenge in the medical field.

[0003] The enteric nervous system (ENS) is an independent nervous system composed of enteric neurons and enteric glial cells, playing a crucial role in regulating intestinal physiological functions, including controlling intestinal peristalsis, fluid absorption and secretion, and immune and hemodynamic regulation. Studies have found that STC (strain-induced thrombosis) is often accompanied by changes in the ENS. Oxidative stress is an imbalance between free radicals produced by oxidation reactions in an organism and the antioxidant system. Reactive oxygen species (ROS) are the main endogenous oxygen free radicals. When ROS production exceeds the body's antioxidant capacity, it can cause lipid peroxidation, damage to protein structure and function, DNA damage, and ultimately irreversible cell death. Studies have found that oxidative stress can induce the translocation of high-mobility group box 1 (HMGB1) in myenteric neurons, causing colonic neuropathy. However, inhibiting the APE1 / Ref-1 redox signaling pathway can inhibit HMGB1 translocation, thereby alleviating myenteric neuron damage and intestinal dysfunction in mice. Currently, research on the relationship between oxidative stress damage to colonic myoneurons and STC is still lacking. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a Cistanche deserticola polysaccharide, its preparation method, and its application in slow-transit constipation.

[0005] The technical solution adopted in this invention is: a method for preparing Cistanche deserticola polysaccharide, comprising the following steps:

[0006] Step 1: Take the dried Cistanche deserticola medicinal material, crush it, add 70% ethanol solution and reflux extract three times, and then defatt it;

[0007] Step 2: After defatting, the medicinal materials are refluxed with water three times for extraction;

[0008] Step 3: Combine the three water extracts, add ethanol for alcohol precipitation, and collect the precipitate;

[0009] Step 4: Dissolve the alcohol precipitate in water, concentrate by dialyzing, and freeze-dry to prepare the crude polysaccharide CDPs of Cistanche deserticola.

[0010] Preferably, in step three, the combined solution of the three water extractions is concentrated, 95% ethanol is added to a final concentration of 80%, and alcohol precipitation is carried out overnight at 4°C.

[0011] Preferably, in step four, the molecular cutoff during dialysis is 3500 Da.

[0012] Preferably, the prepared Cistanche deserticola crude polysaccharide CDPs include polysaccharides in four molecular weight ranges, with polysaccharides of 11.2 kD being the main component, and others including polysaccharides of 45.5 kD, 4.0 kD and 1.8 kD.

[0013] Preferably, the prepared Cistanche deserticola crude polysaccharide CDPs are mainly composed of Glc residues, and also contain small amounts of Rha, GalA, Gal, and Ara residues.

[0014] Preferably, the prepared Cistanche crude polysaccharide CDPs mainly include (1→4)-Glcp, and also contain (1→4,6)-Glcp, (1→4)-Galp and t-Rhap residues.

[0015] A Cistanche deserticola polysaccharide is mainly composed of Glc residues, with small amounts of Rha, GalA, Gal, and Ara residues. It mainly includes (1→4)-Glcp, and also contains (1→4,6)-Glcp, t-Glcp, (1→4)-Galp, and t-Rhap residues. The polysaccharide with a molecular weight distribution of 11.2 kD is the main component, and other polysaccharides include those with molecular weights of 45.5 kD, 4.0 kD, and 1.8 kD.

[0016] Preferably, it is prepared by the method for preparing Cistanche deserticola polysaccharide.

[0017] Application of Cistanche deserticola polysaccharide in the preparation of drugs for the treatment or prevention of slow transit constipation.

[0018] The advantages and positive effects of this invention are: it provides a desert Cistanche deserticola polysaccharide CDPs and its preparation method. This type of Cistanche deserticola crude polysaccharide CDPs can be used to prepare drugs for treating or preventing slow-transit constipation. Compared with traditional laxatives, the bioactive natural compounds have no side effects and low drug dependence; and can effectively relieve constipation symptoms. Attached Figure Description

[0019] Figure 1 Structural characterization of CDPs from Cistanche deserticola: (A) HPGPC chromatogram; (B) Infrared spectrum; (C) Monosaccharide composition chromatogram; (D) Total ion chromatogram for methylation analysis.

[0020] Figure 2 Changes in parameters related to slow-transit constipation. (A) Number of fecal particles; (B) Fecal water content; (C) Time to first melena; (D, E) Serum levels of gastrointestinal motility hormones (SP and VIP). Data are presented as mean ± sem (SEM). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. CON: Control group; LOP: Loperamide-induced constipation model group; LOP+PL: Low-dose group (100 mg / kg); LOP+PM: Medium-dose group (200 mg / kg); LOP+PH: High-dose group (400 mg / kg);

[0021] Figure 3 Morphological changes of mouse colon after administration of Cistanche deserticola polysaccharide. (A) HE staining of colon sections (arrows indicate colonic muscle layer thickness; scale bar: 400 or 100 μm); (B) Alcian blue staining of colon sections (arrows indicate goblet cells; scale bar: 400 or 100 μm); (C) Statistical analysis of colonic muscle layer thickness; (D) Statistical analysis of goblet cell number in colonic crypts. Data are presented as mean ± sem (SEM). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. CON: control group; LOP: loperamide-induced constipation model group; LOP+PL: low-dose group (100 mg / kg); LOP+PM: medium-dose group (200 mg / kg); LOP+PH: high-dose group (400 mg / kg);

[0022] Figure 4 Cistanche deserticola polysaccharide administration to mouse myenteric neurons and nNOS + Changes in neuron number. (A) Myointerstitial neurons in colonic LMMP tissue (arrows indicate neurons; scale bar: 300 μm); (B) Myointerstitial neurons and nNOS in colonic LMMP tissue. + Neuron double staining (arrows indicate nNOS) +Neuron; Scale bar: 100μm); (C) Statistical analysis of the number of neurons in the colonic myometrium; (D) Statistical analysis of the number of neurons in the colonic myometrium nNOS + Neuron number; (E) Statistical analysis of intermuscular nNOS in the colon + The proportion of neurons in the total number of neurons. Data are expressed as mean ± sem (SEM). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. CON: control group; LOP: loperamide-induced constipation model group; LOP+PL: low-dose group (100mg / kg); LOP+PM: medium-dose group (200mg / kg); LOP+PH: high-dose group (400mg / kg);

[0023] Figure 5 Antioxidant-related parameters in mouse colon tissue. Changes in (A) SOD activity, (B) GSH content, (C) lipid peroxidation (MDA) level, (DF) iNOS and Cox2 protein expression levels in LMMP tissue, and (GH) IL-1β and TNF-α mRNA expression levels in LMMP tissue. Data are presented as mean ± sem (SEM). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. CON: Control group; LOP: Loperamide-induced constipation model group; LOP+PL: Low-dose group (100 mg / kg); LOP+PM: Medium-dose group (200 mg / kg); LOP+PH: High-dose group (400 mg / kg).

[0024] Figure 6 Effects of Cistanche deserticola polysaccharide on oxidative stress and dysfunction of mitochondria in myointerstitial neurons of the colon in constipated mice. (A) Mitosox (red) labeling of superoxide production levels in mitochondria (scale bar: 100 μm). (B) JC-1 (green) labeling of mitochondrial membrane potential (scale bar: 100 μm). (C,D) Statistical analysis of fluorescence intensity to assess the production levels of Mitosox and JC-1. Data are presented as mean ± sem (SEM). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. CON: Control group; LOP: Loperamide-induced constipation model group; LOP+PL: Low-dose group (100 mg / kg); LOP+PM: Medium-dose group (200 mg / kg); LOP+PH: High-dose group (400 mg / kg). Detailed Implementation

[0025] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0026] This invention relates to a Cistanche deserticola polysaccharide, its preparation method, and its application in slow transit constipation. The Cistanche deserticola medicinal material was collected from the Huiqin Cistanche deserticola planting base in Gansu Province. After processing the dried Cistanche deserticola medicinal material, it was purified by water extraction and alcohol precipitation to obtain crude Cistanche deserticola polysaccharide. The prepared crude Cistanche deserticola polysaccharide CDPs showed a protective effect against loperamide-induced myenteric nerve in the colon of STC mice, and can be used to prepare drugs for the treatment or prevention of slow transit constipation.

[0027] The preparation method of Cistanche deserticola polysaccharide specifically includes the following steps:

[0028] Step 1: Take the dried Cistanche deserticola medicinal material, crush it, add 70% ethanol solution and reflux extract three times, defatting treatment, the extraction time for the three times is 2 hours, 1 hour and 1 hour respectively;

[0029] Step 2: After defatting, the medicinal materials are refluxed with water three times, with extraction times of 2 hours, 1 hour, and 1 hour respectively;

[0030] Step 3: Combine the three water extracts, concentrate under reduced pressure, add 95% ethanol to a final concentration of 80%, precipitate overnight at 4°C, centrifuge to discard the supernatant and collect the precipitate to obtain the ethanol precipitate;

[0031] Step 4: Add an appropriate amount of deionized water to the alcohol precipitate, stir thoroughly at room temperature until completely dissolved, centrifuge again, dialyze the supernatant, retain a molecular cutoff of 3500 Da, concentrate the dialysate and freeze dry to prepare the crude polysaccharide CDPs of Cistanche deserticola.

[0032] The prepared Cistanche deserticola crude polysaccharides (CDPs) were analyzed to determine their molecular weight and monosaccharide composition. The CDPs comprised polysaccharides in four molecular weight ranges, with the 11.2 kDa polysaccharide being the predominant component, followed by polysaccharides of 45.5 kDa, 4.0 kDa, and 1.8 kDa. They were primarily composed of Glc residues, with minor amounts of Rha, GalA, Gal, and Ara residues. Specifically, they mainly included (1→4)-Glcp, along with (1→4,6)-Glcp, t-Glcp, (1→4)-Galp, and t-Rhap residues.

[0033] Crude polysaccharides (CDPs) from Cistanche deserticola can be used to prepare drugs for the treatment or prevention of slow-transit constipation. Compared with traditional laxatives, bioactive natural compounds are valued for their fewer side effects and lower drug dependence. CDP treatment increased the level of antioxidant enzymes in the serum of constipated mice and reduced lipid peroxidation. Based on the antioxidant properties of CDPs, they can also protect colonic myenteric neurons.

[0034] CDPs treatment significantly reduced the levels of oxidative stress-related biomarkers in LMMP tissues and mitochondrial superoxide production in the myenteric plexus of the colon. Under physiological conditions, mitochondria are responsible for most of the ROS production and regulate cellular redox balance. Excessive ROS production is associated with damage to various cellular components, particularly mitochondrial DNA, due to its lack of introns and high transcription rate. ROS-induced oxidative stress damage leads to mitochondrial DNA mutations, affecting mitochondrial function and cellular processes. Furthermore, excessive ROS production may lead to insufficient ATP production, increased mitochondrial permeability, and decreased ΔΨ by opening the permeability transition pore, resulting in cellular dysfunction and even cell death. The results showed that CDPs treatment reduced ΔΨ dissipation in myenteric neurons of the colon in a dose-dependent manner, suggesting that CDPs provide neuroprotection in loperamide-induced STC mice.

[0035] nNOS neurons play a role in regulating intestinal peristalsis in the enteric nervous system. The nitric oxide (NO) they produce is an inhibitory neurotransmitter involved in the relaxation of intestinal smooth muscle. Previous studies have observed that oxidative stress can lead to increased nNOS expression; this upregulation may be related to the inability of neurons to maintain calcium levels. 2+ It is related to homeostasis. During cellular stress, intracellular calcium levels will increase. 2 + Influx of cytoplasmic Ca 2+ Elevated levels activate nNOS. Subsequently, NO release mediated by nNOS activation leads to colonic motility disorders in STC patients. Furthermore, the large amount of NO produced can react with superoxide anions to generate peroxynitrite, a highly reactive and oxidizing compound capable of causing oxidative damage and modifying biomolecules such as proteins, lipids, and DNA. Results showed that, compared to normal mice, STC mice had a higher proportion of nNOS-positive neurons in total neurons. However, CDPs treatment reduced the proportion of nNOS neurons in the colonic plexus, which may have effectively prevented the increase of intracellular oxidative stress, thereby alleviating cellular structural disorder and protecting cellular function. Simultaneously, reduced iNOS expression in LMMPs was observed in CDPs-treated mice, which may have led to reduced NO production, thus alleviating myenteric neuropathy of the colon.

[0036] CDPs are mainly composed of (1→4)-Glucan, with a small amount of pectin-like polysaccharides. Mouse model experiments showed that CDPs significantly affected defecation-related parameters, regulated the levels of intestinal regulatory peptides, and improved colonic pathological damage in loperamide-induced STC mice. Furthermore, CDPs protected colonic myenteric neurons by reducing oxidative stress damage and maintaining mitochondrial function. These data reveal that CDPs can alleviate constipation symptoms by improving oxidative stress-induced colonic myenteric neuropathy, providing a theoretical basis for the application of CDPs in the prevention and treatment of STC.

[0037] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0038] Example 1: Preparation of Cistanche deserticola polysaccharides (CDPs)

[0039] The *Cistanche deserticola* medicinal material was collected from the *Cistanche deserticola* planting base in Huiqin, Gansu Province, and the specimens are preserved in the Department of Pharmacognosy, School of Pharmacy, Tianjin Medical University.

[0040] Dried *Cistanche deserticola* medicinal material was pulverized and extracted three times by reflux with 70% ethanol solution for 2 hours, 1 hour, and 1 hour, respectively. The defatted material was then extracted three times by reflux with water for 2 hours, 1 hour, and 1 hour, respectively. The three aqueous extracts were combined, concentrated under reduced pressure, and 95% ethanol was added to a final concentration of 80%. The mixture was precipitated overnight at 4°C. After centrifugation, the supernatant was discarded, and the precipitate was collected. An appropriate amount of deionized water was added to the precipitate, and the mixture was stirred thoroughly at room temperature until completely dissolved. The mixture was centrifuged again, and the supernatant was dialyzed (molecular cutoff 3500 Da). The dialysate was concentrated and freeze-dried to obtain crude polysaccharides (CDPs) from *Cistanche deserticola*. The yield of crude polysaccharides (%) was calculated as (mass of crude polysaccharides from *Cistanche deserticola* / mass of *Cistanche deserticola* medicinal material) × 100%. The prepared product was weighed, and the mass of crude polysaccharides extracted from *Cistanche deserticola* was 55.5 g, with a calculated yield of 2.78%.

[0041] In studies on the polysaccharide content of Cistanche deserticola, the polysaccharide content obtained by different laboratories using the water extraction and alcohol precipitation method varies greatly. This is mainly due to differences in extraction temperature, extraction time, origin of the medicinal material, and measurement methods. In this example, the final crude polysaccharide obtained is the mass of water-soluble Cistanche deserticola polysaccharides after multiple centrifugations and dialysis to remove impurities.

[0042] Example 2: Study on the physicochemical characteristics of CDPs

[0043] The molecular weight distribution of CDPs was determined using HPGPC. Pullulan series standards (molecular weights of 642, 337, 194, 107, 47.1, 21.1, 9.6, and 6.1 kDa) were used as standards. 5 mg of each standard and sample were weighed and dissolved in ultrapure water to prepare 5 mg / mL pullulan standard and sample solutions, which were then filtered through a 0.22 μm filter membrane. Separately, 5 mg of the CDPs sample was weighed and dissolved in pure water to prepare a 5 mg / mL sample solution. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane to obtain the sample solution. 10 μL of each standard and sample solution were injected into the HPGPC system, eluting with CH3COONH4 solution as the mobile phase. A standard curve was plotted with retention time (A) on the ordinate and the logarithm of molecular weight (C) on the abscissa. The molecular weight distribution range of CPCDs was calculated using the standard curve.

[0044] Depend on Figure 1 As shown in A, the crude polysaccharides (CDPs) of Cistanche deserticola are mainly distributed in the range of 1.1-2743 kDa, and are mainly composed of polysaccharides in four molecular weight ranges (P1, 45.5 kDa; P2, 11.2 kDa; P3, 4.0 kDa; P4, 1.8 kDa), with the 11.2 kDa polysaccharide being the majority. In recent years, scholars have isolated several polysaccharides from Cistanche deserticola, all with molecular weights distributed within this range. For example, the molecular weight of arabinogalactan extracted from cold water soaking solution is 201 kDa, the average molecular weight of 1,4-linked glucan is 10 kDa, and the molecular weight of rhamnogalacturonic acid polysaccharide is 870 kDa. The molecular weights of three pectin-like polysaccharides (CDP-A, CDP-B, and CDP-C) isolated and purified from Cistanche deserticola by continuous membrane filtration are 400 kDa, 240 kDa, and 120 kDa, respectively. As can be seen, the molecular weight distribution of the isolated acidic polysaccharides is in the higher range, while that of the neutral dextran is mainly in the lower range. The CDPs isolated in this embodiment have a lower molecular weight.

[0045] Total sugar content was determined using the sulfuric acid-phenol method. A 1 mg / mL galactose standard solution was prepared and successively diluted to 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL. 0.2 mL of 5% phenol solution was added, followed by the rapid addition of 1.0 mL of concentrated sulfuric acid. After thorough shaking and standing, the absorbance was measured at 490 nm. A standard curve was plotted with concentration (C) on the x-axis and absorbance (A) on the y-axis. Dry samples were prepared into 100 μg / mL and 50 μg / mL solutions. After color development using the above method, the absorbance was measured. The results were substituted into the standard curve, and the average total sugar content of the Cistanche deserticola polysaccharide samples was calculated. The results are shown in Table 1.

[0046] The content of uronic acid was determined by the m-hydroxybiphenyl method. A 1 mg / mL galacturonic acid standard solution was prepared and diluted to a series of standard solutions of 100 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL. 200 μL of each galacturonic acid standard solution was added to a sulfuric acid-sodium tetraborate solution, cooled in an ice bath, and then heated in a 100℃ water bath for 5 min. After cooling, 20 μL of m-hydroxybiphenyl solution was added, and the mixture was shaken thoroughly and allowed to stand for 5 min. The absorbance was measured at 520 nm. A standard curve was plotted with concentration (C) on the x-axis and absorbance (A) on the y-axis. Dried Cistanche deserticola polysaccharide samples were prepared into 100 μg / mL and 50 μg / mL solutions. After color development using the above method, the absorbance was measured and the results were substituted into the standard curve to calculate the average uronic acid content of the Cistanche deserticola polysaccharide samples. The results are shown in Table 1.

[0047] Table 1

[0048]

[0049]

[0050] The structural characteristics of CDPs were initially determined by spectroscopic analysis, and the major functional groups of CDPs were preliminarily analyzed by FT-IR spectroscopy. 2 mg of the polysaccharide CPCD sample was taken, and 100 mg of dry KBr was added. The mixture was dried in a drying oven, then ground and mixed in an agate mortar. The mixture was then compressed into tablets using a hydraulic press. The compressed KBr tablets were then analyzed using an infrared spectrometer at 4000-400 cm⁻¹. -1 Infrared scanning is performed within the area. Figure 1 B is the infrared spectrum of CDPs, 3428 cm⁻¹. -1 and 2927cm -1 The absorption peaks at 1027 cm⁻¹ are attributed to the stretching vibrations of the OH group on the sugar ring and the CH stretching vibrations of the methylene group on the sugar ring, respectively, and are typical absorption peaks for polysaccharides. -1 The stretching vibration of COC on the sugar ring suggests the presence of the pyranose ring in CDPs. Additionally, at 1748 cm⁻¹... -1 The signal at the point is generated by the (C=O) vibration of the carbonyl group, indicating the presence of a certain amount of uronic acid, which is consistent with the test results of uronic acid content in the chemical composition.

[0051] Example 3: Analysis of monosaccharide composition and glycosidic bond composition of CDPs

[0052] The monosaccharide composition of CDPs was analyzed using a PMP derivatization combined with HPLC. 1 mg each of monosaccharide standards (Fuc, Xyl, Rha, Ara, Man, Gal, Glc, GlcA, GalA) and samples were accurately weighed and dissolved in deionized water (prepared to a concentration of 1 mg / mL). 0.3 mL of each sample was added to 0.3 mL of 4 mol / L TFA solution, and the mixture was heated at 120 °C for 2 h, then evaporated to dryness in a 40 °C water bath with N2. The solution was then dissolved again in 0.3 mL of deionized water and transferred to a screw-cap reaction tube. An equal volume of 0.6 mol / L NaOH solution and 0.6 mL of 0.5 mol / L PMP methanol solution were added, mixed thoroughly, and reacted in a metal bath at 70 °C in the dark for 30 min. After cooling to room temperature, the solution was adjusted to neutral with HCl. Extraction was performed 4-5 times with chloroform, and the chloroform layer was discarded. The solution was filtered through a 0.22 μm microporous membrane and transferred to an HPLC vial. Chromatographic conditions: isocratic elution, mobile phase A (CH3COONH4) 83%, mobile phase B (acetonitrile) 17%, total flow rate 1 mL / min, detection wavelength 245 nm, injection volume 5 μL, column temperature 30 ℃.

[0053] Depend on Figure 1 As shown in Table C and Table 1, CDPs are mainly composed of glucose (Glc) residues, with small amounts of rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), and arabinose (Ara) residues. These results suggest that Cistanche deserticola polysaccharides may primarily consist of glucan and pectin-like polysaccharides.

[0054] To further determine the type of glycosidic bond, the glycosidic bond composition of CDPs was analyzed based on the monosaccharide composition, following a series of steps including methylation, hydrolysis-reduction, and acetylation. The results are as follows: Figure 1 As shown in Table D and Table 2, the total ion chromatogram reveals that p2 is the main glycosidic bond component of CDPs, identified as a (1→4)-linked glucose residue ((1→4)-Glcp), accounting for 91.7% of the total. Other components include p1 ((t-Glcp), p3 ((1→4,6)-Glcp), (1→4)-Galp, and t-Rhap residues. These results suggest that CDPs are primarily composed of (1→4)-Glcuan, with branching at positions O-6.

[0055] Table 2

[0056]

[0057] Example 4: Effects of CDPs on constipation parameters in mice

[0058] 4.1 Animals and Grouping

[0059] Eight-week-old C57BL / 6 mice, weighing between 20±2g and free from specific pathogen infection, were used and provided by Beijing Huafukang Biotechnology Co., Ltd. All mice were housed in a temperature-controlled room with 12 / 12-hour light / dark cycles and provided with standard commercial rodent food and water. All animal husbandry and experimental protocols were strictly conducted in accordance with the guidelines of the China Animal Protection Committee and approved by the Animal Ethics and Welfare Committee of Tianjin Medical University General Hospital.

[0060] After a one-week acclimatization period, 25 mice were randomly divided into 5 groups (n=5 per group): normal control group (CON); loperamide-induced STC model group (LOP); loperamide + low-dose CDPs group (LOP+PL); loperamide + medium-dose CDPs group (LOP+PM); and loperamide + high-dose CDPs group (LOP+PH). Except for the CON group, mice in the other groups were orally administered loperamide 10 mg / kg twice daily for 14 consecutive days. During the loperamide gavage, mice were given different concentrations (100, 200, and 400 mg / kg) of CDPs, while the CON and LOP groups were orally administered an equal volume of physiological saline.

[0061] The general physiological condition and fecal condition of the mice were monitored daily. At the end of the animal experiment, the mice were anesthetized with ether and sacrificed, and serum and colon tissue were collected for further experimental analysis.

[0062] 4.2 Testing bowel-related indicators

[0063] Fourteen days after gavage administration, all mice were fasted for 24 hours and then given 0.2 ml of Indian ink (0.4 mg / ml, Shanghai Yuanye Biotechnology Co., Ltd.) to determine the time of first black feces excretion and assess total gastrointestinal transit time. Feces were collected from each mouse within 6 hours of Indian ink administration, and the number of fecal pellets and wet weight were recorded. The collected feces were dried in a 60°C oven until the weight remained constant, which was the dry weight. Fecal water content was calculated using the following formula: Fecal water content (%) = [(wet weight - dry weight) / wet weight] x 100.

[0064] Statistical analysis revealed that the LOP group exhibited significant constipation symptoms, including a reduced number of stool particles per 6 hours and decreased stool water content. Figure 2 AB, LOP group and CON group, P<0.0001). CDPs treatment effectively alleviated the above symptoms, and the relief effect in the LOP+PH group was more significant compared with the LOP+PL group (P<0.05). In addition, the time to excretion of the first black feces was prolonged in the LOP group, while CDPs shortened the defecation time in a dose-dependent manner. Figure 2 C, LOP+PH group and LOP+PL group, P<0.01).

[0065] To better investigate the effect of CDPs on alleviating constipation symptoms in mice, the levels of two peptides related to intestinal motility regulation in serum were measured. The results showed that the LOP group had the lowest SP level and the highest VIP level. However, after CDPs treatment, the changes in related peptide levels were significantly reversed. Figure 2 DE (P<0.01), among which the LOP+PH group showed the most significant therapeutic effect, with SP content increasing by 16.01 ng / L and VIP content decreasing by 12.18 ng / L. CDPs can significantly alleviate constipation symptoms in STC mice.

[0066] 4.3 Histopathological examination

[0067] Distal colon tissue was collected from mice, fixed in 10% paraformaldehyde solution for 2 days, and embedded in paraffin. The colon tissue was then cut into 5 μm thick sections and stained according to standard histological staining procedures using hematoxylin and eosin (H&E, Solarbio, Beijing, China) or Alcian blue (Solarbio, Beijing, China). Morphological changes were assessed under an optical microscope.

[0068] Distal colon tissue sections were stained with hematoxylin and eosin (HE) and alicin blue to assess the effects of different doses of CDPs on colonic morphology and goblet cell number changes. Results are as follows: Figure 3 As shown in Figures AB, the LOP group exhibited disordered epithelial cell arrangement and a less complete colonic mucosa, while CDPs treatment restored the integrity of the colonic mucosa. Furthermore, it was observed that the therapeutic effect significantly improved with increasing CDPs dosage, primarily manifested in a 400 mg / kg dose significantly restoring colonic muscle layer thickness compared to other groups. Figure 2 C, LOP+PH group and LOP+PL group, P<0.05) and increased goblet cell number ( Figure 2 D, LOP+PH group and LOP+PL group, P<0.0001). 4.4 Effects of CDPs on colonic myenteric neuron lesions

[0069] Blood was collected from the orbital margins of mice, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3500 rpm for 10 minutes at 4°C to collect the supernatant serum. The levels of substance P (SP) and vasoactive intestinal peptide (VIP) in the serum were detected using an ELISA kit (48T, Nanjing Senbega Biotechnology Co., Ltd., Nanjing, China) according to the manufacturer's instructions.

[0070] Total protein was extracted from LMMP using RIPA buffer containing protease inhibitors. Protein concentration in the samples was estimated using a BCA protein assay kit (Solarbio, Beijing, China). An equal volume of protein from each sample was diluted in protein loading buffer and heated to denature. The denatured proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% milk powder for 2 hours and then incubated overnight at 4°C with primary antibodies: anti-rabbit iNOS (1:1000, Affinity); Cox2 (1:1500, Affinity); and GAPDH (1:3000, Servicebio). The next day, the PVDF membrane was incubated at room temperature with secondary antibodies (anti-rabbit, 1:3000) for 40 minutes. Finally, Western blots were developed using ECL reagent and calculated using ImageJ software.

[0071] Loperamide-induced loss of colonic myenteric neurons in STC mice. To investigate whether CDPs have the potential to alleviate colonic myenteric neuronal lesions, neurons in LMMP preparations were labeled with β3-tubulin antibody.

[0072] Preparation of the longitudinal muscle and myenteric plexus (LMMP) preparation: Colonic tissue was immersed in oxyphosphate-buffered saline (PBS) and the intestinal lumen was rinsed to remove any remaining contents. The tissue was then stretched and fixed onto a silica plate, and the LMMP was separated from the other layers of the colon using ophthalmic forceps under a microscope to expose the myenteric plexus.

[0073] The LMMP preparation was fixed overnight at 4°C with 4% paraformaldehyde, then incubated in 0.5% Triton for 2 hours. It was blocked with 5% normal goat serum at room temperature for 2 hours, followed by overnight incubation at 4°C with primary antibodies: β3-tubulin (rabbit, 1:300, CellSignalling Technologies), HUC / D (mouse, 1:200, Invitrogen), and nNOS (rabbit, 1:200, CellSignalling Technologies). The next day, it was incubated for 1 hour at room temperature with specific secondary antibodies labeled with different fluorophores: goat-anti-rabbit IgG (1:100, Proteintech) and goat-anti-mouse IgG (1:100, Abclonal). Finally, the slides were mounted with anti-fluorescence quenching mounting medium and observed under a fluorescence microscope. Five regions were randomly selected from each sample, and the number of HuC / D+ and nNOS+ neurons was counted.

[0074] The results are as follows Figure 4A. CDPs improved the sparse density of colonic myenteric nerve fibers induced by loperamide. To quantify this improvement, neuronal cell bodies were labeled with the pan-neuronal antibody HuC / D and the number of neurons was counted. It was found that the number of neurons in the LOP group was significantly reduced compared to the normal group (P<0.0001), while CDPs alleviated the loss of colonic myenteric neurons in a dose-dependent manner. Figure 4 BC, LOP+PH group and LOP+PL group, P<0.01).

[0075] LOP Group nNOS + The number of neurons decreased ( Figure 4 Groups B, D, LOP and CON (P<0.01) but nNOS + The proportion of neurons in the total number of neurons was higher in the CON group than in the CON group. Figure 4 B, E, P < 0.0001). Following CDPs treatment, nNOS in the LOP+PH group... + The number of neurons increased (LOP+PH group vs. LOP group, P<0.01), almost reaching the level of the CON group. Meanwhile, the number of nNOS in the LOP+PL group... + The proportion of neurons decreased significantly (P<0.05).

[0076] Loperamide has been widely validated for its applicability and stability in inducing STC in animal models. All mice in the LOP group exhibited typical constipation phenotypes, while CDP treatment showed a dose-dependent recovery effect. These results demonstrate that CDPs have a significant effect in improving loperamide-induced STC in mice.

[0077] 4.5 Detection of antioxidant enzymes and lipid peroxidation

[0078] CDPs are natural antioxidants that effectively scavenge various free radicals. This example evaluates the ability of CDPs to resist loperamide-induced oxidative stress in colonic and LMMP-induced tissues.

[0079] A suitable amount of colon tissue was homogenized in lysis buffer and centrifuged to obtain the supernatant. The levels of antioxidant enzymes in the colon tissue were assessed using a Superoxide Dismutase (SOD) activity assay kit (Solarbio, Beijing, China) and a Reduced Glutathione (GSH) content assay kit (Solarbio, Beijing, China). The level of lipid peroxidation was assessed using a Malondialdehyde (MDA) content assay kit (Solarbio, Beijing, China). All experimental procedures were performed according to the manufacturer's instructions.

[0080] Quantitative polymerase chain reaction (q-PCR) analysis was performed. Total RNA was extracted and purified from LMMP according to the manufacturer's instructions (Jianshi Biosciences, Beijing, China), and the concentration and purity of RNA were determined using a spectrophotometer (Thermo Fisher Scientific Inc.). Reverse transcription was performed using FastKing gDNA Dispelling RT SuperMix (TIANGEN BIOTECH, Beijing, China). PCR amplification was then performed. Primer sequences are shown in Table 3. -ΔΔCT The method calculates the relative expression level of the target gene.

[0081] Table 3

[0082]

[0083] The results are as follows Figure 5 As shown in AC, compared with the LOP group, CDPs significantly increased SOD activity (P<0.001) and GSH content (P<0.05) and decreased MDA content (P<0.01) in colonic tissue, and the antioxidant capacity of CDPs increased with increasing dosage. Further investigation was conducted into the antioxidant capacity of CDPs in LMMP tissue. The oxidative stress-related protein iNOS (iNOS) was also observed in the treatment group. Figure 5 E, P<0.01) and Cox2( Figure 4 The levels of CDPs (P<0.05) were significantly lower in the LMMP group than in the LOP group. Furthermore, q-PCR analysis showed that the mRNA expression of inflammatory cytokines IL-1β (P<0.05) and TNF-α (P<0.0001) in LMMP tissues was significantly reduced after CDPs treatment. Figure 5 GH) proved that CDPs have anti-inflammatory properties.

[0084] 4.6 Assessment of mitochondrial superoxide production

[0085] Mito-derived superoxide in LMMP preparations was detected using the MitoSOX mitochondrial superoxide red fluorescent probe (YEASEN, Shanghai, China). To observe and quantify the production of mitochondrial superoxide in colonic myenterocytes, LMMP preparations were labeled with the novel fluorescent probe MitoSOX Red. The MitoSOX Red fluorescent probe can be oxidized by superoxide anions (O2-) within the mitochondria and emits red fluorescence.

[0086] The prepared sample was incubated at 37°C for 40 minutes with 5 μM MitoSOX indicator, fixed overnight with 4% paraformaldehyde at 4°C, and then incubated overnight the next day with β3-tubulin at 4°C. After incubation at room temperature for 1 hour with secondary antibody (goat anti-rabbit IgG, 1:100), images were taken using a fluorescence microscope. All images were taken under the same exposure conditions, and the average fluorescence intensity was calculated using ImageJ software.

[0087] The results are as follows Figure 6 As shown, the LOP group had the strongest fluorescence intensity. Figure 6 The results (A, C, P < 0.0001) indicate that loperamide exacerbated mitochondrial oxidative stress. After CDP treatment, the fluorescence intensity of MitoSOX showed a decreasing trend (P < 0.05), with the highest dose of CDPs significantly alleviating mitochondrial oxidative stress in colonic myenterocytes.

[0088] 4.7 Detection of changes in mitochondrial membrane potential

[0089] To further investigate the protective effect of CDPs against mitochondrial damage, changes in mitochondrial membrane potential (ΔΨ) were specifically detected using the JC-1 fluorescent probe. The mitochondrial membrane potential changes in colonic myenterocytes were assessed using a mitochondrial membrane potential detection kit (JC-1, Solarbio, Beijing, China), which reflects the extent of mitochondrial damage. Immediately after dissection, the LMMP preparation was incubated with JC-1 solution at 37°C for 20 minutes. It was then washed with JC-1 buffer (3 × 10 min) and imaged under a fluorescence microscope.

[0090] The results are as follows Figure 6 As shown, due to the transient depolarization of mitochondria caused by operation-related cellular stress, the fluorescence intensity of JC-1 increased in all groups. However, the increase in fluorescence intensity was more significant in the LOP group. Figure 6 B, D, LOP and CON groups, P<0.0001. Compared with the LOP group, CDP administration led to a dose-dependent decrease in JC-1 fluorescence intensity (LOP+PH group and LOP+PL group, P<0.05).

[0091] CDPs possess a dose-dependent ability to scavenge various free radicals, such as hydroxyl radicals, superoxide anion radicals, DPPH radicals, and ABTS radicals. CDPs can eliminate free radicals and inhibit lipid peroxidation. Furthermore, CDPs enhance endogenous antioxidant defense mechanisms, particularly by upregulating SOD activity and GSH levels. CDP treatment significantly reduced the levels of oxidative stress-related biomarkers in LMMP tissues and mitochondrial superoxide production in the myenteric plexus of the colon. CDP treatment reduced ΔΨ dissipation in myenteric neurons of the colon in a dose-dependent manner, suggesting that CDPs provide neuroprotection in loperamide-induced STC mice.

[0092] Compared to normal mice, STC mice exhibited a higher proportion of nNOS-positive neurons in their total neuronal count. However, CDPs treatment reduced the proportion of nNOS neurons in the colonic plexus, which may have effectively prevented the increase of intracellular oxidative stress, thereby alleviating cellular structural disorder and protecting cellular function. Simultaneously, experimental results also showed reduced iNOS expression in the LMMPs of CDPs-treated mice, which led to reduced NO production and thus alleviated myenteric neuropathy of the colon.

[0093] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made in accordance with the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. The application of Cistanche deserticola polysaccharide in the preparation of drugs for treating or preventing slow-transit constipation, characterized in that: Cistanche deserticola polysaccharides comprise four molecular weight segments, with the 11.2 kD polysaccharide being the predominant component. Others include polysaccharides with molecular weights of 45.5 kD, 4.0 kD, and 1.8 kD. They are primarily composed of Glc residues, with minor amounts of Rha, GalA, Gal, and Ara residues. The main component is (1→4)-Glc. p Additionally, there is (1→4,6)-Glc p t-Glc p (1→4)-Gal p and t-Rha p The residues; Cistanche deserticola polysaccharide contains 3.7% Rha, 4.9% GalA, 81.2% Glc, 7.1% Gal, and 3.1% Ara; the preparation method includes the following steps; Step 1: Take the dried Cistanche deserticola, crush it, add 70% ethanol solution and reflux extract three times, and then defatt it; Step 2: After defatting, the medicinal materials are refluxed with water three times for extraction; Step 3: Combine the three water extracts, add 95% ethanol to a final concentration of 80% for alcohol precipitation, and collect the precipitate; Step 4: The alcohol precipitate was dissolved in water, concentrated by dialyzing, and then freeze-dried. The molecular cutoff during dialyzing was 3500 Da, thus obtaining the crude polysaccharide CDPs of Cistanche deserticola. CDPs reduce the proportion of nNOS neurons in the colonic plexus, effectively preventing increased intracellular oxidative stress, alleviating cellular structural disorder, and protecting cellular function; they also reduce iNOS expression in the longitudinal muscle-myenteric plexus, thereby reducing NO production and alleviating myenteric neuropathy of the colon.

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