CN113663068A

CN113663068A - Application of black food extract in photothermal conversion agent and preparation method thereof

Info

Publication number: CN113663068A
Application number: CN202010412612.7A
Authority: CN
Inventors: 袁苗苗; 徐洋; 章荣俊
Original assignee: Eighth Affiliated Hospital of Sun Yat Sen University
Current assignee: Eighth Affiliated Hospital of Sun Yat Sen University
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2021-11-19

Abstract

The invention provides an application of a black food extract in a photothermal conversion agent and a preparation method thereof, wherein the black food extract is extracted from black food through preset process steps, and the black food extract can be applied to preparing the photothermal conversion agent for near-infrared photothermal treatment of tumor cells. The invention discloses a new application of black food extract, which improves the economic value and social value of black food; the black food extract has low cost, simple preparation, no toxicity to organisms and high safety performance.

Description

Application of black food extract in photothermal conversion agent and preparation method thereof

Technical Field

The invention relates to the technical field of photo-thermal conversion agents, in particular to application of a black food extract in a photo-thermal conversion agent and a preparation method thereof.

Background

The morbidity and mortality of malignant tumors are the only diseases related to cardiovascular diseases, causing enormous economic loss and mental stress to people all over the world. Besides the traditional operation treatment, the photothermal treatment gradually becomes a good afterthought for treating tumors, and has wide development potential. The photothermal therapy is to inject the material with high photothermal conversion efficiency into the human body, focus the material near tumor tissue by targeting identification technology, and convert light energy into heat energy under the irradiation of an external light source (generally near infrared light) to kill cancer cells. The advantages of photothermal therapy are that the pain experienced by the patient can be reduced, the treatment time is short (about ten minutes), and the treatment effect is obvious. The key point of photothermal therapy lies in the selection of photothermal materials, mainly comprising: 1) noble metal nanoparticles such as Au, Ag, and Pt; 2) carbon-based materials such as graphene, carbon nanorods; 3) metal and nonmetal compounds such as CuS, ZnS, and cobalt oxide; 4) organic dye substances, such as indocyanine green, prussian blue. The photothermal materials have the advantages of high photothermal conversion efficiency, obvious tumor killing effect, high price, complex synthesis steps and high toxicity, and the development of the photothermal materials is severely limited.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide an application of a black food extract in a photothermal conversion agent and a preparation method thereof, which are used for solving the technical problems of high price of a photothermal material, complex synthesis steps and high toxicity in the prior art.

To achieve the above and other related objects, the present invention provides a black food extract which can be applied to the preparation of a photothermal conversion agent for near-infrared photothermal treatment of tumor cells. Wherein the black food extract comprises one or more of black bean extract, black sesame seed extract, black rice extract, black fungus extract, black mushroom extract, black grape extract, black pine nut extract, black kidney bean extract and black buckwheat extract.

In an alternative embodiment, the photothermal conversion agent comprises the black-colored food extract and a solvent.

In an alternative embodiment, the solvent comprises phosphate balanced physiological saline.

To achieve the above and other related objects, the present invention also provides a method for preparing a black food extract, the method comprising:

extracting the black food by adopting preset process steps to obtain the black food extract. Wherein the black food comprises one or more of black bean, black sesame, black rice, black fungus, black mushroom, black grape, black pine nut, black kidney bean and black buckwheat.

In an alternative embodiment, the black food comprises black rice, and the step of extracting the black food to obtain the black food extract by using the preset process steps comprises:

obtaining seed coats of the black rice;

performing acid water leaching on the seed coat to obtain an extracting solution;

centrifuging the extract to obtain a supernatant;

ultrafiltering the supernatant to obtain a filtrate;

carrying out primary nanofiltration on the filtrate to obtain a first concentrated solution;

carrying out adsorption and desorption treatment on the first concentrated solution to obtain an eluent;

carrying out secondary nanofiltration on the eluent to obtain a second concentrated solution;

and performing vacuum freeze drying on the second concentrated solution to obtain the black rice extract.

In an alternative embodiment, the step of acid leaching the seed coat to obtain an extract comprises: leaching with a disodium hydrogen phosphate-citric acid buffer solution, wherein the material-liquid ratio is 1: 20, the extraction temperature is 48-52 ℃, the pH is 2-4, and the extraction time is 3-4 h.

In an alternative embodiment, the step of subjecting the first concentrated solution to adsorption and desorption processes to obtain an eluate includes:

purifying the first concentrated solution by adopting macroporous resin, wherein the pH of a sample solution is 2-4, the adsorption flow rate is 1.0-3.0mL/min, and the balance time is 2-4 h;

eluting with ethanol to collect eluate.

In an alternative embodiment, the step of vacuum freeze-drying the anthocyanin concentrate to obtain the black rice extract includes: freeze drying the concentrated solution at-40-50 deg.C and-1.0 MPa to obtain fructus Zizaniae Caduciflorae extract powder.

The black food extract has excellent photothermal conversion effect, and can be applied to preparing photothermal conversion agents for tumor photothermal treatment;

the invention discloses a new application of black food extract, which improves the economic value and social value of black food;

the black food extract is used as a photo-thermal treatment preparation, can promote the local temperature rise of a tumor part to 50-65 ℃, achieves the effects of promoting the death of tumor cells and inhibiting the growth of tumors, simultaneously inhibits the lung metastasis in vivo, and can be used for the photo-thermal treatment of solid tumors;

the black food extract has low cost, simple preparation and almost no toxicity.

Drawings

FIG. 1 is a graph of photothermal profiles of black rice extract solutions of varying solids content.

FIG. 2 is a diagram showing the effect of photothermal tumor inhibition and temperature increase in vivo of black rice extract.

FIG. 3 is a graph showing the change of tumor relative volume in vivo photothermal tumor inhibition of black rice extract.

FIG. 4 is a graph showing the change of the photo-thermal anti-tumor effect of black rice extract in vivo and the survival rate thereof.

FIG. 5 is a photograph of an optical photograph of lung tissue metastasis of black rice extract in vivo.

FIG. 6 is a graph showing the inhibition of lung metastasis by black rice extract in vivo, and the staining of lung tissue metastasis HE.

FIG. 7 is a statistical chart of lung metastasis inhibition in vivo by black rice extract.

Fig. 8 is a graph showing the change in red blood cell count (RBC) and white blood cell count (WBC) in vivo after tail vein injection of black rice extract.

FIG. 9 is a graph showing the changes in aspartate Aminotransferase (AST) and Serum Creatinine (SCR) in vivo after tail vein injection of black rice extract.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Please refer to fig. 1-9. It should be noted that the drawings provided in the present embodiment are only for illustrating the basic idea of the present invention, and the components related to the present invention are only shown in the drawings rather than drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of the components in actual implementation may be changed freely, and the layout of the components may be more complicated.

In order to solve the technical problems of high price of a photothermal material, complex synthesis steps and high toxicity in the prior art, the embodiment of the invention discloses a novel black food extract which can be used for preparing a photothermal conversion agent for near-infrared photothermal treatment of tumor cells.

The black food extract is obtained by extracting black foods through certain process steps, wherein the black foods comprise one or more of black beans, black sesame seeds, black rice, black fungus, black mushrooms, black grapes, black pine nuts, black kidney beans and black buckwheat. That is, the black food extract includes, but is not limited to, one or a combination of black bean extract, black sesame extract, black rice extract, black fungus extract, black mushroom extract, black grape extract, black pine nut extract, black kidney bean extract, and black buckwheat extract.

In the preparation of the photothermal conversion agent, the black food extract can be dissolved in a specific solvent to prepare a black food extract solution with a required concentration according to requirements, and the black food extract solution can be used as the photothermal conversion agent for near-infrared photothermal treatment of tumor cells. As an example, the solvent may include phosphate balanced physiological saline.

When tumor treatment is performed by using the black food extract solution as a photothermal conversion agent for near-infrared photothermal treatment of tumor cells, the black food extract solution can be injected into the inside of a living body, focused near tumor tissues by using a targeting recognition technology, and irradiated by an external near-infrared light source (such as a laser), and the black food extract solution converts light energy into heat energy to kill cancer cells. For example, the black food extract solution has a concentration of 1-30mg/mL and an optical density of 0.1-2W/cm when irradiated with an infrared light source²The illumination time is 5-20min, and the local temperature rise of the tumor part can be promoted to 50-65 ℃; the concentration, optical density, illumination time and other parameters of the black food extract can be reasonably selected according to actual needs, and are not limited to the above parameters.

The technical solution and effect of the present invention will be specifically described below with reference to black rice extract.

Preparation of black rice extract

The black rice extract is obtained by taking black rice seed coats as raw materials and adopting certain process steps. The extraction process is as follows:

black rice → impurity removal → seed coat → extraction with acidic hot water → centrifugation → supernatant → ultrafiltration → exudate → nanofiltration → trapped fluid → macroporous resin adsorption → ethanol elution → eluent → second nanofiltration → freeze drying → black rice extract.

The method comprises the following specific steps:

removing impurities and pulp, and keeping seed coats: removing macroscopic impurities in the black rice, processing the black rice after impurity removal by using a pericarp machine, and removing pulp to obtain the seed coat of the black rice.

Acid water leaching: and carrying out acid water leaching on the seed coat to obtain an extracting solution. As an example, leaching with a disodium hydrogen phosphate-citric acid buffer solution to obtain an extract, wherein during leaching, the feed-liquor ratio is 1: 20, extracting at 48-52 deg.C and pH of 3-4 (such as 2.5-3) for 3-4 hr; the acid water leaching solution, the feed-to-liquid ratio, the extraction temperature, the PH, the extraction time, and the like may be set as necessary, and are not limited to the above-mentioned substances or parameters.

Centrifuging: the extract was centrifuged to obtain a supernatant. For example, the extract is centrifuged at 3000-6000r/min for 10-20min (such as 15min) to remove large particulate matters, and the supernatant is obtained; it should be noted that the centrifugation parameters can be reasonably adjusted according to actual needs, and are not limited to the centrifugation parameters.

And (3) ultrafiltration: subjecting the supernatant to ultrafiltration to obtain a filtrate. As an example, the supernatant may be passed through a 30kDa membrane separator to remove macromolecular substances such as starch, proteins, etc., and the filtrate collected; it should be noted that the specification of the membrane separator may be appropriately selected as needed, and is not limited to the above-listed specifications.

Primary nanofiltration: the filtrate is subjected to a nanofiltration to obtain a first concentrate. As an example, the filtrate can be concentrated using a 200Da nanofiltration membrane to obtain a concentrate (also called retentate, defined as first concentrate); the specification of the nanofiltration membrane may be appropriately selected as needed, and is not limited to the above-mentioned specifications.

Adsorption and desorption: and carrying out adsorption and desorption treatment on the first concentrated solution to obtain an eluent. As an example, in the adsorption step, the concentrated solution may be further purified by using macroporous resin, wherein the pH of the sample solution is 2-4 (such as 3.0), the adsorption flow rate is 1.0-3.0mL/min, and the equilibrium time is 2-4 h; in other examples, the material used for the adsorption, the pH of the sample solution, and the adsorption flow rate equilibrium time may be reasonably selected according to the need, and are not limited to the above listed materials and parameters. As an example, in the desorption step, elution is performed with, for example, 70-80% ethanol (other suitable solvent is also possible) to collect the obtained eluate.

And (3) secondary nanofiltration: and carrying out secondary nanofiltration on the eluent to obtain a second concentrated solution. For example, the eluate may be concentrated with a 200Da nanofiltration membrane to obtain an anthocyanin concentrate, i.e., a second concentrate; the specification of the nanofiltration membrane may be appropriately selected as needed, and is not limited to the above-mentioned specifications.

Vacuum freeze drying: and performing vacuum freeze drying on the second concentrated solution to obtain the black rice extract. For example, the concentrated solution is freeze-dried at-40-50 deg.C and-1.0 MPa to water content of 0.5% or less to obtain fructus Zizaniae Caduciflorae extract powder; it should be noted that the parameters of the vacuum freeze-drying can be reasonably selected according to actual needs, and are not limited to the above-mentioned vacuum freeze-drying parameters.

Research on external photothermal effect of black rice extraction object

Respectively weighing 1g of fructus Zizaniae Caduciflorae extract, adding phosphate balanced physiological saline (PBS for short), mixing to obtain fructus Zizaniae Caduciflorae solution with solid content of 100mg/mL, respectively diluting with PBS to obtain fructus Zizaniae Caduciflorae extract solutions with solid contents of 1mg/mL, 2mg/mL, 5mg/mL, 10mg/mL, 15mg/mL and 25mg/mL, respectively placing the above solutions under a laser (Shanghai Xilong photoelectric technology Co., Ltd.) with wavelength of 808nm, and mixing at 1.0W/cm²Power exposure for 10min, temperature changes over time were recorded with a thermal imager, water as control, and the results are shown in fig. 1:

as can be seen from fig. 1: under the illumination condition, the temperature of the black rice extract solution of each group is increased compared with that of the control group, and the concentration of the black rice extract solution is increased along with the increase of the content of the black rice extract, so that the black rice extract has excellent photothermal conversion effect.

Study on the Effect of Black Rice extract on in vivo tumor inhibition

4T1 cells (mouse breast cancer cells) were injected subcutaneously to form tumors, and 50. mu.L of 20mg/mL black rice extract solution was injected intratumorally under a near infrared laser having a wavelength of 808nm and an optical density of 1W/cm, provided that the control group (water), the black rice extract solution group, the PBS + NIR group and the black rice extract + NIR group were used²Irradiating for 10min, wherein NIR refers to near infrared light source irradiation;

1) the PBS + NIR group and black rice extract + NIR group were imaged in vivo in mice on a thermal image 2, wherein in fig. 2 and subsequent fig. 3-9, the test results for black rice extract correspond to the data indicated for black rice in the figures;

2) the results of the relative tumor volumes for each group after treatment are shown in figure 3;

3) the survival results for each group after treatment are shown in figure 4;

as can be seen from fig. 2, 3 and 4: the local tumor temperature of the black rice extract and NIR group is continuously increased along with the prolonging of the near infrared light irradiation time, and is maintained at 55 ℃, the temperature can effectively promote the death of tumor cells so as to inhibit the growth of tumors, the treatment effect of the black rice extract and NIR group is improved, and the survival rate of mice is improved.

Study of black rice extract on inhibition of in vivo lung metastasis:

4T1 cells were injected subcutaneously to form tumors, and 50 μ L of 20mg/mL black rice extract solution was injected intratumorally under a near infrared laser having a wavelength of 808nm and an optical density of 1W/cm²Irradiating for 10 min;

1) the black rice extract solution inhibits lung metastasis in vivo, and the pulmonary tissue metastasis is shown in an optical photograph of 5, and a control group, a PBS + NIR group, a black rice extract group, and a black rice extract + NIR group are sequentially arranged from left to right;

2) the black rice extract solution inhibits lung metastasis in vivo, and the result of lung tissue metastasis hematoxylin-eosin (HE) staining is shown in FIG. 6, and a control group, a PBS + NIR group, a black rice extract group, and a black rice extract + NIR group are sequentially arranged from left to right;

3) the black rice extract solution can inhibit lung metastasis in vivo, and the lung tissue metastasis statistical chart is shown in FIG. 7;

as can be seen from fig. 5, 6 and 7: the lung metastasis foci of the black rice extract + NIR group were significantly less than those of the control group, the black rice extract group, and the PBS + NIR group, indicating that the black rice extract + NIR treatment significantly inhibited the formation of lung metastasis in vivo.

Study of black rice on in vivo toxicity:

normal BABL/c mice, a control group and a black rice extract group are set, the tail vein of the control group is injected with 100 mu L PBS, and the tail vein of the black rice extract group is injected with 100 mu L20 mg/mL black rice extract solution, and the observation is continuously carried out for 20-30 days; taking whole blood for blood routine and serum for blood biochemical analysis.

1) The red blood cell count (RBC) and white blood cell count (WBC) changes after tail vein injection in PBS group and black rice extract group are shown in fig. 8;

2) the changes of aspartate Aminotransferase (AST) and Serum Creatinine (SCR) after tail vein injection in PBS group and black rice extract group are shown in FIG. 9;

as can be seen from fig. 8 and 9: RBC and WBC, AST and SCR in mice of the PBS group and the black rice extract group are not significantly changed after tail vein injection, which shows that the black rice extract has almost no toxicity in vivo, and shows that the safety is high.

Namely, the black rice extract has excellent photothermal conversion effect and can be further applied to photothermal treatment of tumors, so that the invention further develops the new application of the black rice extract and improves the economic benefit and social value of the black rice extract; in addition, the black rice is applied to tumor photothermal therapy, can obviously promote the death of tumor cells and inhibit the growth of tumors, simultaneously inhibit in-vivo lung metastasis and prolong the life cycle of mice, and the black rice extract basically has no toxicity to organisms and has high safety performance.

It should be noted that, although the black rice extract is described in the present embodiment, it is understood that other black food extracts, such as one or a combination of more of black bean extract, black sesame extract, black fungus extract, black mushroom extract, black grape extract, black pine nut extract, black kidney bean extract, and black buckwheat extract, may also be used as a material for preparing a photothermal conversion agent for near-infrared photothermal therapy of tumor cells.

In addition to the above-mentioned method, the black rice extract can be directly purchased as a product of black rice extract, for example, 25% anthocyanin-containing black rice extract purchased from Xi' an Tianxingjian Natural Bio-Products co., ltd.

In conclusion, the black food extract has excellent photothermal conversion effect, and can be applied to preparing photothermal conversion agents for tumor photothermal treatment; the invention discloses a new application of black food extract, which improves the economic value and social value of black food; the black food extract is used as a photo-thermal treatment preparation, can promote the local temperature rise of a tumor part to 50-65 ℃, achieves the effects of inhibiting the growth of tumors and promoting the death of tumor cells, simultaneously inhibits the lung metastasis in vivo, and can be used for the photo-thermal treatment of solid tumors; the black food extract has low cost, simple preparation and almost no toxicity.

In the description herein, numerous specific details are provided, such as examples of components and/or methods, to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that an embodiment of the invention can be practiced without one or more of the specific details, or with other apparatus, systems, assemblies, methods, components, materials, parts, and/or the like. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of embodiments of the invention.

Reference throughout this specification to "one embodiment", "an embodiment", or "a specific embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment, and not necessarily all embodiments, of the present invention. Thus, respective appearances of the phrases "in one embodiment", "in an embodiment", or "in a specific embodiment" in various places throughout this specification are not necessarily referring to the same embodiment. Furthermore, the particular features, structures, or characteristics of any specific embodiment of the present invention may be combined in any suitable manner with one or more other embodiments. It is to be understood that other variations and modifications of the embodiments of the invention described and illustrated herein are possible in light of the teachings herein and are to be considered as part of the spirit and scope of the present invention.

It will also be appreciated that one or more of the elements shown in the figures can also be implemented in a more separated or integrated manner, or even removed for inoperability in some circumstances or provided for usefulness in accordance with a particular application.

Additionally, any reference arrows in the drawings/figures should be considered only as exemplary, and not limiting, unless otherwise expressly specified. Further, as used herein, the term "or" is generally intended to mean "and/or" unless otherwise indicated. Combinations of components or steps will also be considered as being noted where terminology is foreseen as rendering the ability to separate or combine is unclear.

As used in the description herein and throughout the claims that follow, "a", "an", and "the" include plural references unless otherwise indicated. Also, as used in the description herein and throughout the claims that follow, unless otherwise indicated, the meaning of "in …" includes "in …" and "on … (on)".

The above description of illustrated embodiments of the invention, including what is described in the abstract of the specification, is not intended to be exhaustive or to limit the invention to the precise forms disclosed herein. While specific embodiments of, and examples for, the invention are described herein for illustrative purposes only, various equivalent modifications are possible within the spirit and scope of the present invention, as those skilled in the relevant art will recognize and appreciate. As indicated, these modifications may be made to the present invention in light of the foregoing description of illustrated embodiments of the present invention and are to be included within the spirit and scope of the present invention.

The systems and methods have been described herein in general terms as the details aid in understanding the invention. Furthermore, various specific details have been given to provide a general understanding of the embodiments of the invention. One skilled in the relevant art will recognize, however, that an embodiment of the invention can be practiced without one or more of the specific details, or with other apparatus, systems, assemblies, methods, components, materials, parts, and/or the like. In other instances, well-known structures, materials, and/or operations are not specifically shown or described in detail to avoid obscuring aspects of embodiments of the invention.

Thus, although the present invention has been described herein with reference to particular embodiments thereof, a latitude of modification, various changes and substitutions are intended in the foregoing disclosures, and it will be appreciated that in some instances some features of the invention will be employed without a corresponding use of other features without departing from the scope and spirit of the invention as set forth. Thus, many modifications may be made to adapt a particular situation or material to the essential scope and spirit of the present invention. It is intended that the invention not be limited to the particular terms used in following claims and/or to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include any and all embodiments and equivalents falling within the scope of the appended claims. Accordingly, the scope of the invention is to be determined solely by the appended claims.

Claims

1. The black food extract is characterized by being applied to preparing a photothermal conversion agent for near-infrared photothermal treatment of tumor cells.

2. The black food extract according to claim 1, wherein the black food extract comprises one or more of black bean extract, black sesame extract, black rice extract, black fungus extract, black mushroom extract, black grape extract, black pine nut extract, black kidney bean extract, and black buckwheat extract.

3. The black food extract according to any one of claims 1 to 2, wherein the photothermal conversion agent comprises the black food extract and a solvent.

4. The black food extract according to claim 3, wherein the solvent comprises phosphate balanced physiological saline.

5. A method of preparing the black food extract according to claim 1, comprising: extracting the black food by adopting preset process steps to obtain the black food extract.

6. The method for preparing a black food extract according to claim 5, wherein the black food comprises one or more of black beans, black sesame, black rice, black fungus, black mushrooms, black grapes, black pine nuts, black kidney beans and black buckwheat.

7. The method for preparing a black food extract according to claim 5, wherein the black food comprises black rice, and the step of extracting the black food using the predetermined process steps to obtain the black food extract comprises:

obtaining seed coats of the black rice;

centrifuging the extract to obtain a supernatant;

ultrafiltering the supernatant to obtain a filtrate;

8. The method of claim 7, wherein the step of acid leaching the seed coat to obtain an extract comprises: leaching with a disodium hydrogen phosphate-citric acid buffer solution, wherein the material-liquid ratio is 1: 20, the extraction temperature is 48-52 ℃, the pH is 2-4, and the extraction time is 3-4 h.

9. The method of claim 7, wherein the step of subjecting the first concentrated solution to adsorption and desorption to obtain an eluate comprises:

eluting with ethanol to collect eluate.

10. The method for preparing black rice extract according to claim 7, wherein the step of vacuum freeze-drying the anthocyanin concentrate to obtain black rice extract comprises: vacuum freeze drying the concentrated solution at-40-50 deg.C and-1.0 MPa to obtain fructus Zizaniae Caduciflorae extract powder.