Lithospermum oligosaccharides, their preparation methods and applications

Abstract

This invention belongs to the field of pharmaceutical technology, and particularly relates to a lithospermum oligosaccharide, its preparation method, and its application. The lithospermum oligosaccharide is shown in structural formula I or II: I II. This invention also provides a method for preparing the lithospermum oligosaccharide. The lithospermum oligosaccharide compound of this invention can effectively reduce the level of inflammatory factors and significantly enhance immunity, providing an important material basis for the development of novel, efficient, and easily absorbed immunomodulators. The lithospermum oligosaccharide prepared by the method of this invention has high purity, is convenient and simple, and is suitable for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to an oligosaccharide of Lithospermum erythrorhizon, its preparation method and application. Background Technology

[0002] Lithospermum erythrorhizon is the root of *Arnebia euchroma* (Royle) Johnst. or *Arnebia guttata* Bunge., both belonging to the Boraginaceae family. Lithospermum erythrorhizon has a long history of use in my country as a traditional Chinese medicine. It is cold in nature, sweet and salty in taste, and enters the heart and liver meridians. It has the effects of cooling the blood, promoting blood circulation, detoxifying, and relieving rashes. Clinically, it is often used to treat conditions such as blood heat and toxicity, purplish-black rashes, incomplete measles eruption, sores, eczema, and burns.

[0003] With the continuous advancement of modern medical research, numerous studies have shown that the sugar components in Lithospermum erythrorhizon are one of the material bases for its important biological activities, exhibiting significant activities in immune regulation, antioxidation, anti-inflammation, hypoglycemia, and antidepressant effects, with broad application prospects. However, as macromolecules, Lithospermum erythrorhizon polysaccharides generally suffer from problems such as high molecular weight, poor water solubility, and high solution viscosity, resulting in low absorption and utilization rates in vivo and limited bioavailability, which seriously restricts its clinical translation and practical application.

[0004] Many biological activities of polysaccharides, especially their immunomodulatory effects, often originate from oligosaccharide fragments with specific conformations in their structure. However, research on the subdivision of oligosaccharides derived from Lithospermum erythrorhizon, their preparation, and their immunomodulatory functions remains largely unexplored. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a comfrey oligosaccharide, its preparation method and application, which can obtain a comfrey oligosaccharide with a well-defined structure, and the obtained comfrey oligosaccharide can also have anti-inflammatory and immunomodulatory effects.

[0006] To achieve the above objectives, the present invention provides a comfrey oligosaccharide, as shown in structural formula I or II:

[0007]

[0008] I II.

[0009] Of the aforementioned oligosaccharides of Lithospermum erythrorhizon, the molecular weight of the compound represented by Formula I is 666 Da; and the molecular weight of the compound represented by Formula II is 692 Da.

[0010] The present invention also provides a method for preparing the above-mentioned oligosaccharides from Lithospermum erythrorhizon, comprising the following steps:

[0011] Step 1: Use petroleum ether to perform ultrasonic extraction on Lithospermum erythrorhizon. Take the extracted Lithospermum erythrorhizon to obtain defatted Lithospermum erythrorhizon medicinal material. The material-liquid ratio of Lithospermum erythrorhizon to petroleum ether is 1:(5-15).

[0012] Step 2: The defatted Lithospermum erythrorhizon herb is percolated with ethanol to obtain percolate; the percolate is concentrated and then deproteinized with trichloroacetic acid and neutralized with NaOH. The neutralized percolate is then passed through a macroporous resin column and eluted with distilled water as the eluent; the eluted fractions are collected and monitored by TLC; the eluted fractions containing oligosaccharides are combined, concentrated, and dried to obtain the crude oligosaccharide fraction of Lithospermum erythrorhizon.

[0013] Step 3: The crude oligosaccharide fraction of Lithospermum erythrorhizon was first subjected to silica gel column chromatography, using ethyl acetate-methanol as the eluent. Then, the 0:100 ethyl acetate-methanol elution fraction was subjected to Sephadex LH-20 gel column chromatography, using a methanol-water elution system. TLC was used for tracking and detection to separate the two oligosaccharides of Lithospermum erythrorhizon represented by Formula I and II.

[0014] In the above preparation method, preferably, in the step of concentrating the percolate, the percolate is concentrated until there is no alcohol odor.

[0015] In the above preparation method, preferably, the concentration of NaOH is 1 mol / L.

[0016] In the above preparation method, preferably, NaOH is used for neutralization, and the pH value is neutralized to 7.0.

[0017] In the above preparation method, step one is a defatting step; step two is an extraction step; and step three is a purification step.

[0018] In the above preparation method, preferably, the step of ultrasonic extraction of Lithospermum erythrorhizon using petroleum ether involves taking dried Lithospermum erythrorhizon, pulverizing it, and then ultrasonically extracting the pulverized Lithospermum erythrorhizon using petroleum ether.

[0019] In the above preparation method, ultrasonic extraction can be a commonly used ultrasonic extraction method, and preferably, the ultrasonic extraction temperature is room temperature.

[0020] In the above preparation method, preferably, the ultrasonic extraction is performed 2-5 times, and the extraction time for each extraction is 0.2-1 hour; more preferably, the ultrasonic extraction is performed 3 times, and the extraction time for each extraction is 0.5 hours.

[0021] In the above preparation method, preferably, the ratio of comfrey to petroleum ether is 1:10. The ratio refers to the proportion of material to liquid, and here it means that the ratio of comfrey to petroleum ether is 1:(5-15), preferably 1:10.

[0022] In the above preparation method, preferably, the material-liquid ratio is a weight ratio or a volume ratio.

[0023] In the above preparation method, TLC is used for tracking and detection, which is a common colorimetric detection method for sugars in chemical detection.

[0024] In the above preparation method, the eluted components containing oligosaccharides are combined, concentrated, and dried to fully concentrate and dry the eluted oligosaccharides, thereby obtaining dry solid crude oligosaccharide components of Lithospermum erythrorhizon.

[0025] In the above preparation method, the concentration of ethanol is 50%-95%; preferably, the concentration of ethanol is 70%; and the concentration of trichloroacetic acid is 10%-20%.

[0026] In the above preparation method, preferably, the macroporous resin column is an XAD-16N macroporous resin column.

[0027] In the above preparation method, preferably, the drying is freeze drying.

[0028] In the above preparation method, preferably, the step of using ethyl acetate-methanol as the eluent involves gradient elution using ethyl acetate-methanol in a ratio of 100:0 to 0:100.

[0029] In the above preparation method, preferably, the step of eluting using a methanol-water elution system involves gradient elution using methanol and water at a ratio of 100:0 to 0:100.

[0030] In the above preparation method, preferably, the TLC method is used for tracking and detection, which means using TLC (GF254 thin-layer plate, sulfuric acid-vanillin colorimetric method) to track and detect the elution segments that are displayed as a single point at the same position on the GF254 thin-layer plate and then merging them; that is, to obtain the two lithospermum oligosaccharides shown in Formula I and II.

[0031] In the above-described preparation method, preferably, the method further includes a step of detecting and verifying the separated components by mass spectrometry and / or nuclear magnetic resonance. This step involves identifying the structure of the elution fraction that appears as a single point at the same location on a GF254 thin-layer plate using MS and / or NMR analysis, and / or comparison with literature data; these fractions can be confirmed as the oligosaccharides of lithospermum erythrorhizon represented by Formula I and Formula II, respectively.

[0032] The present invention also provides a composition comprising at least one of the oligosaccharides of formula I or formula II described above.

[0033] The present invention also provides the use of comfrey oligosaccharide or a combination thereof in the preparation of anti-inflammatory products.

[0034] The present invention also provides the use of comfrey oligosaccharide or a combination thereof in the preparation of immunomodulatory products.

[0035] As can be seen from the above, the technical solution provided by the present invention has the following significant beneficial effects:

[0036] This invention provides two unique comfrey oligosaccharide compounds that can effectively reduce inflammatory factor levels and significantly enhance immunity, providing an important material basis for the development of novel, efficient, and easily absorbed immunomodulators. The comfrey oligosaccharide preparation method of this invention produces comfrey oligosaccharides with high purity, is convenient and simple, and is suitable for industrial application. Attached Figure Description

[0037] Figure 1a The oligosaccharide AEO-1 of comfrey in Example 1 1 H NMR spectrum

[0038] Figure 1b The oligosaccharide AEO-1 of comfrey in Example 1 13 C10 NMR spectrum

[0039] Figure 2a The oligosaccharide AEO-2 of comfrey in Example 1 1 H NMR spectrum

[0040] Figure 2b The oligosaccharide AEO-2 of comfrey in Example 1 13 C10 NMR spectrum

[0041] Figure 3 This is a parallel control diagram showing the levels of IL-1β, IL-6, and TNF-α in mouse serum among the normal group, model group, AEO-1 group, and AEO-2 group in Example 1.

[0042] Figure 4a These are fluorescence micrographs comparing the neutrophils in the tails of zebrafish juveniles from the normal group, model group, positive group, AEO-1 group, and AEO-2 group in Example 1.

[0043] Figure 4b This is a comparison of neutrophil counts in the tails of zebrafish juveniles in Example 1 among the normal group, model group, positive group, AEO-1 group, and AEO-2 group.

[0044] Figure 5a These are comparative micrographs of macrophages in the heads of zebrafish juveniles from the normal group, model group, positive group, AEO-1 group, and AEO-2 group in Example 1.

[0045] Figure 5bThis is a comparison of the number of macrophages in the heads of zebrafish juveniles in Example 1, including the normal group, model group, positive group, AEO-1 group, and AEO-2 group. Detailed Implementation

[0046] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described, unless otherwise specified, are commercially available.

[0047] Example 1

[0048] This embodiment provides two oligosaccharide compounds of Lithospermum erythrorhizon, as shown in structural formula I or II:

[0049]

[0050] I II.

[0051] The two structural oligosaccharide compounds of comfrey in this embodiment were prepared by the following method:

[0052] Step 1: Take 1 kg of dried Lithospermum erythrorhizon, crush it, and then use petroleum ether (10 kg) at a material-to-liquid ratio (by weight) of 1:10 to perform ultrasonic extraction on the crushed Lithospermum erythrorhizon 3 times, each extraction lasting 0.5 hours; take the extracted Lithospermum erythrorhizon to obtain defatted Lithospermum erythrorhizon medicinal material.

[0053] Step 2: The defatted Lithospermum erythrorhizon herb was percolated with 70% ethanol to obtain percolate. The percolate was concentrated until there was no alcohol odor, and then deproteinized with 15% trichloroacetic acid and neutralized to pH 7.0 with 1 mol / L NaOH. The neutralized percolate was then eluted through an XAD-16N macroporous resin column with distilled water as the eluent. The eluted fractions were collected and monitored by TLC. The eluted fractions containing oligosaccharides were combined, concentrated, and dried to obtain the crude oligosaccharide fraction of Lithospermum erythrorhizon.

[0054] Step 3: The obtained crude oligosaccharide fraction of Lithospermum erythrorhizon was first subjected to silica gel column chromatography using an ethyl acetate-methanol elution system with a gradient elution ratio of 100:0 to 0:100. Then, the 0:100 ethyl acetate-methanol elution fraction was subjected to Sephadex LH-20 gel chromatography using a methanol-water elution system with a gradient elution ratio of 100:0 to 0:100. TLC was used for tracking and detection; on a GF254 thin-layer plate, each oligosaccharide was identified as a single spot. The two oligosaccharides of Lithospermum erythrorhizon, represented by Formulas I and II, were obtained after separation and purification. The purities of the two oligosaccharides were 96% and 95%, respectively.

[0055] For ease of representation, the two lithospermum oligosaccharides shown in Formula I and Formula II are named as follows: the lithospermum oligosaccharide shown in Formula I is named AEO-1; the lithospermum oligosaccharide shown in Formula II is named AEO-2.

[0056] The structures of the two comfrey oligosaccharides obtained in this embodiment were characterized and verified:

[0057] (1) Monosaccharide composition analysis: The monosaccharide composition of the oligosaccharides AEO-1 and AEO-2 from Lithospermum erythrorhizon was determined by HPLC-RID after acid hydrolysis. 3 mg of oligosaccharide sample was weighed, dissolved in 1 mL of 2 mol / L TFA, and hydrolyzed in an oil bath at 100℃ for 2 h. After complete acid hydrolysis, a small amount of methanol was added, and the mixture was evaporated under reduced pressure at 55℃. This process was repeated 4-5 times to remove all TFA. Then, 200 μL of distilled water was added to reconstitute the solution, filtered through a 0.22 μm organic phase filter membrane, and analyzed by HPLC-RID. 3 mg each of fructose and glucose monosaccharide standards were weighed and prepared into a 3 mg / mL monosaccharide standard solution, which was then analyzed by HPLC-RID along with the oligosaccharide sample. The results showed that both AEO-1 and AEO-2 were composed of glucose and fructose.

[0058] (2) Infrared spectroscopy analysis: Take about 1 mg each of dried oligosaccharide samples AEO-1 and AEO-2, compress them into KBr pellets, and incubate them at 4000 ~ 400 cm⁻¹. -1 Infrared spectral scanning was performed within the specified range. The results showed that the infrared spectrum was within the range of 3385.02 cm⁻¹. -1 The peak at 2943.33 cm⁻¹ shows the stretching vibration of the OH group in sugar molecules. -1 The peak observed is the CH stretching vibration absorption peak at 1406.09 cm⁻¹. -1 The peak at 1060.83 cm⁻¹ represents the alternating angular vibration of the CH₂-OH ring of fructose. -1 It is the characteristic absorption peak of the CO stretching vibration of the sugar ring, at 869.88 cm⁻¹. -1 and 821.66 cm -1 These are the characteristic absorption peaks of the stretching and angular vibrations of the CH bonds in the furanose ring, respectively, at 781.16 cm⁻¹. -1 The absorption peaks are symmetric stretching vibrations of COC on the sugar ring, suggesting that the AEO-1 and AEO-2 structures contain characteristic absorption peaks of furanose and pyranose rings.

[0059] (3) Nuclear magnetic resonance (NMR) measurement: Oligosaccharide AEO-1 and AEO-2 samples were dissolved in D2O and NMR measurements were performed. 1 H NMR, 13 C NMR, DEPT-135 1 H- 1 H COSY、 1 H- 1H TOCSY, HSQC, HSQC-TOCSY, HMBC, and ROESY). (e.g.) Figure 1a , 1b As shown in Figures 2a and 2b, the spectral analysis results are the chemical structural formulas I and II of the aforementioned Lithospermum erythrorhizon oligosaccharide.

[0060] The efficacy of the oligosaccharides AEO-1 and AEO-2 from the comfrey plant in this embodiment was verified:

[0061] Efficacy Verification 1: The effect of AEO-1 and AEO-2 on improving immune function in immunosuppressed mice

[0062] Effects of AEO-1 and AEO-2 on Spleen and Thymus Indices in Immunosuppressed Mice: Fifty mice were randomly divided into five groups of ten each: a normal control group, a model group (cyclophosphamide, 80 mg / kg), a positive control group (levamisole hydrochloride, 40 mg / kg), an AEO-1 group (10 mg / kg), and an AEO-2 group (10 mg / kg). After three days of acclimatization, except for the normal control group which received intraperitoneal injections of saline, the other groups received intraperitoneal injections of cyclophosphamide once daily for three consecutive days starting on day 2 to establish an immunosuppressed mouse model. The AEO-1 and AEO-2 groups received intraperitoneal injections once daily for seven consecutive days after successful model establishment. After the experiment, the mice in each group were weighed, blood was collected from the eyeballs, and the thymus and spleen were collected. The organ indices of the thymus and spleen were calculated using the formula: Organ Index = m 胸腺 / 脾 / m 体质量 .

[0063] Table 1 shows that, compared with the control group, the spleen index and thymus index of the model group mice were significantly decreased (P < 0.0001); compared with the model group, the spleen index and thymus index of the AEO-1 and AEO-2 groups were significantly increased (P < 0.001, P < 0.0001). The results indicate that AEO-1 and AEO-2 have a significant enhancing effect on the immune function of immunocompromised mice and play a protective role against damaged immune organs.

[0064] Table 1. Effects of AEO-1 and AEO-2 on thymus and spleen indices in immunocompromised mice (n=10)

[0065]

[0066] Compared with the blank group: * P < 0.001, ** P < 0.0001;

[0067] Compared with the model group: # P < 0.001, ## P < 0.0001

[0068] Effects of AEO-1 and AEO-2 on serum inflammatory factor levels in immunosuppressed mice: Mouse blood was allowed to stand at room temperature for 2 h, centrifuged at 3000 r / min for 15 min, and serum was obtained. The levels of IL-1β, IL-6, and TNF-α in the serum were detected using an ELISA kit. Figure 3 As shown, Figure 3 This is a parallel control diagram showing the serum IL-1β, IL-6, and TNF-α levels in mice from the normal group, model group, AEO-1 group, and AEO-2 group. The experiment was conducted in triplicate. The results showed that compared with the normal group, the serum IL-1β, IL-6, and TNF-α levels in the model group were significantly increased (P < 0.0001); compared with the model group, the serum IL-1β, IL-6, and TNF-α levels in the AEO-1 and AEO-2 groups were decreased (P < 0.0001).

[0069] Efficacy Verification 2: Effects of Lithospermum erythrorhizon oligosaccharides AEO-1 and AEO-2 on the number of immune cells in zebrafish

[0070] This embodiment validated the effects of immunofluorescence-labeled transgenic Tg(lyz:DsRed) zebrafish and wild-type AB zebrafish on the number of neutrophils and macrophages in zebrafish. Healthy juvenile zebrafish were randomly transferred to 24-well plates (20 juveniles per well) at day 2 post-fertilization (dpf). The juveniles were divided into four groups: normal group, model group (rapamycin, RAPA, 10 μg / mL), positive control group (levamisole hydrochloride, LH, 80 μg / mL), AEO-1 group (50 μM), and AEO-2 group (50 μM). The cells were cultured for 24 h in a 28°C incubator (light / dark: 14 h / 10 h). Transgenic zebrafish juveniles (Tg(lyz:DsRed)) were collected, anesthetized, and placed on agarose gel. Neutrophils in the tail region of each group of juvenile zebrafish were observed and photographed using a stereomicroscope. The fluorescence intensity of the tail neutrophils was quantified using ImageJ. The results are as follows: Figure 4a , 4b As shown. Wild-type AB zebrafish juveniles were collected and washed twice on E3 medium. Then, 1 mL of 10 mg / L neutral red solution (PTU final concentration 0.2 mmol / L) was added, and the fish were cultured and stained in a 28℃ incubator in the dark for 6 hours. The solution was discarded, the fish were anesthetized, and photographed using a stereomicroscope. The number of macrophages in the head of each group of zebrafish was recorded. The results are shown below. Figure 5a , 5b As shown.

[0071] Depend on Figure 4a , 4b and Figure 5a , 5b As can be seen, compared with the normal group, the number of macrophages and neutrophils in the zebrafish in the model group decreased by 28.82% and 19.76%, respectively. Compared with the model group, after AEO-1 and AEO-2 treatment, the number of macrophages in zebrafish increased significantly by 79.28% and 83.78%, respectively, and the number of neutrophils increased by 75.00% and 106.1%, respectively.

[0072] Example 2

[0073] This embodiment provides two oligosaccharide compounds of Lithospermum erythrorhizon, as shown in structural formula I or II:

[0074]

[0075] I II.

[0076] The two structural oligosaccharide compounds of comfrey in this embodiment were prepared by the following method:

[0077] Step 1: Take 1 kg of dried Lithospermum erythrorhizon, crush it, and then use petroleum ether (5 kg) at a material-to-liquid ratio (by weight) of 1:5 to perform ultrasonic extraction twice, each time for 1 hour; take the extracted Lithospermum erythrorhizon to obtain defatted Lithospermum erythrorhizon medicinal material.

[0078] Step 2: Percolation extraction of defatted Lithospermum erythrorhizon material was performed using 50% ethanol to obtain percolate. The percolate was concentrated and then deproteinized with 20% trichloroacetic acid and neutralized to pH 7.0 with 1 mol / L NaOH. The neutralized percolate was then eluted through an XAD-16N macroporous resin column using distilled water as the eluent. The eluted fractions were collected and monitored by TLC. The eluted fractions containing oligosaccharides were combined, concentrated, and dried to obtain the crude oligosaccharide fraction of Lithospermum erythrorhizon.

[0079] Step 3: The obtained crude oligosaccharide fraction of Lithospermum erythrorhizon was first subjected to silica gel column chromatography using an ethyl acetate-methanol elution system with a gradient elution ratio of 100:0 to 0:100. Then, the 0:100 ethyl acetate-methanol elution fraction was subjected to Sephadex LH-20 gel chromatography using a methanol-water elution system with a gradient elution ratio of 100:0 to 0:100. TLC was used for tracking and detection; on a GF254 thin-layer plate, each oligosaccharide was identified as a single spot. The two oligosaccharides of Lithospermum erythrorhizon, represented by Formulas I and II, were obtained after separation and purification. The purities of the two oligosaccharides were 97% and 97%, respectively.

[0080] The two oligosaccharides of Lithospermum obtained in the examples were characterized and verified. The spectral conclusions are shown in Example 1. The spectral analysis results are the above-mentioned chemical structural formulas I and II of the Lithospermum oligosaccharides.

[0081] Example 3

[0082] This embodiment provides two oligosaccharide compounds of Lithospermum erythrorhizon, as shown in structural formula I or II:

[0083]

[0084] I II.

[0085] The two structural oligosaccharide compounds of comfrey in this embodiment were prepared by the following method:

[0086] Step 1: Take 1 kg of dried Lithospermum erythrorhizon, crush it, and then use petroleum ether (15 kg) at a material-to-liquid ratio (by weight) of 1:15 to perform ultrasonic extraction on the crushed Lithospermum erythrorhizon 5 times, each extraction lasting 0.2 hours; take the extracted Lithospermum erythrorhizon to obtain defatted Lithospermum erythrorhizon medicinal material.

[0087] Step 2: Percolation extraction of defatted Lithospermum erythrorhizon material was performed using 85% ethanol to obtain percolate. The percolate was concentrated and then deproteinized with 10% trichloroacetic acid and neutralized to pH 7.0 with 1 mol / L NaOH. The neutralized percolate was then eluted using an XAD-16N macroporous resin column with distilled water as the eluent. The eluted fractions were collected and monitored by TLC. The eluted fractions containing oligosaccharides were combined, concentrated, and dried to obtain the crude oligosaccharide fraction of Lithospermum erythrorhizon.

[0088] Step 3: The obtained crude oligosaccharide fraction of Lithospermum erythrorhizon was first subjected to silica gel column chromatography using an ethyl acetate-methanol elution system with a gradient elution ratio of 100:0 to 0:100. Then, the 0:100 ethyl acetate-methanol elution fraction was subjected to Sephadex LH-20 gel chromatography using a methanol-water elution system with a gradient elution ratio of 100:0 to 0:100. TLC was used for tracking and detection; on a GF254 thin-layer plate, each oligosaccharide was identified as a single spot. The two oligosaccharides of Lithospermum erythrorhizon, represented by Formulas I and II, were obtained after separation and purification. The purities of the two oligosaccharides were 97% and 97%, respectively.

[0089] The two oligosaccharides of Lithospermum obtained in the examples were characterized and verified. The spectral conclusions are shown in Example 1. The spectral analysis results are the above-mentioned chemical structural formulas I and II of the Lithospermum oligosaccharides.

[0090] Example 4

[0091] This embodiment provides a composition comprising the oligosaccharides of comfrey shown in Formulas I and II of Example 1. It also comprises levamisole hydrochloride. The weight ratio of the three is 1:1:1.

[0092] Efficacy verification of the composition showed that the levels of IL-1β, IL-6, and TNF-α in mouse serum were decreased, while the number of macrophages in zebrafish was significantly increased.

[0093] As can be seen, the embodiments of this invention provide two unique comfrey oligosaccharide compounds that can effectively reduce the level of inflammatory factors and significantly enhance immunity, providing an important material basis for the development of novel, efficient, and easily absorbed immunomodulators. The comfrey oligosaccharide preparation method of this invention produces comfrey oligosaccharides with high purity, is convenient and simple, and is suitable for industrial application.

[0094] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention (including the claims) is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of the embodiments of the invention as described above, which are not provided in the details for the sake of brevity.

[0095] The embodiments of this invention are intended to cover all such substitutions, modifications, and variations falling within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the embodiments of this invention should be included within the protection scope of this invention.

Claims

1. A lithospermic oligosaccharide, as shown in structural formula I or II: Ⅰ Ⅱ。 2. The method for preparing the oligosaccharide from Lithospermum erythrorhizon according to claim 1, comprising the following steps: Step 1: Use petroleum ether to perform ultrasonic extraction on Lithospermum erythrorhizon. Take the extracted Lithospermum erythrorhizon to obtain defatted Lithospermum erythrorhizon medicinal material. The material-liquid ratio of Lithospermum erythrorhizon to petroleum ether is 1:(5-15). Step 2: The defatted Lithospermum erythrorhizon herb is percolated with ethanol to obtain percolate; the percolate is concentrated and then deproteinized with trichloroacetic acid and neutralized with NaOH. The neutralized percolate is then passed through a macroporous resin column and eluted with distilled water as the eluent; the eluted fractions are collected and monitored by TLC; the eluted fractions containing oligosaccharides are combined, concentrated, and dried to obtain the crude oligosaccharide fraction of Lithospermum erythrorhizon. Step 3: The crude oligosaccharide fraction of Lithospermum erythrorhizon was first subjected to silica gel column chromatography and eluted using an ethyl acetate-methanol elution system. Then, the 0:100 ethyl acetate-methanol elution fraction was subjected to column chromatography using a Sephadex LH-20 gel column and eluted using a methanol-water elution system. The two Lithospermum erythrorhizon oligosaccharides represented by Formula I and II were separated by TLC.

3. The method of claim 2, wherein: The feed-liquid ratio is a weight ratio or a volume ratio.

4. The method of claim 2, wherein: The concentration of the ethanol is 70%; the concentration of the trichloroacetic acid is 10%-20%.

5. The method of claim 2, wherein: The macroporous resin column is an XAD-16N macroporous resin column.

6. The method of claim 2, wherein: The drying process is freeze-drying.

7. The method of claim 2, wherein: The preparation method also includes a step of detecting and verifying the separated components by mass spectrometry and / or nuclear magnetic resonance.

8. A composition comprising at least one of the oligosaccharides of formula I or formula II as described in claim 1.

9. The use of the comfrey oligosaccharide of claim 1 or the composition of claim 6 in the preparation of an anti-inflammatory product.

10. The use of the comfrey oligosaccharide of claim 1 or the composition of claim 6 in the preparation of immunomodulatory products.

Images