Extraction method of guanine nucleoside of cornu cervi pantotrichum and application of guanine nucleoside in medicine

Abstract

The invention discloses an extraction method of guanine nucleoside of pilose antler, and further provides medicinal application of the guanine nucleoside. The method comprises the following steps: extracting a new substance, namely Guanosine of Antler (Guan A for short) from cornu cervi pantotrichum, and ensuring that the compound is not influenced by severe external conditions by adopting a membrane separation technology; pure water is used as a solvent, so that the problem of doping of other ions does not exist; depigmenting by using an ion exchange chromatography technology, and purifying active ingredients to obtain a total extract; active ingredients can be reserved to the maximum extent, and use and residues of organic solvents are reduced.

Description

Technical Field

[0001] This invention discloses a method for extracting guanine nucleosides from deer antler, relating to guanine nucleosides. (Guanosine) This invention relates to a novel extraction technique for bioactive substances with bond angle isomerism, and further provides information on their medicinal applications. It belongs to the field of extraction technology for bioactive compounds derived from animal bodies. Background Technology

[0002] As a health-preserving medicine, deer antler has significant effects in enhancing metabolism, protecting and renewing damaged organs and tissues, regulating immune function, activating phagocyte function, slowing down the aging process, lowering blood pressure, and improving gonadotropin and thyroid function.

[0003] There are numerous medicinal products related to deer antler, and their preparation methods vary. If the active ingredients of deer antler are not fully understood, and their chemical properties are unknown, the preparation and extraction process will be largely unpredictable, potentially leading to the loss of active ingredients and waste of resources. Furthermore, the quality standards for medicines and food products using deer antler as a raw material should be based on the chemical properties of the active ingredients contained in the deer antler to ensure the quality and safety of related products.

[0004] Traditional extraction processes for deer antler preparations have primarily employed techniques such as alcohol maceration, boiling, supercritical CO2 extraction, and ultrafiltration concentration. However, due to a lack of understanding of the chemical structure, molecular weight, and physicochemical properties of the active ingredients in deer antler, each extraction process has its own limitations. Furthermore, the unknown effective dosage range of the pharmacological effects of the active ingredients in deer antler makes it impossible to quantify the dosage of various preparations, leading to some uncertainty in clinical application. Additionally, it is impossible to establish quality assessment standards for deer antler medicinal materials based on the chemical properties of the active ingredients. Summary of the Invention

[0005] The purpose of this invention is to provide a guanosine of antler (GuanA) and its extraction method, which is a new active ingredient.

[0006] A further objective of this invention is to provide the application of the guanine nucleoside active ingredient of deer antler in the pharmaceutical field.

[0007] The technical solution of this invention is as follows: 1. Membrane separation technology is used to extract and concentrate the extract to ensure that the compound is not affected by severe external conditions. 2. Pure water is used as the solvent, eliminating the problem of other ion incorporation. 3. Ion exchange chromatography is used to depigment and purify the active ingredients to maximize the retention of active ingredients and reduce the use and residue of organic solvents.

[0008] Specific separation operation steps: Add 5-10% (g / v) distilled water to the deer antler powder, stir for 4 hours at room temperature to 40℃, and refrigerate overnight. Centrifuge the extract at 8000 rpm for 40 minutes and discard the sediment. Filter the supernatant of the extract through a 0.8 μm filter membrane. Ultrafilter the filtrate through a 3KD ultrafiltration membrane. Discard the ultrafiltration external liquid (internal liquid) and then nanofilter through a nanofiltration membrane to obtain the nanofiltration internal liquid. Concentrate the nanofiltration internal liquid by rotary evaporation at 56℃ to obtain the nanofiltration concentrate. Pass it through a CM-cellulose cation exchange column with distilled water as the eluent and collect the active peak. Concentrate the active peak solution by rotary evaporation. Then, perform DEAE-sephadex A25 anion exchange chromatography with distilled water as the mobile phase and collect the active peak. Concentrate by rotary evaporation and freeze-dry to obtain a lyophilized powder (i.e., the total deer antler extract, abbreviated as LR, hereinafter the same), which contains Guan A, and its spectrum is shown in the figure. Figure 1 As shown in peak number 4.

[0009] Simultaneously, in the above operation, after passing the solution through a CM-cellulose cation exchange column and eluting with distilled water, the active peak was collected, and the active peak solution was concentrated by rotary evaporation. A DEAE-Sephadex A25 anion exchange column was equilibrated with Tris hydrochloric acid buffer at pH 9.0, and the concentrated solution was chromatographically analyzed, eluted with buffer, and the breakthrough peak was discarded. Then, elution was performed with 2% formic acid, and the desorbed fraction was collected. The solution was concentrated to dryness by rotary evaporation at 56℃, and then dissolved in distilled water. Freeze-drying also yielded a lyophilized powder (total deer antler extract, LR), which contains Guan A.

[0010] If, during the above extraction steps, the DEAE-Sephadex A25 anion exchange column is equilibrated with Tris hydrochloric acid buffer at pH 9.0, eluted with buffer, and the breakthrough peak is discarded, followed by elution with 2% formic acid, the desorbed fraction is collected, concentrated to dryness by rotary evaporation at 56°C, and then dissolved in distilled water, the main active ingredient contains Guan A.

[0011] Based on the liquid chromatography characteristics of the extract, a single peak was prepared from the lyophilized powder, yielding the compound (Guanosine of Antler, abbreviated as Guan A); molecular formula: C4H4N2O2; molecular weight: 112.3; white powder, m.p 302~304℃; structural formula as follows:

[0012] The asterisk (*) indicates that the 3D key is not defined.

[0013] Analysis of the one-dimensional and two-dimensional NMR spectra revealed that Guan A is a bond-angle isomer of the standard (guanine nucleoside). The sample differs from the standard in terms of molar absorption coefficient, melting point, optical rotation, NMR spectrum, and activity. This difference is manifested in the change in the angle of the NH ring in the stereostructure, resulting in discrepancies between the instrumental analysis data and existing products.

[0014] Furthermore, Guan A exhibits significant differences in bioactivity compared to the standard. Guan A expresses the pharmacological activity of deer antler, with an effective dose range similar to that of corresponding cell-stimulating factors and growth factors. Guan A stimulates the proliferation of mouse bone marrow mesenchymal stem cells treated with 5-fluorouracil (5-FU), similar to macrophage-granulocyte colony-stimulating factor (GM-CSF), while the standard shows no activity. Guan A can stimulate the metabolic activity of cardiomyocytes damaged by hypoxia / reoxygenation. Guan A can activate the phagocytic function of mouse peritoneal macrophages, while guanosine shows no activity. Guan A can promote the proliferation of rabbit limbal stem cells, similar to the effect of fibroblast growth factor (FGF). Therefore, Guan A has potential applications in treating anemia, recovery from trauma and fatigue, the repair period after angina pectoris and coronary heart disease attacks, immunodeficiency, and "increasing white blood cell count" after chemotherapy.

[0015] The positive effects of this invention are as follows:

[0016] A novel substance, guanosine of antler (Guan A), is extracted from deer antler using membrane separation technology to ensure the compound is not affected by harsh external conditions; pure water is used as the solvent to avoid the introduction of other ions; ion exchange chromatography is used to depigment and purify the active ingredient to obtain the total extract; this invention can maximize the retention of active ingredients and reduce the use and residue of organic solvents.

[0017] This invention, based on a clear understanding of the active ingredients in deer antler, quantifies their content in medicinal materials and experimentally verifies the effective dosage range. A novel active ingredient underwent chemical structure analysis and pharmacological activity verification, and its potential significance in pharmaceutical applications was elucidated. Attached Figure Description

[0018] Figure 1 This is the guanine nucleoside spectrum of the deer antler described in this invention. Detailed Implementation

[0019] The present invention is further illustrated by the following embodiments, which are not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention. Example 1

[0020] Add 30g of deer antler powder to 600ml of distilled water, stir at room temperature to 40℃ for 4 hours, and refrigerate overnight. Centrifuge the extract at 8000rpm for 40 minutes and discard the sediment; filter the supernatant of the extract through a 0.8μm filter membrane. Ultrafilter the filtrate through an ultrafiltration membrane (3KD) to obtain the ultrafiltration external solution; then nanofilter through a nanofiltration membrane to obtain the nanofiltration internal solution. Concentrate by rotary evaporation at 56℃ to obtain the nanofiltration concentrate. Elute with distilled water on a CM-cellulose cation exchange column and collect the active peak; concentrate the active peak solution by rotary evaporation and precipitate it on a DEAE-sephadex A25 anion exchange column with distilled water as the mobile phase, and collect the active peak. Concentrate by rotary evaporation and freeze-dry to obtain 53.67mg of lyophilized powder (total deer antler extract, LR), with a yield of 0.178%; the total extract was prepared by liquid chromatography to obtain the active ingredient, accounting for 11.99% of the total weight; among which, Guan A (0.81mg); its chromatogram is shown below. Figure 1 Peak 4 is shown in the middle; molecular formula is C4H4N2O2; molecular weight = 112.3; white powder, m.p 302~304℃; structural formula is as follows:

[0021] The asterisk (*) indicates that the 3D key is not defined. Example 2

[0022] Add 30g of deer antler powder to 600ml of distilled water, stir at room temperature to 40℃ for 4 hours, and refrigerate overnight. Centrifuge the extract at 8000rpm for 40 minutes and discard the sediment. Filter the supernatant of the extract through a 0.8μm filter membrane. Ultrafilter the filtrate through an ultrafiltration membrane (3KD) to obtain the ultrafiltration solution. Nanofilter the solution through a nanofiltration membrane to obtain the ultrafiltration solution. Concentrate the solution by rotary evaporation at 56℃ to obtain the nanofiltration concentrate. Elute the solution onto a CM-cellulose cation exchange column with distilled water and collect the active peak. Concentrate the active peak solution by rotary evaporation. Perform DEAE-sephadex A25 anion exchange chromatography, equilibrate the column with Tris hydrochloric acid buffer at pH 9.0, elute with buffer, and discard the breakthrough peak. Elute with 2% formic acid and collect the desorbed fraction. Concentrate the solution to dryness by rotary evaporation at 56℃, and dissolve it again in distilled water. Freeze-dry to obtain 22.8 mg of lyophilized powder (total deer antler extract, LR), with a yield of 0.076%. The active ingredient accounts for 28.25%, the same proportion as above. Example 3

[0023] Add 30g of deer antler powder to 600ml of distilled water, stir at room temperature to 40℃ for 4 hours, and refrigerate overnight. Centrifuge the extract at 8000rpm for 40 minutes and discard the sediment. Filter the supernatant of the extract through a 0.8μm filter membrane. Ultrafilter the filtrate through an ultrafiltration membrane (3KD) to obtain the ultrafiltrate. Concentrate the ultrafiltrate through a nanofiltration membrane to obtain the ultrafiltrate inner solution. Concentrate the ultrafiltrate by rotary evaporation at 56℃ to obtain the nanofiltration concentrate. Elute the concentrate onto a CM-cellulose cation exchange column with distilled water and collect the active peak. Concentrate the active peak solution by rotary evaporation. Perform DEAE-sephadex A25 anion exchange chromatography, equilibrate the column with Tris hydrochloric acid buffer at pH 9.0, elute with buffer, and discard the breakthrough peak. Elute with distilled water and collect the breakthrough peak to obtain 10.27mg of lyophilized powder, with an active ingredient content of 42%. It mainly contains uracil and uridine-like substances from deer antler. The sample was further eluted with 2% formic acid, and the desorbed portion was collected and concentrated to dryness by rotary evaporation at 56°C. Distilled water was then added to dissolve the residue. The main active ingredients were inosine and Guan A from deer antler. Freeze-drying yielded 12.81 mg of lyophilized powder, with the active ingredient accounting for 10.92%.

[0024] The following experimental examples demonstrate the medicinal uses of this invention.

[0025] Experimental Example 1: Effect of Guan A on the proliferation of bone marrow mesenchymal stem cells (MSCs) in 5-fluorouracil-treated model mice 1. The experimental methods were based on "Methodology of Cytokine Research" edited by Sun Weimin et al.

[0026] 2. Results: Guan A had a strong effect on stimulating the proliferation of MSCs in model mice, while the standard had no activity (see Table 1).

[0027] Table 1. Effects of various components of deer antler on the proliferation of bone marrow mesenchymal stem cells (MSCs) in mice treated with 5FU (x(_)±s, n=3).

[0028]

[0029] Note: Control group 0.267±0.020, compared with control group *P<0.05,** P<0.01,*** P<0.001, other P>0.05.

[0030] 3. Conclusion The Guan A sample showed a strong effect on stimulating the proliferation of MSCs in model mice, similar to the effect of colony-stimulating factor (GM-CSF). The effective dose range is between 100.0-1.0 ng / ml. Guanosine is inactive.

[0031] Experimental Example 2: Effect of Guan A on the proliferation of limbal stem cells 1. The experimental method is the same as above; 2. Results: Guan A at doses ranging from 100.0 to 1.0 ng / ml stimulated the proliferation of limbal stem cells, with effects similar to those of fibroblast growth factor (FGF) (see Table 2).

[0032] Table 2. Stimulating effect of various components of deer antler on the proliferation of limbal stem cells (x(_)±s, n=4).

[0033]

[0034] Note: The control group was 0.0428±0.0047. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0035] 3. Conclusion Guan A, at doses ranging from 100.0 to 1.0 ng / ml, has the same stimulating effect on the proliferation of limbal stem cells as FGF.

[0036] Experimental Example 3: Protective effect of Guan A on hypoxia / reoxygenation injury of neonatal rat cardiomyocytes 1. Experimental methods (refer to relevant literature) Cardiomyocyte culture in neonatal rats: Hearts of 3-day-old neonatal rats were aseptically harvested, the pericardium removed, and myocardial tissue from the apex was taken for cell culture. The cells were subjected to nitrogen hypoxia followed by reoxygenation with air to induce cell damage.

[0037] 2. Results showed that Guan A had a protective effect on hypoxia / reoxygenation injury of neonatal rat cardiomyocytes, while the standard had no effect (see Table 3).

[0038] Table 3. Effects of Guan A on mitochondrial dehydrogenase activity in neonatal rat cardiomyocytes after hypoxia / reoxygenation injury (x(_)±s, n=3)

[0039] Note: The control group was 0.221±0.022. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0040] 3. Conclusion Compared with the control group, Guan A significantly increased the activity of mitochondrial dehydrogenases in neonatal rat cardiomyocytes after hypoxia / reoxygenation injury. Guanosine, however, had no significant effect on this indicator.

[0041] Example 4: Effect of Guan A on phagocytic function of mouse peritoneal macrophages.

[0042] 1. The experimental methods were based on "Methodology of Cytokine Research" edited by Sun Weimin et al.

[0043] 2. Results showed that Guan A could enhance the phagocytic activity of mouse peritoneal macrophages, while the standard had no effect (see Table 4).

[0044] Table 4. Effects of Guan A on phagocytic function of peritoneal macrophages in mice (x(_)±s, n=3).

[0045]

[0046] Note: Control group 0.066±0.003, compared with control group *P<0.05, **P<0.01, ***P<0.001.

[0047] 3. Conclusion Table 4 shows that, compared with the control group, Guan A significantly enhanced the phagocytic activity of mouse peritoneal macrophages. The standard had no significant effect on this indicator.

[0048] The above pharmacological activity assays suggest that Guan A in deer antler extract has potential therapeutic effects in the pharmaceutical field. These include repair of corneal damage; promoting the repair of immunosuppression caused by antitumor drugs and enhancing the body's immune function; repairing damaged myocardial cells and treating angina pectoris, myocardial ischemia, etc.; and regenerative repair of skin injuries.

[0049] Preparations containing deer antler extract (containing Guan A component) can be combined with appropriate pharmaceutical carriers to prepare various dosage forms for use in treating inflammatory ulcers of the skin, mucous membranes, and cornea; the recovery period after a heart attack; anemia and leukopenia; and post-exercise recovery. Drugs containing the guanine nucleoside activity of deer antler can be formulated into any dosage form.

Claims

1. A guanosine of antler (abbreviated as Guan A); molecular formula C4H4N2O2; molecular weight = 112.3; white powder, m.p 302~304℃; structural formula as follows: ； in, "*" indicates that the stereo key is not defined.

2. The method for extracting guanine nucleosides from deer antler as described in claim 1, comprising the following steps: 1) Take deer antler powder, add distilled water at 5-10% (g / v), stir at room temperature to 40℃ for 4 hours, refrigerate overnight, centrifuge the extract at 8000rpm for 40 minutes, and discard the sediment. 2) The supernatant of the leachate is filtered through a 0.8 μm filter membrane, the filtrate is ultrafiltered through a 3 KD ultrafiltration membrane, and the superfiltration solution is then concentrated by nanofiltration through a nanofiltration membrane to obtain nanofiltrate; 3) The nanofiltration solution was concentrated by rotary evaporation at 56°C to obtain a concentrated solution; 4) Pass it through a CM-cellulose cation exchange column with distilled water as the eluent, and collect the active peak. The active peak solution is concentrated by rotary evaporation. Then, it is subjected to DEAE-sephadex A25 anion exchange chromatography with distilled water as the mobile phase, and the active peak is collected. The solution is concentrated by rotary evaporation and freeze-dried to obtain a lyophilized powder containing the total extract of the active ingredients of deer antler. Single components are prepared by liquid chromatography.

3. The method for extracting guanine nucleosides from deer antler according to claim 2, characterized in that: In step 4), the solution was passed through a CM-cellulose cation exchange column with distilled water as the eluent. The active peak was collected and concentrated by rotary evaporation. Then, it was subjected to DEAE-sephadex A25 anion exchange chromatography equilibrated with Tris hydrochloric acid buffer at pH 9.

0. After elution with buffer and discarding the breakthrough peak, the solution was eluted with 2% formic acid. The desorbed portion was collected, concentrated to dryness by rotary evaporation at 56°C, dissolved in distilled water, and freeze-dried to obtain a lyophilized powder containing the total compounds.

4. The method for extracting guanine nucleosides from deer antler according to claim 2, characterized in that... exist: Step 4) involves passing the solution through a CM-cellulose cation exchange column with distilled water as the eluent, collecting the active peak, and concentrating the solution by rotary evaporation. Then, it is subjected to DEAE-Sephadex A25 anion exchange chromatography equilibrated with Tris hydrochloric acid buffer at pH 9.

0. After elution with buffer and discarding the breakthrough peak, the solution is eluted with distilled water and collected. Elution is then continued with 2% formic acid, and the desorbed portion is collected and concentrated to dryness by rotary evaporation at 56°C. The solution is then dissolved in distilled water and freeze-dried to obtain Guan A-containing lyophilized powder.

5. The use of the guanine nucleoside of deer antler as described in claim 1 in the preparation of a drug for treating immunodeficiency.

6. The use of the guanine nucleoside of deer antler as described in claim 1 in the preparation of a medicament for treating skin wounds.

7. The use of the guanine nucleoside of deer antler as described in claim 1 in the preparation of a drug for treating angina pectoris and coronary heart disease.

8. The use of the guanine nucleoside of deer antler as described in claim 1 in the preparation of a drug for increasing white blood cell count.

9. The drug containing guanine nucleoside activity of deer antler as described in claims 5 to 8 can be formulated into any dosage form of the drug.

Images