Bletilla striata polysaccharide capable of delaying senescence and preparation method of bletilla striata polysaccharide

Abstract

The invention relates to the field of medicines, in particular to rhizoma bletillae polysaccharide capable of delaying senescence and a preparation method thereof. The rhizoma bletillae polysaccharide is obtained by methods of water extraction, alcohol precipitation, deproteinization, membrane separation and the like. Particularly, the rhizoma bletillae polysaccharide is glucomannan composed of mannose and glucose, the ratio of the mannose to the glucose is 64.0: 30.7, the rhizoma bletillae polysaccharide also contains a small amount of galactose (3.0%), arabinose (1.0%), galacturonic acid (0.8%) and xylose (0.5%), and the weight-average molecular weight of the glucomannan is 159.8 kDa. In-vivo experiment results show that the bletilla striata polysaccharide has a good anti-aging effect, the preparation method is simple, non-toxic and pollution-free, and the bletilla striata polysaccharide can be industrially produced and has a wide market prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a Bletilla striata polysaccharide for delaying aging and a preparation method thereof. Background Art

[0002] Aging is defined as the continuous decline in body function and resilience over time, accompanied by an increased probability of illness and death. Aging results from a complex interplay between molecular events and biochemical pathways triggered by genetic and environmental factors. An important cause of accelerated aging and the onset of age-related diseases is the oxidative damage to cells caused by excessive reactive oxygen species (ROS). Excessive ROS exposure can damage cellular components such as proteins, lipids, and DNA, acting both directly as mutagens and indirectly as messengers. In addition, the accumulation of age-related stressors can lead to an imbalance in cellular homeostasis, ultimately accelerating the aging process.

[0003] Aging is associated with decreased food intake, impaired exercise capacity, and decreased reproductive capacity. With accelerated aging, the body's ability to neutralize ROS decreases, leading to a decrease in the activity of enzymes such as superoxide dismutase (SOD) and catalase (CAT), and a consequent weakening of antioxidant defenses.

[0004] Bletilla striata, also known as "Baiji" in Chinese, is a traditional Chinese medicinal herb of the Orchidaceae family. It has been widely used in ancient and modern China as a medicine for hemostasis, promoting wound healing and tissue regeneration, and alleviating inflammation. Bletilla striata polysaccharides (BSPs), the main component of Bletilla striata, possess various pharmacological properties, such as immunomodulatory, anti-aging, and anti-inflammatory effects. BSPs were found to possess significant antioxidant activity as determined by an in vitro 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) / ABTS free radical scavenging activity assay. BSPs have been shown to possess antioxidant capacity by inhibiting ROS production in mouse myoblasts and to have protective effects against Alzheimer's disease and lipopolysaccharide-induced sarcopenia in rats. BSPs also extend lifespan and improve locomotor behavior in Caenorhabditis elegans by activating the insulin / insulin-like growth factor (IGF) signaling pathway.

[0005] Bletilla striata itself contains a large amount of polysaccharides, but there are relatively few studies on Bletilla striata polysaccharides. The main reason is that Bletilla striata polysaccharides have poor water solubility, which limits their application in food and medicine. The water-soluble Bletilla striata polysaccharides prepared by the present invention through multiple technical means have outstanding effects on the fruit fly model and have good application prospects and development potential. Summary of the Invention

[0006] The present invention provides a Bletilla striata polysaccharide for delaying aging and a preparation method thereof. The present invention uses Bletilla striata as a raw material and is prepared through water extraction, alcohol precipitation, deproteinization, ultrafiltration and other process steps. The main purpose is to provide a natural and safe food or pharmaceutical raw material for delaying aging. The Bletilla striata polysaccharide of the present invention can effectively delay aging; the Bletilla striata polysaccharide of the present invention is safe, non-toxic and has no side effects; the present invention has a simple formula, low cost and significant effect.

[0007] In order to achieve the above-mentioned purpose, the present invention provides a Bletilla striata polysaccharide for delaying aging, wherein the monosaccharide composition of the Bletilla striata polysaccharide includes: mannose and glucose; the ratio of mannose to glucose is 64.0:30.7; and the weight average molecular weight of the Bletilla striata polysaccharide is 150-180 kDa.

[0008] Preferably, the monosaccharide composition of the Bletilla striata polysaccharide further comprises:

[0009] Galactose 3.0%;

[0010] Arabinose 1.0%;

[0011] Galacturonic acid 0.8%

[0012] and xylose 0.5%.

[0013] Preferably, the Bletilla striata polysaccharide is prepared by the following steps: obtaining a Bletilla striata extract, concentrating it, adding an organic solvent to form a precipitate, redissolving it after centrifugation, and adding an oxidant to deproteinize it.

[0014] Preferably, the Bletilla striata polysaccharide is used to prepare anti-aging food, cosmetics or anti-aging drugs.

[0015] Another aspect of the present invention provides a method for preparing Bletilla striata polysaccharide for delaying aging, comprising the following steps:

[0016] S1: extracting Bletilla striata with water once or multiple times to obtain a Bletilla striata extract;

[0017] S2: concentrating the Bletilla striata extract, adding an organic solvent to make the organic solvent concentration reach 60-90%, and centrifuging to obtain a precipitate;

[0018] S3: re-dissolving the precipitate in water, and then adding an oxidant to deproteinize, thereby obtaining an aqueous solution of a Bletilla striata extract;

[0019] S4: ultrafiltration is performed on the aqueous solution of the Bletilla striata extract through a 3.5K ultrafiltration membrane, and the retentate is collected and concentrated and dried to obtain Bletilla striata polysaccharide.

[0020] Preferably, in step S1, the water extraction temperature is 60-100°C, the number of extractions is 1-3 times, the extraction time is 0.5-3h, and the solid-liquid ratio is 5:1 to 30:1.

[0021] Preferably, in step S2, the organic solvent is selected from at least one of methanol, ethanol, propanol and acetone.

[0022] Preferably, in step S3, the oxidant is selected from one of hydrogen peroxide, peracetic acid, and sodium hypochlorite, the material ratio of the oxidant to Bletilla striata is 1:1 to 30:1, and the degradation time is 0.5 to 2 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a chromatogram of the monosaccharide composition of the Bletilla striata polysaccharide in Example 4 of the present invention;

[0024] Figure 2 is the chromatogram of the mixed standard solution of monosaccharides;

[0025] Figure 3 The figure shows the effects of different doses of Bletilla striata polysaccharide (BSP) on the lifespan of Drosophila in Example 5 of the present invention.

[0026] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0029] The experimental methods in the following examples, unless otherwise specified, are conventional methods; the test materials and test strains used in the following examples, unless otherwise specified, are purchased from commercial channels.

[0030] Example 1

[0031] Bletilla striata polysaccharide for delaying aging and preparation method thereof

[0032] 100g of Bletilla striata was extracted with 500g of H2O and extracted three times for 30 minutes each time at 80°C. The combined extracts were filtered through a 50-mesh sieve and concentrated. Ethanol was added to the concentrate to reach 90% alcohol content. The concentrate was centrifuged to obtain a precipitate. The precipitate was redissolved in water and 100g of hydrogen peroxide was added. The reaction was degraded for 0.5h at 30°C. The hydrolyzate was passed through a 3.5K ultrafiltration membrane, and the retentate was collected and concentrated to dryness to obtain Bletilla striata polysaccharide 1, whose molecular weight was measured to be 150-180kDa.

[0033] Example 2

[0034] Bletilla striata polysaccharide for delaying aging and preparation method thereof

[0035] 100 kg of Bletilla striata was extracted with 1000 kg of H2O and extracted twice, each time for 3 hours at 100°C. The combined extracts were filtered through a 50-mesh sieve and concentrated. Ethanol was added to the concentrate to increase the alcohol content to 60%. The concentrate was centrifuged to obtain a precipitate, which was redissolved in water and degraded with 1000 kg of peracetic acid at 45°C for 2 hours. After the reaction was completed, the hydrolyzate was passed through a 3.5K ultrafiltration membrane, and the retentate was collected and concentrated to dryness to obtain Bletilla striata polysaccharide 2, whose molecular weight was measured to be 150-180 kDa.

[0036] Example 3

[0037] Bletilla striata polysaccharide for delaying aging and preparation method thereof

[0038] 100g of Bletilla striata was added to 3000g of H2O and extracted for 2h at 60°C. The combined extracts were filtered through a 50-mesh sieve and concentrated. Ethanol was added to the concentrate to reach 65% alcohol content. The concentrate was centrifuged to obtain a precipitate. The precipitate was redissolved in water and 500g of hydrogen peroxide was added. The reaction was degraded for 1h at 65°C. The hydrolyzate was passed through a 3.5K ultrafiltration membrane, and the retentate was collected and concentrated to dryness to obtain Bletilla striata polysaccharide 3, whose molecular weight was measured to be 150-180kDa.

[0039] Example 4

[0040] Bletilla striata polysaccharide for delaying aging and preparation method thereof

[0041] 10 kg of Bletilla striata was taken and 100 kg of H2O was added. The extraction was carried out twice, each time for 1 hour at 100°C. The extracts were combined and filtered through a 50-mesh sieve and concentrated. Ethanol was added to the concentrate to make the alcohol content reach 70%. The concentrate was centrifuged to obtain a precipitate. The precipitate was redissolved in water and 100 kg of sodium hypochlorite was added. The reaction was degraded for 2 hours at 40°C. After the reaction was completed, the hydrolyzate was passed through a 3.5K ultrafiltration membrane, and the retentate was collected and concentrated to dryness to obtain Bletilla striata polysaccharide 4 (BSP).

[0042] The present invention selects the Bletilla striata polysaccharide 4 prepared in Example 4 for structural characterization, which includes the following steps:

[0043] (1) The monosaccharide composition determination method is as follows:

[0044] Ion chromatography was used to determine the monosaccharide composition of Bletilla striata polysaccharides. A 5 mg / mL solution was prepared, and 0.5 mL was placed in an ampoule. 0.5 mL of 4 M TFA solution was added, and the vial was sealed with an alcohol burner. Hydrolysis was performed in a 105°C oven for 6 hours, followed by drying with a nitrogen blower. Repeat this 3-5 times. A mixed standard sample was prepared to a 100 ppm solution.

[0045] Chromatographic conditions:

[0046] Chromatographic column: Dionex CarboPac™ PA1 BioLC™ 4×250 mm;

[0047] Mobile phase: A: H2O, B: 10 mM NaOH;

[0048] Column temperature: 35°C;

[0049] Flow rate: 0.8 mL / min;

[0050] Injection volume: 25 μL;

[0051] Detector: Ampere detector;

[0052] Data collection time: 90 minutes.

[0053] The results of the 4 monosaccharide compositions of Bletilla striata polysaccharide are shown in Figure 1 Bletilla striata polysaccharide 4 is a glucomannan composed of mannose and glucose, with a mannose-glucose ratio of 64.0:30.7. It also contains small amounts of galactose (3.0%), arabinose (1.0%), galacturonic acid (0.8%), and xylose (0.5%).

[0054] (2) The molecular weight determination method is as follows:

[0055] The sample was prepared into a 10 mg / mL solution using the mobile phase and filtered through a 0.22 μm filter membrane to detect the molecular weight of BSP.

[0056] Chromatographic conditions:

[0057] Column: TSK-GEL GMPWXL;

[0058] Mobile phase: 0.1 M NaNO3;

[0059] Column temperature: 30°C;

[0060] Flow rate: 1 mL / min;

[0061] Injection volume: 20 μL;

[0062] Detector: differential detector, multi-angle laser detector;

[0063] Data collection time: 30 minutes.

[0064] The molecular weight of Bletilla striata polysaccharide 4 is 159.8 kDa.

[0065] Example 5

[0066] Anti-aging effect experiment

[0067] Experimental drug: Bletilla striata polysaccharide 4 (BSP), prepared in Example 4.

[0068] Experimental animals: w 1118 Drosophila (Drosophila breeding center (BDSC), Bloomington, Indiana, USA).

[0069] Experimental method: 0.5mg / ml, 3.0mg / ml, 9.0mg / ml and 18mg / ml were selected to optimize the BSP concentration. Fruit flies were separated by sex and grouped, with at least five groups of each sex. Each group consisted of 150 flies, housed in five plastic tubes, with 30 flies per tube. Deaths were counted every two days, and surviving flies were transferred to new tubes. Lifespan curves were statistically analyzed using GraphPad Prism software.

[0070] Experimental results: In order to exclude the toxicity of BSP and screen the optimal BSP concentration for extending the lifespan of Drosophila, we studied the effects of BSP supplementation at different concentrations (0.5, 3, 9, and 18 mg / ml) on the lifespan of Drosophila. We found that compared with the control group, the lifespan of male Drosophila fed with 0.5 mg / ml and 3 mg / ml BSP was significantly prolonged (p<0.05, p<0.01), while the lifespan of male Drosophila fed with higher concentrations of BSP was significantly shortened ( Figure 3 ). Figure 3 (A) is the male survival curve; (B) is the female survival curve; data are expressed as mean ± SEM, *p < 0.05, **p < 0.01 and ***p < 0.001. The mean lifespan, median lifespan and maximum lifespan of male fruit flies supplemented with 0.5 mg / ml BSP were extended by 5.88%, 8.33% and 5.88%, respectively (p < 0.05) (Table 1). Similarly, when treated with 3 mg / ml BSP, the lifespan was extended by 14.32%, 18.75% and 11.76%, respectively, compared with the control group (p < 0.001) (Table 1). At the same time, the lifespan of female fruit flies was extended only at 3 mg / ml BSP and shortened at higher concentrations ( Figure 3The mean lifespan, median lifespan and maximum lifespan of female fruit flies taking 3 mg / ml BSP were significantly prolonged by 7.17%, 4.34% and 2.94% respectively (p<0.05).

[0071] Table 1 Effects of Bletilla striata polysaccharide 4 on the lifespan of w1118 male Drosophila

[0072]

[0073] Note: 150 flies were used in each group. *p<0.05, ***p<0.001.

[0074] Table 2 Effects of Bletilla striata polysaccharide 4 on the lifespan of w1118 female Drosophila melanogaster

[0075]

[0076] Note: 150 flies were used for each group. ns p>0.05, *p<0.05

[0077] Experimental conclusion: At the tested dose, supplementation with Bletilla striata polysaccharide 4 (BSP) can delay the aging of fruit flies, and with the increase of the dosage, it shows a good dose-dependent relationship; therefore, Bletilla striata polysaccharide 4 (BSP) can be used as a raw material to prepare foods, cosmetics or anti-aging drugs that delay aging.

Claims

1. A Bletilla striata polysaccharide for delaying aging, characterized in that: The monosaccharide composition of the Bletilla striata polysaccharide includes: mannose and glucose; the ratio of mannose to glucose is 64.0:30.7; and the weight average molecular weight of the Bletilla striata polysaccharide is 150-180 kDa.

2. The Bletilla striata polysaccharide according to claim 1, characterized in that The monosaccharide composition of the Bletilla striata polysaccharide also includes: Galactose 3.0%; Arabinose 1.0%; Galacturonic acid 0.8% and xylose 0.5%.

3. The Bletilla striata polysaccharide according to claim 1, characterized in that The Bletilla striata polysaccharide is prepared by the following steps: obtaining Bletilla striata extract, concentrating, adding an organic solvent to form a precipitate, redissolving after centrifugation, and adding an oxidant to deproteinize.

4. The Bletilla striata polysaccharide according to claim 1, characterized in that The bletilla striata polysaccharide is used for preparing anti-aging food, cosmetics or anti-aging medicine.

5. A method for preparing Bletilla striata polysaccharide for delaying aging, characterized in that: The following steps are involved: S1: extracting Bletilla striata with water once or multiple times to obtain a Bletilla striata extract; S2: concentrating the Bletilla striata extract, adding an organic solvent to make the organic solvent concentration reach 60-90%, and centrifuging to obtain a precipitate; S3: re-dissolving the precipitate in water, and then adding an oxidant to deproteinize, thereby obtaining an aqueous solution of a Bletilla striata extract; S4: ultrafiltration is performed on the aqueous solution of the Bletilla striata extract through a 3.5K ultrafiltration membrane, and the retentate is collected and concentrated and dried to obtain Bletilla striata polysaccharide.

6. The preparation method according to claim 5, characterized in that In step S1, the water extraction temperature is 60-100° C., the number of extractions is 1-3 times, the extraction time is 0.5-3 hours, and the material-liquid ratio is 5:1 to 30:

1.

7. The preparation method according to claim 5, characterized in that In step S2, the organic solvent is selected from at least one of methanol, ethanol, propanol and acetone.

8. The preparation method according to claim 5, characterized in that In step S3, the oxidant is selected from one of hydrogen peroxide, peracetic acid, and sodium hypochlorite, the material ratio of the oxidant to Bletilla striata is 1:1 to 30:1, and the degradation time is 0.5 to 2 hours.

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