LU500548B1 - Application of syringic acid(3,5-dimethoxy-4-hydroxybenzoic acid) in preparing medicaments for promoting peripheral nerve repair - Google Patents

Abstract

The invention relates to the technical field of new indications of medicaments, and particularly discloses an application of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) in preparing medicaments for promoting peripheral nerve repair. The invention explores and studies the effect of syringic acid, a traditional Chinese medicine monomer, on promoting peripheral nerve repair, and the results show that syringic acid promotes the repair and regeneration of peripheral nerves by promoting Schwann cell migration, proliferation and differentiation. The invention provides a new idea for promoting peripheral nerve repair by traditional Chinese medicine and experimental basis for the treatment of peripheral nerve injury.

Description

DESCRIPTION Application of syringic acid(3,5-dimethoxy-4-hydroxybenzoic acid) in preparing medicaments for promoting peripheral nerve repair

TECHNICAL FIELD The invention relates to the technical field of new indications of medicaments, in particular to an application of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) in preparing medicaments for promoting peripheral nerve repair.

BACKGROUND More than 1 million patients suffer from peripheral nerve injury caused by various trauma or surgery (such as tumor resection) every yearn in China, which will suffer permanent loss of neurological function in relevant parts without timely and effective repairing treatment. With scar tissue that grows in the nerve gap after nerve injury being an main obstacle to the self-repair of peripheral nerves, large gap of peripheral nerve injury can be repaired by autologous nerve transplantation, which still has many defects including nerve dysfunction in the donor area, neuroma formation in the donor area, limited donor area nerves, and difficulty in matching the donor area nerves with the recipient area nerves, etc. While some of Chinese medicine such as Radix Astragali seu Hedysari and Radix Salviae Miltiorrhizae has been found having a promoting effect in nerve repair, 1t is not clear which component plays the role in nerve repair and what effects other components of Chinese medicine may have on the human body due to the complex components of Chinese medicine, Schwann cells (SCs) are the most important glial cells unique to the peripheral nerve system, which play an important role in the generation of peripheral nerves and regeneration after injury. It 1s the sheath cells of the peripheral nerve fibers arranged in a string and wrapping the axons of the peripheral nerve fibers, and a myelin sheath formed by repeatedly enveloping the concentric layers. Schwann cells secrete cell adhesion molecules, various neurotrophic factors (NTFs) and extracellular matrix to promote and guide the regeneration of peripheral axons. In summary, Schwann cells participate in myelin formation and nerves repairment through functions of migration, proliferation and differentiation.

Syrinic acid (SYRA), also known as 3,5-dimethoxy-4-hydroxybenzoic acid, is a naturally occurring gallic acid o-methylated product with the molecular formula of CoH1005, which can be extracted from Ardisia elliptica and Euterpe oleracea (also known as acai berry), it can also be prepared by selective hydrolysis (demethylation) of trimethyl gallate with 20% sulfuric acid. Studies showed that syringic acid could promote the migration, proliferation and differentiation of Schwann cells, which indicates that syringic acid could promote nerve repair.

Chinese patent 201610155201 discloses the application of syrinic acid(4-hydroxy-3,5- dimethoxybenzoic acid) in the preparation of drugs for termination of pregnancy, which belongs to the field of pharmaceutical technology, the medicine disclosed is prepared from the active ingredient which is composed of syrinic acid(4-hydroxy-3,5- dimethoxybenzoic acid) as the only component and pharmaceutically acceptable excipients, and has the advantages of high pregnancy termination efficiency, no damage to viscera, small toxic and side effects, good safety and reliability, which is suitable for termination of early pregnancy. Chinese patent 01210098200.6 discloses an application of rhizoma gastrodiae and its extract in preparing a medicament for peripheral nerve injury treatment and re-repair, and is characterized in that rhizoma gastrodiae or rhizoma gastrodiae extract 1s independently prepared into powder, capsule, tablet or injection, or is prepared into a composite preparation with other medicaments being applied in medicaments for treating peripheral nerve injury and re-repair. As for the present invention, the positive effects are that it provides a new medicament for treating peripheral nerve injury and re-repair with a medicament of low cost and reliable in effect. However, no report has been seen about the application of the syringic acid, a traditional Chinese medicine monomer, in preparing the medicament for treating peripheral nerve repair.

SUMMARY The invention provides evidence that traditional Chinese medicine monomer syringic acid promotes peripheral nerve repair, and provides new ideas and methods for the treatment of peripheral nerve repair.

The first object of the invention is to provide an application of syringic acid (3,5- dimethoxy-4-hydroxybenzoic acid) in preparing a medicament for promoting peripheral nerve repair aiming at the defects in the prior art.

And the second object of the present invention is to provide a medicament for promoting peripheral nerve repair.

In order to achieve the first one, the invention adopts the technical scheme that: Application of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) in preparation of medicament for promoting peripheral nerve repair.

As a preferred embodiment of the present invention, the syringic acid (3,5-dimethoxy-4- hydroxybenzoic acid) 1s used as the only active ingredient of a medicament for promoting peripheral nerve repair.

In order to achieve the second object, the invention adopts the technical scheme that: A medicament for promoting peripheral nerve repair, wherein the active component contained is syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid). As a preferred technical scheme of the invention, the medicament for promoting peripheral nerve repair further comprises pharmaceutically acceptable excipients, namely fillers, disintegrants, adhesive, flavoring agents, lubricants and the like.

The filler 1s selected from one or more of pregelatinized starch, lactose, mannitol and microcrystalline cellulose; the disintegrant is selected from one or more of sodium carboxymethyl starch, crospovidone, croscarmellose sodium and low-substituted hydroxypropyl cellulose; the adhesive is selected from one or more of starch slurry, hydroxypropyl cellulose solution, povidone solution; the lubricant is one or two of magnesium stearate and talcum powder.

As a preferred embodiment of the present invention, the medicament or pharmaceutical composition of the present invention is conventional in the art in various dosage forms, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non- aqueous solution or a suspending agent, more preferably a powder, granule, tablet, capsule, pill, solution, suspension or injection, etc.

A preferred route of administration of the medicament or pharmaceutical composition is by injection or oral administration.

The injectable administration preferably includes methods of intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous.

In a embodiment of the present invention, Schwann cells were incubated with DMSO culture solution containing 0 and 500 uM syringic acid, respectively.

The results showed that syringic acid of 500 uM significantly promoted the migration of Schwann cells.

In a embodiment of the present invention, DMSO culture solution containing 0, 300, 400, 500, and 600 uM syringic acid were added into Schwann cells respectively.

The results showed that syringic acid had no significant effect on the proliferation of Schwann cells at low concentration, while it showed a significantly increased promoting effect in the cells at the concentration of 600 uM.

As the time went on, the promoting effect became more obvious.

In a embodiment of the present invention, DMSO medium containing 0, 400, 500, and 600 uM syringic acid were added into Schwann cells, respectively.

The results showed that the proliferation of Schwann cells increases as the concentration rises.

In a embodiment of the present invention, DMSO culture solution containing 0 and 600 uM syringic acid were added into Schwann cells respectively.

The results showed that Schwann cells increased in volume and elongated antennae with long spindle shape under the effect of syringic acid.

In the invention, syringic acid powder with different concentrations was added into the DMSO culture solution of Schwann cells to observe the migration ability, proliferation ability and differentiation ability of the schwann cells, and results showed that syringic acid significantly promoted the migration, proliferation and differentiation of Schwann cells.

The invention explores and studies the effect of syringic acid, a traditional Chinese medicine monomer, on promoting peripheral nerve repair, and the results show that syringic acid promotes the repair and regeneration of peripheral nerves by promoting

Schwann cell migration, proliferation and differentiation. The invention provides a new idea for promoting peripheral nerve repair by traditional Chinese medicine and experimental basis for the treatment of peripheral nerve injury.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is the molecular structure of a traditional Chinese medicine monomer syringic acid. Figure 2 shows the ability of syringic acid to promote Schwann cell migration, wherein syringic acid at 500 uM significantly promotes Schwann cell migration as compared to syringic acid at a drug concentration of 0 um. Fig. 3 is the proliferation curve of Schwann cells. Among them, syringic acid has no significant effect on the proliferation of Schwann cells at low concentration, but promots the proliferation of cells significantly at concentration of 600 uM. With the prolongation of time, the promotion became more obvious. Fig. 4 shows the colony-forming ability of Schwann cells. The effect of syringic acid on the proliferation of Schwann cells is more obvious with the increase of concentration. Fig. 5 is a morphological diagram of Schwann cells after treatment with syringic acid. The Schwann cells became larger with elongated antennae and a long spindle shape as a whole under the treatment with syringic acid.

DESCRIPTION OF THE INVENTION The invention is further illustrated below in conjunction with the detailed description. It is to be understood that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention; Furthermore, it should be understand that various changes or modifications may be made by those skilled in the art after reading the foregoing description of the invention, and that such equivalents may fall within the scope of the appended claims.

Embodiment 1 Effect of Syringic Acid on Schwann Cell Migration In this embodiment, Schwann cells were taken and re-suspended in DMEM culture medium containing 2% FBS of syringic acid after digestion, and the cell density was adjusted to 2x10° cells/ml. Each small chamber was added with 100 ul and 700 pl DMEM culture medium containing 10%FBS was added into the lower chamber. The drug concentrations were 0 and 500 uM in sequence, with two secondary wells for each concentration, and then they were cultured in an incubator at 37°C and 5% CO» with saturated humidity for 20 h. The medium was discarded, and the remained was rinsed with appropriate amount of PBS for two times, and the cells in the chamber were removed by cotton swabs. Then the cells were stained with 4% ethanol crystal violet and observed under the microscope after treatment. Three fields of view were randomly selected for each sample and photographed. The number of migrated cells was counted and graphpad5.0 was used for statistical analysis. The results showed that syringic acid of 500 uM can significantly promote the migration of Schwann cells (Figure 2). Embodiment 2 Effect of Syringic Acid on Proliferation of Schwann Cells Schwann cells in this embodiment were inoculated at a density of 40,000 cells/ml into 96- well plates with 100 ul per well, and incubated overnight at 37°C in a incubator with 5% CO: of saturated humidity. The DMEM complete medium containing syringic acid was replaced with concentrations of 0, 300, 400, 500, and 600 uM, respectively, and six secondary wells were set for each concentration for 1, 3, 5, and 7 days. The OD value of each group of cells at 492 nm was measured by MTT, and the proliferation rate was calculated as: proliferation rate =OD value of sample/OD value of control group. The proliferation curve was drawn with graphpad5.0 and statistical analysis was performed. The results showed that the effect of syringic acid on the proliferation of Schwann cells was not significant at low concentration, but increased at concentration of 600 uM. With the prolongation of time, the promoting effect was more obvious (Fig. 3).

Embodiment 3 Effect of Syringic Acid on Colony Forming Ability of Schwann Cells Schwann cells in this example were inoculated at a density of 500 cells/ml into 6-well plates, 2ml per well, and incubated overnight at 37°C in a incubator with 5% CO; anf of saturated humidity. On the next day, syringic acid was added at the concentrations of 0, 400, 500, and 600 uM in sequence, with two secondary pores for each concentration, and the treatment was conducted for seven days. The cells were stained with 4% ethanol crystal violet and photographed, it was counted by Photoshop, and plotted by graphpad5.0 for statistical analysis. The results showed that, with the increase of concentration, syringic acid had a more significant effect on the proliferation of Schwann cells (Fig. 4).

Embodiment 4 Effect of Syringic Acid on Promoting Differentiation of Schwann Cells Schwann cells in this example were inoculated at a density of 40,000 cells/ml into 96- well plates, 100 ul per well, and incubated overnight at 37°C in a incubator with 5% CO» of saturated humidity. On the next day, DMEM complete medium containing 0 and 600 uM syringic acid was replaced, with six secondary wells for each concentration. The medium was cultured for two days and photographed under a microscope every day. The results showed that under the action of syringic acid, Schwann cells became larger in size, and elongated in antennae with long spindle shape as a whole (Fig. 5).

While the foregoing is only a description of the preferred embodiments of the present invention, it should be noted that modifications and additions may be made by those skilled in the art without departing from the method of the present invention, and such modifications and additions should also be considered within the scope of the present invention.

Claims (6)

CLAIMS:

1. An application of syringic acid(3,5-dimethoxy-4-hydroxybenzoic acid) in preparation of medicament for promoting peripheral nerve repair.

2. The application according to claim 1, characterized in that the syringic acid(3,5- dimethoxy-4-hydroxybenzoic acid) 1s used as the only active ingredient of a medicament for promoting peripheral nerve repair.

3. A medicament for promoting peripheral nerve repair, which is characterized in that the active component of the medicament for promoting peripheral nerve repair is syringic acid(3,5-dimethoxy-4-hydroxybenzoic acid).

4. The medicament according to claim 3, wherein that medicament for promoting peripheral nerve repair further comprises pharmaceutically acceptable excipients.

5. The medicament of claim 4, wherein the pharmaceutically acceptable excipients comprise a filler, a disintegrant, a adhesive, a flavoring agent and a lubricant.

6. The medicament according to claim 3, wherein the dosage form of the medicine for promoting peripheral nerve repair is powder, granule, tablet, capsule, pill, solution, suspension or injection.