A method for preparing 6-o-l-arabinopyranosyl-beta-d-glucopyranoside and its use in the treatment of malaria

Abstract

This invention relates to a 6- O -L-arabinopyranosyl- β The preparation of β-D-glucopyranoside and its application in the preparation of antimalarial products belong to the field of traditional Chinese medicine and natural drug pharmaceuticals. Compound 6- of this invention... O -L-arabinopyranosyl- β This invention relates to an antimalarial drug with β-D-glucopyranoside as the active ingredient. It expands the application of aromatic disaccharide compounds 6- from *Hydrangea macrophylla*. O -L-arabinopyranosyl- β The medicinal value of D-glucopyranoside.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine and natural drug manufacturing technology, specifically relating to an aromatic disaccharide compound 6- O -L-arabinopyranosyl- β Methods for preparing β-D-glucopyranoside, pharmaceutical compositions thereof as active ingredients, and their use as antimalarial agents. Background Technology

[0002] Malaria is a major global public health threat, primarily transmitted to humans through the bite of infected female Anopheles mosquitoes. Five species of Plasmodium can cause malaria in humans, with Plasmodium falciparum being the most prevalent, widely found in Africa. Plasmodium falciparum Malaria is the most deadly disease. According to the latest malaria report released by the World Health Organization in December 2025, there are 282 million cases of malaria worldwide, with approximately 610,000 deaths. The efficacy of single-drug antimalarial drugs has declined due to long-term widespread use or improper administration, further worsening the control situation in malaria-endemic areas. Therefore, there is an urgent need to find lead drugs with antimalarial potential to supplement treatment options.

[0003] Traditional medicinal plants are an important resource for the research and development of antimalarial drugs; Southwest Hydrangea ( Hydrangea davidii *Hydrangea* Franch. is a plant belonging to the genus *Hydrangea* in the family Hydrangeaceae. Other names include David's Hydrangea, Yunnan Hydrangea, and Dian Hydrangea. It is found in Sichuan, Yunnan, and Guizhou provinces, growing in dense forests in valleys, sparse forests on mountain slopes, roadsides, or forest edges at altitudes of 1400-2400 meters. According to *A Compendium of Chinese Medicinal Resources*, the roots and leaves of *Hydrangea santalinus* are used for malaria, while the stem pith is used for measles and painful urination. *A Compendium of Chinese Ethnic Medicines* records that *Hydrangea santalinus* is a Lisu medicine, with its roots and leaves primarily used to treat malaria. Although this plant has clear medicinal records in ethnic medicine systems, research on its active ingredients has long been lacking. Currently, no information on *Hydrangea santalinus* or its compounds 6- has been found domestically or internationally. O -L-arabinopyranosyl- β A study report on the antimalarial activity of β-D-glucopyranoside. Summary of the Invention

[0004] The purpose of this invention is to provide an aromatic disaccharide compound 6- O -L-arabinopyranosyl- β Methods for preparing β-D-glucopyranoside, pharmaceutical compositions thereof as active ingredients, and their use as antimalarial agents.

[0005] To achieve the above objectives, the present invention employs the following technical solution: The aromatic disaccharide compounds shown below are 6- O -L-arabinopyranosyl- β-D-glucopyranoside,

[0006] 6- O -L-arabinopyranosyl- β Preparation method of -D-glucopyranoside: Take Hydrangea ( Hydrangea The roots, branches, leaves, flowers, or whole plant of a plant are directly extracted by hot reflux or cold soaking with organic solvents such as ethyl acetate, acetone, methanol, ethanol, or water. Alternatively, the plant can be first extracted by hot reflux or cold soaking with the above-mentioned organic solvents or water, and then extracted with n-butanol to obtain a total extract. The total extract is then subjected to repeated column chromatography to obtain the compound of the present invention.

[0007] The method for preparing the compounds of this invention more specifically uses: A: The total extract is obtained by reflux extraction or cold soaking of the roots, branches, leaves, flowers or whole plant of Hydrangea species with acetone, methanol or ethanol or hydrothermal reflux, followed by n-butanol extraction to obtain n-butanol extract. The compound of the present invention can be obtained by repeated column chromatography.

[0008] B: The total extract is obtained by direct hot reflux extraction or cold soaking of the roots, branches, leaves, flowers or whole plant of Hydrangea plants using organic solvents (such as methanol, ethanol, n-butanol, acetone, ethyl acetate). The compound of the present invention can be obtained by repeated column chromatography of the total extract.

[0009] More specifically, the preparation method of the compound of the present invention involves drying the roots, branches, leaves, flowers, or whole plant of a Hydrangea plant in the shade, pulverizing them to 20-30 mesh, and extracting them four times with 95% ethanol at 75 °C under reflux. The first and second extractions are each for 1 h, and the third and fourth extractions are each for 1.5 h. After extraction, the filtrates are combined and concentrated to obtain a total extract. The total extract is dispersed in water and then sequentially extracted with ethyl acetate or n-butanol as the extraction solvent to obtain the corresponding ethyl acetate extract or n-butanol extract. The n-butanol extract is dissolved in water and subjected to coarse separation using D101 macroporous resin. First, it is eluted with 5 column volumes of purified water, then eluted with 5 column volumes of 30%, 70%, and 100% methanol solutions to obtain four fractions. Fraction 2 is repeatedly purified by RP-18 and preparative HPLC to obtain compound 6-. O -L-arabinopyranosyl- β -D-glucopyranoside.

[0010] This invention relates to the use of the compounds of this invention and their pharmaceutically acceptable salts for the production of pharmaceutical products to treat patients with diseases caused by Plasmodium, etc. This invention comprises pharmaceutical compositions comprising an effective therapeutic amount of the compounds of this invention combined with at least one pharmaceutically acceptable carrier, excipient, diluent, adjuvant, or medium. The pharmaceutical compositions can be used as antimalarial drugs.

[0011] In another aspect, the present invention also provides the aromatic disaccharide compound 6- O -L-arabinopyranosyl- β The use of -D-glucopyranoside or pharmaceutical compositions in the preparation of antimalarial drugs. Furthermore, the compounds of this invention can also be used in combination with antimalarial drugs, including but not limited to artemisinin-based drugs, quinoline-based drugs, and pyrimethamine.

[0012] In another aspect, the present invention relates to the use of a method of using the compounds of the present invention as active ingredients or pharmaceutical compositions thereof to prevent or treat various diseases caused by Plasmodium or the like in animals or humans, the use comprising administering to humans or animals a pharmaceutically acceptable and effective therapeutic amount of the compounds of the present invention as active ingredients or pharmaceutical compositions thereof.

[0013] The malaria described in this invention refers to malaria caused by Plasmodium falciparum (… Plasmodium falciparum ), Plasmodium vivax ( Plasmodium vivax ), Plasmodium malariae ( Plasmodium malariae ), Plasmodium ovale ( Plasmodium ovale ), Plasmodium norotri ( Plasmodium knowlesi Diseases caused by ).

[0014] The present invention also includes a method for treating or alleviating a patient’s illness caused by malaria parasites or other diseases, or for which the patient is sensitive, the method comprising treating the patient with a therapeutically effective amount of the compound described in the present invention.

[0015] The present invention has been described in detail above, but the above embodiments are merely illustrative in nature and are not intended to limit the present invention. Attached Figure Description

[0016] Appendix Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the compound in this invention; Appendix Figure 2 This is the carbon NMR spectrum of the compound in this invention; Detailed Implementation

[0017] The present invention will be further illustrated below through specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also cover the scope defined by this invention without departing from its spirit and scope.

[0018] Example 1: The dried roots and branches of *Hydrangea macrophylla* (13.15 kg) were pulverized to 20-30 mesh and extracted four times with 95% ethanol at 75 °C under reflux. The first and second extractions were each for 1 h, and the third and fourth extractions were each for 1.5 h. After extraction, the filtrates were combined, concentrated, and dried to obtain the total extract of *Hydrangea macrophylla* (1276.5 g). The extract was dispersed in an appropriate amount of purified water and then subjected to liquid-liquid extraction with n-butanol. The n-butanol fraction was concentrated under reduced pressure to obtain the n-butanol extract (574 g). The n-butanol extract was subjected to gradient elution on a D101 macroporous resin column (the eluent was initially purified water, followed by 30%, 70%, and 100% methanol). The fraction A (192.5 g) obtained by elution with purified water, the fraction B (114 g) obtained by elution with 30% methanol aqueous solution, and the fraction C (108 g) obtained by elution with 70% methanol aqueous solution were collected.

[0019] Fraction B was eluted with a gradient of 0 to 35% methanol using an RP-18 column. TLC analysis identified identical fractions, resulting in five fractions (Fr. B-1 ~ Fr. B-5). Fr. B-2 was eluted with a gradient of 0 to 100% ethanol using an HP 20 macroporous resin column to obtain two fractions (Fr. B-2-1 ~ Fr. B-2-2). Fr. B-2-1 was eluted with a semi-preparative column (YMC-ODS-A) at constant temperature using 32% methanol to obtain ten fractions (Fr. B-2-1-1 ~ Fr. B-2-1-10). Fr. B-2-1-2 was eluted with a semi-preparative column (YMC-ODS-A) at constant temperature using 26% methanol to obtain five fractions (Fr. B-2-1-2-1 ~ Fr. B-2-1-2-5). Compound 6- was obtained by constant elution with a 26% methanol aqueous solution using a semi-preparative chromatographic column (YMC-ODS-A). O -L-arabinopyranosyl- β -D-glucopyranoside.

[0020] Example 2: Compound 6 of this invention O -L-arabinopyranosyl- β Partial physical and spectroscopic data of -D-glucopyranoside: 6- O -L-arabinopyranosyl- β -D-glucopyranoside: Reddish-brown solid. UV (MeOH)λ max (log ε ): 204 (4.15) nm; IR (KBr) ν max: 3422, 2831, 1605, 1364, 1082, 1007 cm -1 . 1 H-NMR (400 MHz, CD3OD-D2O) δ H : 7.20 (4H, m, H-2, 3, 5, 6), 7.11 (1H, m, H-4), 4.31 (2H, m, H-1', H-1''), 4.08 (2H, m, H-8), 3.85 (1H, dd, J = 12.4, 3.0 Hz,H-5'b), 3.80 (1H, m, H-4''), 3.75 (2H, m, H-6'), 3.59 (1H, dd, J = 8.9, 6.9Hz, H-2''), 3.43-3.52 (3H, m, H-5', 3'', 5''a), 3.38 (2H, m, H-3', 4'); 3.21(1H, m, H-2'), 2.94 (2H, m, H-7); 13 C-NMR (100 MHz, CD3OD-D2O) δ C : 140.0 (s, C-1), 129.3 (d, C-2), 130.0 (d, C-3), 127.2 (d, C-4), 130.0 (d, C-5), 129.3 (s, C-6), 37.1 (t, C-7), 71.8 (t, C-8), 104.3 (d, C-1'), 74.9 (d, C-2'), 77.7 (d,C-3'), 71.3 (d, C-4'), 76.8 (d, C-5'), 69.4 (t, C-6'), 105.1 (t, C-1''), 72.3(d, C-2''), 74.1 (d, C-3''), 69.5 (d, C-4''), 66.8 (t, C-5'').

[0021] Example 3: Antimalarial activity test of the compounds of this invention: The anti-malarial activity of the compounds of this invention against Plasmodium falciparum was evaluated in vitro using the SYBR Green I method. Plasmodium falciparumThe activity of strain 3D7 was investigated. Plasmodium was cultured in RPMI 1640 medium with a human hematocrit of 2%, at 37 °C and a CO2 concentration of 5%. The Plasmodium was synchronized to the cyclic stage with 5% sorbitol. The final detection system showed a Plasmodium infection rate of 1% and a hematocrit of 2%. The above-mentioned cyclic Plasmodium and the tested extract were co-cultured in 96-well plates for 72 h, followed by the addition of the fluorescent dye SYBR Green I. Finally, the fluorescence value (excitation: 485 nm, emission: 535 nm) was measured using a microplate reader to detect the growth of Plasmodium. The inhibition rate was calculated using the following formula: Malaria parasite inhibition rate (%) = ×100% Table 1. Results of the antimalarial activity of the compounds of this invention. Group <![CDATA[IC 50 (µM)]]> Artemisinin 0.03 6-L-arabinopyranosyl-D-glucopyranoside 5.37 The antimalarial activity data of the compounds of this invention are shown in Table 1. Compound 6- O -L-arabinopyranosyl- β -D-glucopyranoside has significant antimalarial activity and shows good application prospects.

[0022] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. The compound 6- described by the following structural formula O -L-arabinopyranosyl- β Preparation method of -D-glucopyranoside; 2. An antimalarial drug, characterized in that, It contains the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

3. A pharmaceutical composition, characterized in that... It contains the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient; the pharmaceutical composition further contains at least one pharmaceutically acceptable excipient.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition also contains at least one other antimalarial drug formulation, such as artemisinin, chloroquine, quinine, or pyrimethamine.

5. The pharmaceutical composition according to claim 3 or 4, wherein it is an antimalarial drug.

6. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating malaria.

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