A conjugated compound of phytochemical substances coated with gold nanoparticles and its preparation method and application

Abstract

The present invention discloses a conjugated compound of phytochemicals coated with gold nanoparticles and a preparation method and application thereof. The conjugated compound GNPs-F is formed by coating gold nanoparticles GNPs with phytochemicals, wherein the phytochemicals are F.arabica Extract F. F.arabica Extracts from F.arabica The plant raw material is dissolved in a solvent and filtered, wherein the solvent includes a polar solvent or a non-polar solvent to obtain a polar plant extract F 极 , Non-polar plant extract F 非极 , the polar solvent includes water, and the non-polar solvent includes methanol or hexane. F.arabica The extract was combined with GNPs against MTB isolates and showed significant inhibitory capacity against Mycobacterium tuberculosis strains.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a conjugated compound of phytochemical substances coated with gold nanoparticles, and a preparation method and application thereof. Background Art

[0002] Tuberculosis (TB) is a major public health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis (MTB). Drug-resistant TB is resistant to any drug, and multidrug-resistant TB (MDR-TB) is resistant to isoniazid (INH) and rifampicin (RIF). Technically, all drug-resistant MTB isolates will take adequate time to treat, but some will fail, leading to death. Alternative drug development approaches and improved targeted delivery are therefore urgently needed.

[0003] Nanomedicine is a good alternative anti-TB drug for targeted delivery, treatment and diagnosis. Multi-drug resistant tuberculosis is a major problem and obstacle for global tuberculosis control programs. The present invention is very effective against sensitive MDR MTB isolates. Although the Mycobacterium tuberculosis isolates are resistant, sometimes the drug cannot reach the target site of Mycobacterium tuberculosis.

[0004] Currently, only synthetic drugs are used for tuberculosis treatment, to which MTB isolates (the causative agent of tuberculosis) show resistance, and there are no literature reports on the use of F. arabica in combination with GNPs against MTB isolates.

[0005] The present invention uses gold nanoparticles to deliver phytochemical components of Fagonia arabica (F.arabica) in a targeted manner. This will also reduce the spread time of mycobacterium in the human population. Summary of the invention

[0006] Purpose of the invention: In order to overcome the deficiencies in the prior art, the present invention provides a phytochemical-coated gold nanoparticle conjugated compound and a preparation method and application thereof, specifically a new formulation of a biologically and chemically synthesized conjugated compound of gold nanoparticles (GNPs-F), which is combined with phytochemical components of F. arabica in hexane, methanol and water to evaluate its inhibitory ability against Mycobacterium tuberculosis.

[0007] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0008] The first object of the present invention is to provide a method for preparing a conjugated compound GNPs-F, which comprises forming a conjugated compound GNPs-F by coating gold nanoparticles GNPs with phytochemicals, wherein the phytochemicals are F. arabica extracts, denoted as F.

[0009] Optionally, in one embodiment of the present invention, the F. arabica extract is obtained by dissolving a F. arabica plant raw material in a solvent and filtering it, wherein the F. arabica plant raw material includes F. arabica plant powder or a plant paste obtained by grinding the F. arabica plant, and the solvent includes a polar solvent or a non-polar solvent, respectively, to obtain a polar plant extract F 极 , Non-polar plant extract F 非极 .

[0010] Optionally, in one embodiment of the present invention, the polar solvent includes water, and the non-polar solvent includes methanol or hexane.

[0011] Optionally, in one embodiment of the present invention, the polar plant extract F 极 The preparation method comprises: washing the green plant F.arabica and grinding it into a paste to obtain a plant paste, adding the plant paste into a polar solvent at a ratio of 0.1-1.0 mg / ml, heating for 10-30 minutes, cooling at room temperature, and filtering to obtain a polar base plant extract F 极 .

[0012] Preferably, the plant paste is added to the polar solvent at a ratio of 0.1-0.5 mg / ml, more preferably, at a ratio of 0.4 mg / ml.

[0013] Optionally, in one embodiment of the present invention, when the polar solvent is water, the F. arabica extract is a water-based plant extract F3.

[0014] Optionally, in one embodiment of the present invention, the non-polar plant extract F 非极 The preparation method comprises: adding F.arabica plant powder to a non-polar solvent at a ratio of 0.05-0.5 mg / ml, shaking continuously for one week at room temperature, and filtering to obtain a non-polar plant extract F 非极 Preferably, shaking is continued at room temperature for one week.

[0015] Preferably, the F. arabica plant powder is added to the non-polar solvent at a ratio of 0.1-0.3 mg / ml, more preferably, at a ratio of 0.2 mg / ml.

[0016] Optionally, in one embodiment of the present invention, when the non-polar solvent includes methanol or hexane, methanol plant extract F2 and hexane plant extract F1 are obtained accordingly.

[0017] Optionally, in one embodiment of the present invention, the polar plant extract F 极The gold nanoparticles GNPs were coated by a green synthesis method, comprising the following steps: 3-6 ml of polar plant extract F 极 Mix with 0.7-1.0M gold salt solution at room temperature to synthesize polar-based conjugated compound GNPs-F 极 , the gold salt includes sodium chloroaurate.

[0018] Optionally, in one embodiment of the present invention, the non-polar plant extract F 非极 The gold nanoparticles GNPs are coated by chemical synthesis, including the following steps:

[0019] (1) Covering GNPs with cysteine ​​Cys as a linker to obtain gold nanoparticles with linkers GNPs-Cys;

[0020] (2) In an organic solvent, the gold nanoparticles GNPs-Cys with a linker are combined with the non-polar plant extract F through the linker. 非极 The carboxyl, carbonyl or hydroxyl groups in the synthesis of non-polar conjugated compounds GNPs-Cys-F 非极 , wherein the organic solvent includes any one of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, sodium cyanoborohydride, and carbonyldiimidazole-tetrahydrofuran suspension.

[0021] Further optionally, in one embodiment of the present invention, in step (2), the non-polar plant extract F 非极 The method comprises: adding 0.5-5 ml of 20 mg / ml EDC aqueous solution and 30-80 mg of freeze-dried non-polar plant extract F to 2.5 ml of GNPs-Cys aqueous solution. 非极 Cross-linking is performed, stirring continuously for 1-5 hours in the dark at 25-45°C, centrifuged, washed, and the non-polar plant extract F is obtained. 非极 Carboxyl-bound nonpolar conjugated compound GNPs-Cys-F 非极-羧基 .

[0022] Further optionally, in one embodiment of the present invention, 0.5-3 ml of EDC aqueous solution is added, preferably 1 ml.

[0023] Further optionally, in one embodiment of the present invention, the pH of the EDC aqueous solution is 6-7, preferably pH 6.4.

[0024] Further optionally, in one embodiment of the present invention, 40-60 mg of freeze-dried non-polar plant extract F is added 非极 , preferably 50 mg is added.

[0025] Further optionally, in one embodiment of the present invention, stirring is performed at 30-40°C, preferably 37°C.

[0026] Further optionally, in one embodiment of the present invention, stirring is continued at a rotation speed of 70-150 rpm, preferably 100 rpm.

[0027] Further optionally, in one embodiment of the present invention, the stirring time is 1-3 hours, preferably 2 hours.

[0028] Further optionally, in one embodiment of the present invention, in step (2), the non-polar plant extract F 非极 The carbon-based method includes: GNPs-Cys, non-polar plant extract F 非极 The conjugate solution was dissolved in sodium carbonate-sodium citrate buffer at a molar ratio of 1:(1-5) at a concentration of 1-10 mg / ml, and the pH was maintained at 7-10. Each ml of the conjugate solution containing 5M sodium cyanoborohydride was added to sodium hydroxide. After standing for 1-3 hours, ethanolamine was added to block the unreacted aldehyde. After purification by dialysis, the non-polar plant extract F was obtained. 非极 Carbon-based non-polar conjugated compound GNPs-Cys-F 非极-碳基 .

[0029] Further optionally, in one embodiment of the present invention, the sodium carbonate-sodium citrate buffer is prepared from 0.05 M sodium carbonate and 0.1 M sodium citrate at a pH of 9.5.

[0030] Further optionally, in one embodiment of the present invention, the GNPs-Cys, non-polar plant extract F 非极 The molar ratio of 1:4 is 1:(3-5), preferably 1:4.

[0031] Further optionally, in one embodiment of the present invention, 3-6 M sodium cyanoborohydride, preferably 5 M, is added to each milliliter of the conjugate solution.

[0032] Further optionally, in one embodiment of the present invention, the standing time is 2 hours.

[0033] Further optionally, in one embodiment of the present invention, after standing, 1-5 M ethanolamine is added to each milliliter of the conjugate solution to block unreacted aldehyde, preferably 3 M ethanolamine.

[0034] Further optionally, in one embodiment of the present invention, in step (2), the non-polar plant extract F 非极The method comprises: freeze-dried non-polar plant extract F 非极 The mixture was suspended in tetrahydrofuran suspension containing 30-70 mg / ml carbonyldiimidazole at 3-8% w / v, stirred at room temperature for 1-3 hours, washed with THF and water in turn, and resuspended in cold coupling buffer at 8-20 mg / ml; then 1-10 mg GNPs-Cys was added, stirred at 0-10°C for at least 18 hours, and finally 0.1-0.3 M ethanolamine was added, stirred for 1-3 hours, and the particles were centrifuged and washed to obtain the non-polar plant extract F. 非极 Hydroxyl-bound nonpolar conjugated compound GNPs-Cys-F 非极-羟基 .

[0035] Further optionally, in one embodiment of the present invention, washing the particles after centrifugation specifically includes suspending the particles in 0.1M sodium carbonate coupling buffer after centrifugation, and washing with 0.1M sodium carbonate coupling buffer, wherein the sodium carbonate coupling buffer is 0.1-0.2M sodium carbonate coupling buffer with a pH of 9.5, preferably 0.1M sodium carbonate coupling buffer.

[0036] Further optionally, in one embodiment of the present invention, the cold coupling buffer is 0.01 M pyridine-HCL, 0.1 M NaCl, pH 6.0.

[0037] Further optionally, in one embodiment of the present invention, the concentration of carbonyldiimidazole in the tetrahydrofuran suspension of carbonyldiimidazole is 40-55 mg / ml, preferably 50 mg / ml.

[0038] Further optionally, in one embodiment of the present invention, the non-polar plant extract F 非极 Suspended in a suspension of carbonyldiimidazole in tetrahydrofuran at 4-6% w / v, preferably 5% w / v.

[0039] Further optionally, in one embodiment of the present invention, after adding GNPs-Cys, stirring is performed at 2-6°C, preferably at 4°C.

[0040] Further optionally, in one embodiment of the present invention, the tetrahydrofuran suspension is resuspended in cold coupling buffer at 8-15 mg / ml, preferably 10 mg / ml.

[0041] Further optionally, in one embodiment of the present invention, the added ethanolamine is 0.1-0.2 M, preferably 0.1 M ethanolamine.

[0042] The second object of the present invention is to provide a conjugated compound GNPs-F prepared according to any of the above methods.

[0043] The third object of the present invention is to provide a pharmaceutical composition comprising the conjugated compound GNPs-F as described above or a pharmaceutically acceptable form thereof.

[0044] The fourth object of the present invention is to provide a pharmaceutical kit comprising the conjugated compound GNPs-F as described above or a pharmaceutically acceptable form thereof and one or more pharmaceutically acceptable carriers, or comprising the pharmaceutical composition as described above or a pharmaceutically acceptable form thereof and one or more pharmaceutically acceptable carriers.

[0045] The fifth object of the present invention is to provide the conjugated compound GNPs-F, pharmaceutical composition, and pharmaceutical kit as described above, which are used for anti-Mycobacterium tuberculosis activity or for preparing drugs for treating tuberculosis.

[0046] Beneficial effects:

[0047] (1) Our novel F. arabica phytochemical model and its coupling with GNPs for the treatment of tuberculosis showed good inhibitory effects against drug-resistant and MDR strains of Mycobacterium tuberculosis.

[0048] (2) This plant extract does not have any obvious toxic effects on the liver, heart, and kidneys.

[0049] (3) F. arabica phytochemicals are easily available and cost-effective.

[0050] (4) The current model can reduce the burden of drug-resistant cases. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0052] Figure 1 UV-visible absorption spectra of GNPs and their conjugated compounds, where (A) is pure GNPs, (B) is cysteine-encapsulated GNPs, and (C) is green-synthesized GNPs-F3;

[0053] Figure 2The UV-Vis absorption spectra of the conjugated compounds formed by GNPs-Cys combined with different plant extracts, wherein (D) is the carboxyl group in the methanol plant extract, (E) is the hydroxyl group in the hexane plant extract, (F) is the carbonyl group in the methanol plant extract, (G) is the carbonyl group in the hexane plant extract, (H) is the hydroxyl group in the methanol plant extract, and (I) is the hydroxyl group in the hexane plant extract;

[0054] Figure 3 , Figure 4 FTIR transmission graphs of GNPs and their conjugated compounds, where (A) is pure GNPs, (B) is cysteine-capped GNPs, (C) is green synthesized GNPs-F3, (D) is the carboxyl group of phytochemicals in methanol plant extracts, (E) is the carboxyl group of phytochemicals in hexane plant extracts, (F) is the carbonyl group of phytochemicals in methanol plant extracts, (G) is the carbonyl group of phytochemicals in hexane plant extracts, (H) is the hydroxyl group of phytochemicals in methanol plant extracts, and (I) is the FTIR transmission graph of GNPs conjugated with the hydroxyl group of phytochemicals in hexane plant extracts;

[0055] Figure 5 DLS graphs of pure GNPs (A), cysteine-capped GNPs (B), and green synthesized GNPs-F3 (C), and DLS graphs of GNPs conjugated with carboxyl groups of phytochemicals in methanol plant extracts (D), carboxyl groups of phytochemicals in hexane plant extracts (E), carbonyl groups of phytochemicals in methanol plant extracts (F), carbonyl groups of phytochemicals in hexane crop extracts (G), hydroxyl groups of phytochemicals in methanol plant extracts (H), and hydroxyl groups of phytochemicals in hexane plant extracts (I);

[0056] Figure 6 XRD diffraction patterns of pure GNPs (A), cysteine-capped GNPs (B), and green synthesized GNPs (C);

[0057] Figure 7 is the SEM image of GNPs;

[0058] Figure 8 SEM images of cysteine-capped GNPs at 1 μm, 400 nm, 200 nm, 100 nm, and 30 nm scales;

[0059] Fig. 9 SEM images of green synthetic GNPs at 1μm, 400nm, 200nm, 100nm and 30nm scales;

[0060] Fig.10 The histopathological results of the liver.

[0061] Fig.11 .Histopathology results of one kidney. PIC2423: Slide part 1. PIC2424: Slide part 2 PIC2425: Slide part 4 PIC2426: Slide part 4. PIC2587: Normal slide part 1. PIC2588: Normal slide part 2.

[0062] Fig.12 .Histopathological examination of the heart.

[0063] Fig.13 :LJ slant shows growth in positive control (P). No growth in negative control (N). F1, F2, F3, F4: plant extracts. M2 and M6: GNPs combined with phytochemicals showing MTB growth in doses from 5 to 50 μl. Vials labeled M1, M3, M4 and M5 showed no growth in the presence of 50 μl GNPs.

[0064] Fig.14 : LJ vials showed no growth in F1, F2, F3, F4, M1, M2, M3, M4, M5, and M6 with 100 μl of nanoconjugated plant extract and GNPs at a concentration of 4.28 μg / μl.

[0065] Fig.15 . Antimycobacterial effect of green synthetic GNPs particles at 150 μl and 200 μl concentration (4.28 μg / μl) against MDR strains. F1, F2, F3, F4, F5, F6 represent M1, M2, M3, M4, M5, M6 (see Table 1). GC: Growth control (positive). DETAILED DESCRIPTION

[0066] The present invention will be further described below in conjunction with the accompanying drawings and Examples. According to the following examples, the present invention can be better understood. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0067] The embodiments of the present invention use Fourier transform infrared spectroscopy (FTIR), ultraviolet visible spectroscopy (UV-Vis), energy dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), scanning electron microscopy (SEM) and X-ray diffraction (XRD) to characterize the synthesized GNPs, and design an in vitro model to determine the anti-tuberculosis ability of F. arabica conjugated GNPs using the Lowenstein Jensen slope method and the MGIT culture method recommended by the World Health Organization.

[0068] Example

[0069] 1. Preparation of Plant Extracts

[0070] Phytochemicals with antimicrobial activity were extracted by decoction, maceration and infusion. F. arabica was dried in a cool place for 1 to 2 weeks. The dried plant was ground into a fine powder using a mortar. F. arabica extracts were prepared in water, methanol and hexane.

[0071] 1.1 Water-based extracts

[0072] Green plants (F. arabica) were collected from the central Pakistani town of Derighazi Khan, washed with deionized water, and then ground into a paste. About 40 mg of the plant paste was added to 100 ml of deionized water and heated on a hot plate for 20 minutes. The mixture was cooled at room temperature. About 60 ml of the plant extract was collected and purified by filtration. The plant aqueous extract was stored at -20°C for future use.

[0073] 1.2 Methanol extract

[0074] About 100 g of powdered F. arabica plant extract from the Institute of Molecular Biology and Biotechnology Laboratory, University of Lahore, Pakistan (F. arabica was dried in a cool place for 1 to 2 weeks and the dried plant was ground into a fine powder using a mortar) was added to 500 ml of methanol. The mixture was kept at room temperature with continuous shaking for one week. The mixture was filtered and 80 ml of methanol plant extract was freeze-dried and stored at -20°C for future use.

[0075] 1.3 Hexane extract

[0076] About 100 g of powdered F. arabica plant extract from the Institute of Molecular Biology and Biotechnology Laboratory, University of Lahore, Pakistan (F. arabica was dried in a cool place for 1 to 2 weeks and the dried plant was ground into a fine powder using a mortar) was added to 500 ml of hexane. The mixture was placed at room temperature and continuously shaken for one week. The mixture was filtered and 40 ml of the hexane plant extract was freeze-dried and stored at -20°C for future use.

[0077] 2. Synthesis of conjugated compound GNPs-F

[0078] The conjugated compound GNPs-F was prepared by two methods: green synthesis and chemical synthesis. In the first method, i.e., green synthesis, GNPs were capped with phytochemicals, and cysteine ​​was not used as a linker, because there are many phytochemicals attached to GNPs at different positions and in different proportions.

[0079] In the second method, chemical synthesis, GNPs are first capped with cysteine, which then acts as a linker to connect the functional groups of phytochemicals in a fixed ratio.

[0080] These two methods are described below.

[0081] 2.1 Polar-based conjugated compound GNPs-F 极 Green synthesis

[0082] The green synthesis method is to add plant extracts to a gold salt solution (tetrachloroauric acid, HAuCl 4 ) was used to prepare GNPs-F. This example optimizes the procedure (concentration, pH value and temperature).

[0083] The specific method is as follows:

[0084] (1) The whole F. arabica plant was used for the biotechnology of preparing GNPs-F, including leaves, bark, stems, roots, etc. The plant was washed with distilled water, ground into a paste, and then boiled in a universal solvent (deionized water) to prepare a water-based plant extract F3, which was purified by filtration.

[0085] (2) GNPs were synthesized using different ratios of gold salt and extract (0.93 mol / 5 ml, 0.95 mol / 4 ml, 0.75 mol / 3 ml, 0.83 mol / 3 ml and 0.85 mol / 6 ml) at different temperatures (100°C to 110°C) and pH levels (pH 5 to 6.4). About 3 ml of plant extract was mixed with 0.83 M gold salt solution at room temperature to synthesize GNPs. Phytochemicals are reducing and stable, therefore, no external sealant is required. The color change of the reaction mixture was visually monitored, and then GNPs-F3 was collected and further used after high-speed centrifugation at 10,000 rpm.

[0086] According to the test results, in the subsequent related test experiments, the water-based conjugated compound GNPs-F3 was obtained by using a ratio of 0.83M:3ml. That is, 3ml of plant extract was mixed with 0.83M gold salt solution, pH = 6.4, to synthesize the water-based conjugated compound GNPs-F3 (denoted as F4), and then the synthesized conjugated compound was characterized as described in Section 2.1.1. The results are as follows Figure 1 (C) shown.

[0087] 2.1.1 Feature description;

[0088] The synthesized GNPs were characterized by UV-Vis spectroscopy, FTIR, DLS, SEM and XRD. The liquid samples of GNPs were characterized by UV-Vis spectroscopy and DLS, and the powdered GNPs were characterized by XRD, SEM and FTIR.

[0089] 2.1.1.1 UV-Vis Spectroscopy

[0090] This quantitative technique measures the intensity of light passing through a sample compared to the intensity of light passing through a blank. The spectrophotometer used for nanocomposite analysis (BK-UV1900PC, BIOBASE) provides accurate, high-performance results.

[0091] 2.1.1.2 FTIR spectroscopy

[0092] FTIR spectroscopy, known as infrared spectroscopy, is used to identify organic, inorganic and polymer materials. Infrared light is passed through the test sample to measure its chemical properties. The infrared spectrum shows various functional groups and provides the chemical composition and physical state of the test sample.

[0093] 2.1.1.3DLS spectroscopy

[0094] DLS is a method of assessing how much light is reflected by particles in a solution. As particles move away from their starting position due to Brownian motion, the reflected light will change over time. This will be at different rates depending on the hydrodynamic radius of the particle. Therefore, the size of the suspended particles can be determined from the measurement of the rate of change of reflected light. A graph of reflected light intensity versus particle size is used to display the results of DLS.

[0095] 2.1.1.4XRD technology

[0096] XRD is a technique used to determine the crystalline properties of materials based on Bragg's law (n = 2dsin). XRD is a broad and non-discriminatory method for characterizing the crystalline properties of materials. It detects structural performance data of nanocomposites, including preferred crystal orientation (texture), phase purity, and other structural indicators, including average grain size, crystallinity, strain, and crystal defects. XRD peaks are generated by positive interference when monochromatic X-rays in the sample diffuse from each pair of lattice planes at a specific angle. The peak intensity is determined based on the atomic position within the lattice plane. Powder XRD analysis is used to detect unknown crystalline compounds in fields such as geology, materials science, environmental science, and engineering.

[0097] 2.1.1.5 SEM microscope

[0098] This technique is very useful for characterizing the size of particles and generating magnified images. The electron beam scans the sample to produce a magnified image for analysis. It is also known as SEM microscopy. It provides information about the surface morphology and composition of the sample and has several advantages. Because it can analyze the size of at least 10nm in transmission mode (T-SEM) compared to analyzing nanoparticles on bulk substrates. SEM microscopy is highly recommended, valuable, traceable and accurate for characterizing nanocomposites. During the electron-sample interaction, SEM shows information about the structure, chemical composition, size and surface morphology. EDX analyzes the composition of nanoparticles (energy dispersive X-rays) and the sharp peaks represent nanoparticles.

[0099] 2.1.1.6 EDX spectroscopy

[0100] EDX microanalyzers can determine the elements and their concentrations in a sample. This method relies on the unique X-ray signature emitted by a material subjected to accelerated electrons. Atoms in a sample may be broken apart by the high-energy incident electrons, which causes the atoms to ionize. When the outer electrons fill the inner shell, X-ray photons are produced.

[0101] 2.2 Polar-group conjugated compound GNPs-F 极 Chemical synthesis

[0102] First, it is necessary to synthesize gold nanoparticles GNPs. In this embodiment, as an example, the turkevich method is used to prepare chemical GNPs. In the embodiment of the present invention, the synthesis method of gold nanoparticles GNPs is not limited.

[0103] As described by Turkevich and Frens, the standard method is to reduce citrate at 100°C, specifically comprising: reducing 12.5 ml of 25 mM gold hydrochloride solution with 12.5 ml of 1% sodium citrate by boiling the gold solution in a beaker on a hot plate. In this example, reflux was not used to avoid temperature gradients in the liquid, and when the solution began to boil, about 12.5 mL of warm 1% sodium citrate solution was added (in order to produce different particle sizes, we adjusted the citrate content (replacing cysteine ​​with citrate)). After 15 minutes, when the solution turned brick red, the liquid was cooled to room temperature to obtain gold nanoparticles GNPs, recorded as pure GNPs, and the synthesized GNPs were then characterized as described in Section 2.1.1. The results are shown in Figure 2. Figure 1 (A) shown.

[0104] 2.2.1 Covering GNPs with Linkers

[0105] Cysteine ​​Cys was used as a linker in chemically prepared GNPs.

[0106] About 49.95 M cysteine ​​solution (adjusted to pH 7.4) was prepared, and about 2.5 ml of cysteine ​​solution was added to the chemically synthesized GNPs and kept in the dark at 37 °C for 2 h with continuous shaking at 100 rpm, followed by standing for 24 h to obtain cysteine-covered GNPs, i.e., GNPs-Cys, which were characterized as described in the previous section.

[0107] The GNPs were capped with cysteine ​​to bind the carboxyl, aldehyde, and hydroxyl groups of phytochemicals.

[0108] 2.2.2 Binding of phytochemicals to carboxyl groups:

[0109] Phytochemicals were cross-linked to cysteine-linked GNPs using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) technique.

[0110] The specific method includes: dissolving 50 mg of cysteine-capped GNPs in 1 ml of deionized water (adjusting pH to 8 using 1 M sodium hydroxide); for carboxyl activation, adding 1 ml of EDC (20 mg / ml deionized water) (adjusting pH to 6.4 using 1 M HCl). After the reaction mixture is continuously stirred at 100 rpm in the dark for 30 minutes, 1 ml of EDC (10 mg / ml deionized water) and 50 mg of freeze-dried methanol plant extract F2 or hexane plant extract F1 are added, and the mixture is continuously stirred at 100 rpm in the dark at 37°C for 2 hours, and then the conjugated particles are separated by centrifugation at 14,000 rpm for 20 minutes, and then washed twice with deionized water. The washed particles are conjugated with non-polar plant extract F2. 非极 The carboxyl-bound nonpolar conjugated compounds GNPs-Cys-F2-carboxyl (denoted as M1) or GNPs-Cys-F1-carboxyl (denoted as M2) were stored at 4°C and then characterized as described in Section 2.1.1.

[0111] 2.2.3 Binding of phytochemicals to aldehyde groups

[0112] A buffer solution was prepared using 0.05 M sodium carbonate and 0.1 M sodium citrate at a pH of 9.5. The amine containing GNPs that had to be conjugated with the phytochemicals having aldehyde groups were dissolved in the buffer solution at a molar ratio of 1:4 with methanol plant extract F2 or hexane plant extract F1 at a concentration of 1-10 mg / ml, and the pH was maintained at 7-10. Sodium cyanoborohydride was added to the conjugated solution to obtain a conjugated solution containing 5 M sodium cyanoborohydride per ml, which was then placed in sodium hydroxide. After standing for 2 hours, 3 M ethanolamine was added to each ml of the conjugated solution to block the unreacted aldehyde. The particles were purified by dialysis for 15 minutes and then stored at 4°C. These synthesized plant extracts F with non-polar groups 非极 The carbon-based conjugated non-polar group conjugated compounds GNPs-Cys-F2-carbon-based (denoted as M3) or GNPs-Cys-F1-carbon-based (denoted as M4) were characterized according to the method described in the previous section.

[0113] 2.2.4 Binding of phytochemicals to hydroxyl groups

[0114] Prepare approximately 0.1M sodium carbonate coupling buffer at a pH of 9.5. Frozen methanol plant extract F2 or hexane plant extract F1 was suspended at 5% w / v in a tetrahydrofuran (THF) suspension containing 50mg / ml carbonyldiimidazole (CDI) and stirred at room temperature for 2 hours. The activated particles were washed with THF to remove excess CDI and byproducts, and then washed with ice-cold deionized water to remove any solvent, and then resuspended in cold coupling buffer (0.01M pyridine-HCL, pH: 6.00, 0.1M NaCl) at 10mg / ml. Then 1-10mg of amine-containing GNPs (i.e., GNPs-Cys) was added and stirred at 4°C for at least 18 hours for reaction. Finally, 0.1M ethanolamine was added and stirred for 2 hours. The particles were centrifuged and suspended in 0.1M sodium carbonate coupling buffer and washed 3 times with 0.1M sodium carbonate coupling buffer. The non-polar plant extract F was obtained. 非极 The hydroxyl-bound non-polar group conjugated compounds GNPs-Cys-F2-hydroxyl (denoted as M5) or GNPs-Cys-F1-hydroxyl (denoted as M6) were then characterized as described in Section 2.1.1 above.

[0115] 3. Results

[0116] 3.1 UV-Vis spectrophotometry

[0117] Nine samples (pure GNPs (A), cysteine ​​encapsulated GNPs i.e. GNPs-Cys (B), green synthesized GNPs-F3 i.e. F4 (C), carboxyl groups of phytochemicals in methanol plant extracts i.e. M1 (D), carboxyl groups of phytochemicals in hexane plant extracts i.e. M2 (E), aldehyde groups of phytochemicals in methanol plant extracts i.e. M3 (F), aldehyde groups of phytochemicals in hexane crop extracts i.e. M4 (G), hydroxyl groups of phytochemicals in methanol plant extracts i.e. M5 (H) and hydroxyl groups of phytochemicals in hexane plant extracts i.e. M6 (I)) were tested by UV-Vis.

[0118] A peak was observed at 500.5 nm for GNPs prepared from sodium tricitrate ( Figure 1 A), the peak of GNPs coated with cysteine ​​is at 529 nm ( Figure 1 B). GNPs synthesized using F. arabica reduced gold showed an increase in the wavelength at 493 nm ( Figure 1 C). Considering the error, the maximum absorbance of GNPs is at 500nm, 529nm and 493nm ( Figure 1 The absorption peaks of GNPs combined with phytochemicals showed significant changes ( Figure 2 ).

[0119] It was found that the monodispersity of M1 increased (Table 1) with maximum absorbance at 312nm, 352nm, 546nm and 672nm ( Figure 2 D). M2 conjugate shows peaks at 276nm and 294nm ( Figure 2 E). In contrast, M3 showed maximum absorbance at 426 nm and 384 nm ( Figure 2 F). The absorption peaks of M4 and M5 are at 286nm and 400nm respectively ( Figure 2 G) and 274nm and 292nm ( Figure 2 In M6, GNPs were conjugated with hydroxyl groups of hexane plant extracts and the maximum absorbance was obtained at 282 nm ( Figure 2 I).

[0120] 3.2 FTIR analysis of GNPs

[0121] FTIR analysis of GNPs was performed on nine samples to analyze the different functional groups of F. arabica extract involved in the coupling of phytochemicals synthesized in cysteine-capped GNPs.

[0122] The spectrum shows that GNPs are at 1621.85 cm -1 The peak at 3261.91 cm shows C=C stretching (conjugated olefin group). -1, showing OH stretching (carboxylic acid group) ( Figure 3 A). Cysteine-terminated GNPs showed many peaks ( Figure 3 B), including 3564.39cm -1 The OH peak at 1733.13 cm -1 The C=O stretch at 2884.59 cm -1 The CH peak and 1715.24cm -1 The C=O peak at 3617 cm-1 confirmed the presence of aldehyde groups. In addition to carboxyl and aldehyde groups, we also observed hydroxyl groups at 3617 cm-1. -1 The peak is formed at Figure 3 ).

[0123] During the coupling of phytochemicals to cysteine-terminated GNPs, carboxyl groups were activated using carbodiimide. It was conjugated with methanol and hexane extracts of F. arabica ( Figure 3 D; Figure 3 E), during which we observed OH stretching (3564.39 cm -1 ) disappears and at 3255.25cm -1 and 3393.68cm -1 At the same time, the C=C peak is at 1734.11cm -1 and 1734.20cm -1 remains unchanged ( Figure 4 ).

[0124] Sodium cyanoborohydride was used to activate the aldehyde groups of cysteine-terminated GNPs. When these activated GNPs were conjugated with methanol and hexane extracts of F. arabica ( Figure 3 F and Figure 4 G), we are at 2884.59cm -1 A CH peak was observed.

[0125] In contrast, the GNPs bound to the methanol extract showed only a peak at 1734.42 cm -1 The C=O peak was observed at Figure 4 ). Following activation of the hydroxyl groups of cysteine-terminated GNPs using carbonyl imidazole, the conjugated methanol and hexane extracts ( Figure 4 H, Figure 4 I) shows that OH from 3617cm -1 Down to 3391.06cm -1 and 3247.91cm -1 , confirming the disappearance of hydroxyl groups. The FTIR spectrum of green synthesized GNPs confirmed the presence of amines and phenols, with the NH peak at 3232.17 cm -1, CN peak is located at 1579.58cm -1 , OH peak is located at 3444.42cm -1 , CO peak is located at 1077.55cm -1 (confirm Figure 3 C).

[0126] DLS analysis

[0127] DLS was used to determine the size and dispersion of GNPs.

[0128] The particle size is spherical with a diameter of 34 nm ( Figure 5 A), while the smaller peak at 282 nm shows the aggregation of particles due to delayed characterization after particle formation. Similarly, due to aggregation, the cysteine-capped GNPs ( Figure 5 ) showed a size range of 25nm to 168.8nm. In contrast, the green synthesized GNPs with the reducing power of F. arabica plant were 2nm ( Figure 5 C). There are very small particles and more aggregation as they are all cysteine-capped GNPs coated with different phytochemicals showing the GNPs conjugated with the carboxyl groups of methanolic plant extract phytochemicals ( Figure 5 DH). Due to conjugation and particle aggregation, the diameters of all these conjugated GNPs vary from 0.2 nm to thousands of nm ( Figure 5 ).

[0129] 3.4 XRD

[0130] The XRD diffraction pattern shows that the 2θ values ​​in the (111), (200), (220) and (311) planes are 38.12°, 44.32°, 64.54° and 77.72° ( Figure 6 ). Crystalline GNPs have been characterized by four peaks corresponding to the standard Bragg reflections (111), (200), (220) and (311) of the face-centered cubic lattice. The strong peak at 38.12 represents the preferential growth in the (111) direction.

[0131] In addition, the characteristic peak of Au+ signal at 2θ = 38.12° confirmed the presence of Au+ in the GNPs nanocomposite. However, there is an overlap of Au+ signal with the wurtzite plane at (311). The peaks at 2θ = 38.12°, 44.32°, and 64.54° are assigned to the (111), (200), and (220) planes of metallic gold ( Figure 6 ). The XRD peaks of GNPs indicate the formation of clear and distinct phases. The appearance of discrete phases of GNPs reveals the synthesis of crystalline GNPs. The identified peaks represent the purity of GNPs.

[0132] 3.5 SEM

[0133] Figure 8 , 9 and 10 illustrate the morphological characteristics of the nanocomposites and the elemental composition using SEM equipped with EDX. They show the surface morphology of the nanocomposites at different magnifications of 30nm, 100nm, 200nm, 400nm and 1μm. The magnified image shows irregular spherical shapes of GNPs with a diameter of approximately 30-40nm ( Figure 7 ). SEM reveals information about solid samples when a high-energy electron beam forms a signal array focused on a target sample. Cysteine-terminated GNPs observed under SEM showed an irregular cubic morphology with a diameter of approximately 35-40 nm ( Figure 8 ). The green synthesized GNPs showed aggregated particles of different sizes. The approximate diameter of the particles was less than 10 nm ( Fig. 9 ).

[0134] 3.6 EDX

[0135] The elemental composition of the synthesized GNPs was obtained using EDX analysis. EDX confirmed the presence of Au and other elements (Table 1).

[0136] Gold present in pure GNPs constitutes 13.5% of the total weight of pure GNPs formed; other elements present in GNPs (in larger weight percentages) are oxygen, nitrogen and carbon.

[0137] The total weight percentage of gold present in cysteine-capped GNPs (GNPs-Cys) was 53.19%, while the weight percentages of carbon, oxygen, and sodium elements present were 12.9%, 14.5%, and 14.3%, respectively.

[0138] The weight percentage of gold in green synthesized GNPs-F3 is 4.3%, and along with GNPs, other elements such as carbon, oxygen, and sodium are also present in the weight percentages of 18.0%, 25.6%, and 2.6%.

[0139] Table 1. Elements and their weight % within GNPs types

[0140]

[0141] 4. In vivo cytotoxicity evaluation of F. arabica and GNPs nanoconjugates

[0142] The cytotoxicity of the synthesized F.arabica and GNPs nanoconjugates was studied in a rat model. The effects of nanoconjugate toxicity were examined in the rat liver, heart, and kidney. Blood tests and histopathological examinations were performed to observe the toxicity levels compared to standard controls. All rat groups were treated with F.arabica extracts and synthesized GNPs at different concentrations (5μl, 50μl, 100μl, and 150μl). 15 albino rats were arranged in groups A, C, and D, each containing 5 rats. Four rats in each group were treated with F.arabica and synthesized GNPs, and one rat in each group (total = 5) was kept as a spare in case of rat death.

[0143] Table 2. Elements and their weight % within GNPs types

[0144] Group Number of rats Dosage type Group A 5 Nanocoupling of F.arabica and GNPs Group C 5 F.arabica plant extract Group D 5 Negative control group (not treated with F.arabica and / or GNPs nanoconjugate)

[0145] Histopathological findings of the liver Fig.10 As shown, each slide is divided into different sections during microscopic examination. PIC2409: Section 1 of the slide. PIC2410: Section 2 of the slide. PIC2411: Section 3 of the slide. PIC2412: Section 4 of the slide. The other slides below are slides for the normal / control group PIC2394: Section 1 of the normal slide and PIC2394: Section 2 of the normal slide.

[0146] The histopathological results of the liver showed no significant difference in the effects on the rat liver between the normal group (control group) and the rats treated with GNPs conjugated with F. arabica phytochemicals.

[0147] Histopathological findings of one kidney Fig.11 Shown, PIC2423: Slide Part 1. PIC2424: Slide Part 2 PIC2425: Slide Part 1 PIC2426: Part 4. PIC2587: Common Slide Part 1. PIC2588: Common Slide Part 2.

[0148] The histopathological results of the kidneys showed no significant difference in the effects on the liver between the normal group (control group) and the rats treated with GNPs conjugated with F. arabica phytochemicals.

[0149] Histopathological examination of the heart Fig.12 As shown,

[0150] The top four sections are: PIC2439: Slide Section 1; PIC2441: Slide Section 2; PIC2438: Slide Section 3; PIC2440: Slide Section 4. The bottom two are: PIC2582: Regular Slide Section 1. PIC2581: Regular Slide Section 2

[0151] The histopathological results of the heart showed no significant difference in the effects on the liver between the normal group (control group) and the rats treated with GNPs conjugated with F. arabica phytochemicals.

[0152] 5. Antifungal (inhibitory) activity of Arabica powder and GNPs nanoconjugates on Lowenstein-Jenson medium

[0153] To avoid contamination, a biosafety level 3 (BSL-3) laboratory was used to examine the antifungal activities of the synthetic GNPs and F. arabica extracts.

[0154] The laminar flow cabinet was disinfected with ethanol and sterilized with UV light. After putting on a lab coat, mask, and gloves and completing all the protocols of the BSL-3 laboratory, some autoclaved bottles were removed and labeled. Then LJ medium was prepared using the standard protocol and slants containing 8 ml of LJ culture were prepared. The solid slants of LJ medium were inoculated with H37Rv and resistant strains of MTB, except for the negative control. The slants were kept horizontal for almost 48 to 72 hours to allow the inoculated MTB to be evenly distributed on the loose LJ medium.

[0155] MTB showed non-pigmented colonies with a rough and dry texture, which was further confirmed by smear microscopy and immunochromatographic (ICT) testing. In smear microscopy, zinc-stained smears were prepared and observed under a microscope; the presence of cord factor (CF) confirmed MTB. For ICT testing, 200 μl of cultured MTB was added to the sample pad using a TB Ag MPT64 rapid test device, and after 15 minutes, the presence of MTB was confirmed by the appearance of the test line and the control line.

[0156] After observing the growth of MTB on the culture medium, the culture was induced with F. arabica extract and GNPs were synthesized at concentrations of 5 μl, 50 μl, and 100 μl as shown in Table 3. The growth of MTB was observed after 25 to 45 days.

[0157] The LJ slope shows the growth of the positive control group (P) Fig.13Negative control group showed no growth (N); F1, F2, F3, F4: plant extracts. M2 and M6: GNPs combined with phytochemicals showed MTB growth at doses from 5 to 50 μl. Vials labeled M1, M3, M4 and M5 showed no growth in the presence of 50 μl GNPs.

[0158] The antimycobacterial activity results of F. arabica crude extract and GNPs combined with phytochemicals are shown in ( Fig.13 , 14 ). Hexane, methanol, and F. arabica aqueous extracts inhibited bacterial growth at a dose of 100 μl. In contrast, doses less than 100 μl had no significant inhibitory effect on mycobacteria grown on LJ medium. When used in amounts of 5 μl and 50 μl, green synthesized and conjugated GNPs showed lower inhibitory effects, except for F4, M2, and M6, while all LJ vial slants showed no growth of resistant Mtb when a concentration of 100 μl was used.

[0159] like Fig.14 As shown, LJ vials showed no growth in F1, F2, F3, F4, M1, M2, M3, M4, M5, and M6 with 100 μl of nanoconjugated plant extract and GNPs at a concentration of 4.28 μg / μl.

[0160] like Fig.15 Shown is the anti-mycobacterial effect of green synthetic GNPs particles at 150 μl and 200 μl concentration (4.28 μg / μl) against MDR strains, F1, F2, F3, F4, F5, F6 represent M1, M2, M3, M4, M5, M6 (see Table 3). GC: Growth control (positive).

[0161] The F. arabica extracts bound to different GNPs were poured into each MGIT tube at 150 μl and 200 μl volumes (concentration 4.28 μg / μl). The results of each MGIT tube culture showed that the F. arabica phytochemicals bound to GNPs had good inhibitory activity against MDR strains. Growth control (GC) ( Fig.15 ) showed more Mtb growth, while other MGIT tubes containing F. arabica extract combined with GNPs showed no Mtb growth even after 8 to 25 days. These results suggest that our new formulation GNPs combined with F. arabica phytochemicals have potential inhibitory activity against MDR strains of M. tuberculosis and therefore have the potential to be used as an anti-TB agent to better control drug-resistant strains.

[0162] 6. Antibacterial (inhibitory) activity of F. arabica and GNPs nanoconjugates targeting multidrug resistance in MGIT

[0163] To further confirm the antifungal activity, multidrug resistant strains (MDR) of MTB were cultured and induced in MGIT tubes, and GNPs with two different concentrations of 150 μl and 200 μl (4.28 μg / μl) were synthesized with extracts of F. arabica (Table 3). The growth of MTB in MGIT tubes was monitored daily for 16 days.

[0164] Table 3. Description of F. arabica and GNPs extracts

[0165] Serial number Label describe 01 F1 Hexane Plant Extracts 02 F2 Methanol plant extract 03 F3 Water plant extract 04 F4 F.arabica (green synthesis) GNPs 05 M1 GNPs conjugated with carboxymethanol 06 M2 Binding of GNPs to carboxyl groups of hexane 07 M3 GNPs combined with carbon groups in methanol 08 M4 Carbon-based binding of GNPs to hexane 09 M5 GNPs bind to hydroxyl groups in methanol 10 M6 GNPs bind to the hydroxyl groups of hexane

[0166] Pour 200 μl of MDR MTB positive culture into each MGIT tube ( Fig.15 ). F. arabica extracts coupled with different GNPs in 150 μl and 200 μl volumes (concentration 4.28 μg / μl) were also poured into each MGIT tube. The results of each MGIT tube culture showed that the F. arabica phytochemicals combined with GNPs had good inhibitory activity against MDR strains.

[0167] Growth Control (GC) Fig.14 , Fig.15 ) had adequate MTB growth, whereas other MGIT tubes containing F. arabica extracts combined with GNPs showed no MTB growth even after 8 to 25 days (including blank). These results suggest that our novel GNPs coupled with F. arabica phytochemicals have potential inhibitory activity against MDR strains of MTB. They can be used as anti-TB agents to better manage drug-resistant strains.

[0168] In summary, the present invention used a novel formulation of biologically and chemically synthesized gold nanoparticle conjugate compounds (GNPs-F) to conjugate with phytochemicals from F. arabica in hexane, methanol, and water solvents to determine its inhibitory potential against MTB. In vivo toxicity was tested in a rat model, verifying that this plant extract did not have any significant toxic effects on the liver, heart, and kidneys. In addition, when used as targeted delivery with GNPs in Lowenstein-Jenson medium and World Health Organization MGIT medium, the anti-MTB potential of T. arabica extract was significantly increased ( Fig.11 , 12, 13). It fully shows that the F. arabica phytochemicals coupled with GNPs have a good inhibitory effect on drug-resistant and MDR strains of Mycobacterium tuberculosis. These results show that our new GNPs formulation combined with F. arabica phytochemicals can be used to combat MTB resistant strains.

[0169] Through the above experimental analysis, the present invention can draw the following conclusions:

[0170] a. The present invention proposes for the first time that F. arabica has an inhibitory ability against Mycobacterium tuberculosis strains.

[0171] b. Comparative effects of methanol, hexane, and water-based extracts showed that the inhibitory potential of hexane extracts was more significant than that of methanol extracts, and the inhibitory capacity of methanol extracts was greater than that of water-based extracts.

[0172] The ability of cFarabica to fight Mycobacterium tuberculosis can be enhanced by plant-made gold nanoparticles (GNPs) using water-based extracts (green synthesis of GNPs).

[0173] d. Six nanoformulations were synthesized by reacting carboxyl-containing phytochemicals extracted by methanol (M1), carboxyl-containing phytochemicals extracted by hexane (M2), aldehyde-containing phytochemicals extracted by methanol (M3), aldehyde-containing phytochemicals extracted by hexane (M4), hydroxyl-containing phytochemicals extracted by methanol (M5), and hydroxyl-containing phytochemicals extracted by hexane (M6).

[0174] e. All methanol extract-based nanoformulations showed strong inhibitory potential against drug-resistant strains of Mycobacterium tuberculosis.

[0175] f. At low concentration (50 μl), the hexane extract-based nanoformulation M4 (aldehyde-conjugated nanoformulation) had a stronger inhibitory potential than the other two hexane extract-based nanoformulations M2 and M6.

[0176] g. At low concentrations, the methanol extract-based phytochemical nanoformulations (M1, M3, and M5) had stronger effects than the hexane extract-based phytochemical nanoformulations (M2, M4, and M6).

[0177] h. At low concentrations, the specific nanoformulations showed stronger antimycobacterial potential than methanol extract, hexane extract, and plant-prepared GNPs (green synthesis).

[0178] i. At higher concentrations of nanoformulation (100 μl, 150 μl and 200 μl), drug-resistant Mycobacterium tuberculosis did not grow.

[0179] j. All nanoformulations (GNPs and F. arabica phytochemicals) also showed good inhibitory activity against multidrug-resistant strains of Mycobacterium tuberculosis.

[0180] Therefore, the F. arabica extract, the conjugated compound GNPs-F and the pharmaceutical composition thereof of the present invention have the potential to be applied to the anti-Mycobacterium tuberculosis activity or to the preparation of drugs for treating tuberculosis.

[0181] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a conjugated compound GNPs-F for inhibiting Mycobacterium tuberculosis, characterized in that: The method comprises: coating gold nanoparticles GNPs with phytochemicals to form conjugated compounds GNPs-F, wherein the phytochemicals are Fagonia arabica extract, denoted as F; Said Fagonia arabica Extracts from F. arabica The plant material is dissolved in a solvent and filtered, wherein the F. arabica Plant materials include F. arabica Plant powder or F. arabica The plant paste after grinding the plant, the solvent includes a polar solvent or a non-polar solvent, and the polar base plant extract F is obtained respectively. 极 , Non-polar plant extract F 非极 ; The polar solvent is water, and the non-polar solvent is methanol or hexane; The synthesis method of the gold nanoparticles GNPs is to reduce citrate at 100° C., specifically: boiling the gold solution, reducing 12.5 ml of 25 mM hydrochloric acid gold solution with 12.5 ml of 1% sodium citrate, and when the solution turns brick red, cooling to room temperature to obtain the gold nanoparticles GNPs; The polar base plant extract F 极 Gold nanoparticles (GNPs) were coated using a green synthesis method, including the following steps: 3-6 ml of polar plant extract F 极 Mix with 0.7-1.0 M gold salt solution to synthesize polar-based conjugated compound GNPs-F 极 , the gold salt is tetrachloroauric acid; The non-polar plant extract F 非极 The gold nanoparticles GNPs are coated by chemical synthesis, including the following steps: (1) Covering GNPs with cysteine ​​Cys as a linker to obtain gold nanoparticles with a linker GNPs-Cys, specifically comprising: preparing a 49.95 M cysteine ​​solution, adjusting the pH to 7.4, adding 2.5 ml of the cysteine ​​solution to the chemically synthesized GNPs, and keeping it in the dark at 37°C for 2 hours, continuously shaking at a speed of 100 rpm, and then standing for 24 hours to obtain cysteine-covered GNPs, namely GNPs-Cys; (2) In an organic solvent, the gold nanoparticles with linkers GNPs-Cys are combined with the non-polar plant extract F through the linker 非极 The carboxyl, carbonyl or hydroxyl groups in the synthesized non-polar conjugated compound GNPs-Cys-F 非极 , wherein the organic solvent is a suspension containing carboxyl, carbonyl or hydroxyl, wherein the carboxyl-containing suspension is 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide, the carbonyl-containing suspension is sodium cyanoborohydride, and the hydroxyl-containing suspension is carbonyldiimidazole-tetrahydrofuran; The combination of the non-polar plant extract F 非极 The method comprises: adding 0.5-5 ml of 20 mg / ml EDC aqueous solution and 30-80 mg of freeze-dried non-polar plant extract F to 2.5 ml of GNPs-Cys aqueous solution. 非极 Cross-linking was performed, stirring was continued for 1-5 hours in the dark at 25-45 ℃, and after centrifugation and washing, the non-polar plant extract F was obtained. 非极 Carboxyl-bound nonpolar conjugated compound GNPs-Cys-F 非极-羧基 ; The combination of the non-polar plant extract F 非极 The carbon-based method includes: GNPs-Cys, non-polar plant extract F 非极 The conjugate solution was dissolved in a sodium carbonate-sodium citrate buffer at a molar ratio of 1: 1-5 and a concentration of 1-10 mg / ml to obtain a conjugate solution, the pH was maintained at 7-10, sodium cyanoborohydride was added to obtain a conjugate solution containing 5M sodium cyanoborohydride per ml, and then added to sodium hydroxide. After standing for 1-3 hours, 3M ethanolamine was added to each ml of the conjugate solution to block the unreacted aldehyde. After purification by dialysis, a non-polar plant extract F was obtained. 非极 Carbon-based non-polar conjugated compound GNPs-Cys-F 非极-碳基 ; The combination of the non-polar plant extract F 非极 The method comprises: freeze-dried non-polar plant extract F 非极 The mixture was suspended in tetrahydrofuran suspension containing 30-70 mg / ml carbonyldiimidazole at 3% w / v, stirred at room temperature for 1-3 hours, washed with THF and water in turn, and resuspended in cold coupling buffer at 8-20 mg / ml; then 1-10 mg GNPs-Cys was added, stirred at 0-10 °C for at least 18 hours, and finally 0.1-0.3 M ethanolamine was added, stirred for 1-3 hours, and the particles were centrifuged and washed to obtain the non-polar plant extract F. 非极 Hydroxyl-bound nonpolar conjugated compound GNPs-Cys-F 非极-羟基；所述 The cold coupling buffer was 0.01 M pyridine-HCl, pH: 6.00, 0.1 M NaCl.

2. A conjugated compound GNPs-F having anti-tuberculosis activity, characterized in that: Prepared by the method described in claim 1.

3. A pharmaceutical composition having anti-tuberculosis activity, characterized in that: Comprising the conjugated compound GNPs-F according to claim 2 or a pharmaceutically acceptable form thereof.

4. The conjugated compound GNPs-F as claimed in claim 2 or the pharmaceutical composition as claimed in claim 3 is used in the preparation of a drug for treating tuberculosis.

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