Natural carthamin yellow-beta-cyclodextrin inclusion compound with high stability and preparation method of natural carthamin yellow-beta-cyclodextrin inclusion compound

Abstract

The invention provides a natural carthamin yellow-beta-cyclodextrin inclusion compound with high stability and a preparation method of the natural carthamin yellow-beta-cyclodextrin inclusion compound, and belongs to the technical field of stable modification of natural pigments. The natural carthamin yellow-beta-cyclodextrin inclusion compound comprises beta-cyclodextrin and natural carthamin yellow, wherein the molecular ratio of the beta-cyclodextrin to the natural carthamin yellow is 1: 1. The preparation method comprises the following steps: dissolving beta-cyclodextrin in water to form a beta-cyclodextrin saturated solution, adding natural carthamin yellow into the beta-cyclodextrin saturated solution, stirring for 1 hour at the temperature of 50 DEG C, and freeze-drying to obtain the natural carthamin yellow-beta-cyclodextrin. According to the natural safflower yellow-beta-cyclodextrin inclusion compound, beta-cyclodextrin serves as a subject, natural safflower yellow serves as an object, and the inclusion compound with the low pigment loss rate and the high light stability is successfully prepared by optimizing three key process parameters including the subject-object molecular ratio, the stirring temperature and the stirring time. The protection effect on the natural carthamin yellow is improved.

Description

Technical Field

[0001] This invention belongs to the field of natural pigment stabilization and modification technology, and particularly relates to a highly stable natural safflower yellow pigment-β-cyclodextrin inclusion complex and its preparation method. Background Technology

[0002] Natural safflower yellow pigment is a natural pigment extracted from the petals of safflower. Studies have found that it is not only a valuable natural food coloring but also possesses various pharmacological functions, including dilating coronary arteries, protecting the myocardium, lowering blood pressure, immunosuppression, and neuroprotection. As a safe, nutritious, and physiologically active water-soluble pigment, natural safflower yellow pigment shows broad application prospects in the food, cosmetics, and pharmaceutical industries. However, the unsaturated bonds in its molecular structure make it extremely sensitive to external environmental factors such as light, heat, and oxygen. It is prone to degradation and discoloration during production, storage, and sales, leading to a significant decrease in its coloring ability and biological activity. This inherent instability severely restricts its further development and utilization. Therefore, exploring effective technical means to improve the stability of natural safflower yellow pigment is crucial for promoting its industrial application.

[0003] Among the many existing modification methods, cyclodextrin-based inclusion technology is favored due to its simplicity, mild conditions, and effective protection of guest molecules. This technology embeds guest molecules within the hydrophobic cavity of cyclodextrin, forming a host-guest inclusion complex, thereby achieving physical isolation and protection of light- and heat-sensitive molecules. β-Cyclodextrin, as the most widely used inclusion material in the cyclodextrin family, is non-toxic, porous, water-soluble, and its cavity size matches that of many natural pigment molecules, allowing it to form relatively stable inclusion complexes with guest molecules in aqueous solutions. Its characteristics mainly stem from its unique molecular structure. It consists of seven glucose units forming a truncated cone-shaped hollow ring molecule with a moderate cavity diameter of approximately 0.6–0.8 nm, enabling it to selectively include various guest molecules, such as active pharmaceutical ingredients, fragrances, and functional factors. Furthermore, after forming inclusion complexes, β-cyclodextrin can significantly improve the solubility and bioavailability of poorly soluble drugs, effectively mask unpleasant odors and bitterness, and enhance the stability of guest molecules to light, heat, and oxygen, thereby achieving controlled release of the contents and improvement of their physical form. Given its favorable balance between performance, safety, and cost-effectiveness, β-cyclodextrin has become a highly valuable inclusion carrier and functional modifier in the food, pharmaceutical, and cosmetic industries.

[0004] In summary, the preparation of a natural safflower yellow pigment-β-cyclodextrin inclusion complex using β-cyclodextrin to improve the stability of natural safflower yellow pigment is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a highly stable natural safflower yellow pigment-β-cyclodextrin inclusion complex and its preparation method. This invention utilizes β-cyclodextrin to include natural safflower yellow pigment, thereby improving the photostability of natural safflower yellow pigment.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of the present invention: This invention provides a highly stable natural safflower yellow pigment-β-cyclodextrin inclusion complex, wherein the natural safflower yellow pigment-β-cyclodextrin inclusion complex comprises β-cyclodextrin and natural safflower yellow pigment in a molecular ratio of 1:(0.5-1.5).

[0007] Furthermore, the molecular ratio of the β-cyclodextrin to the natural safflower yellow pigment is preferably 1:1.

[0008] Furthermore, the pigment loss rate of the natural safflower yellow pigment-β-cyclodextrin inclusion complex was 4.82% to 5.47%.

[0009] Furthermore, the pigment loss rate is determined by measuring absorbance, and the calculation formula is as follows: A = (A0 - A1) × A0 -1 ×100% In the formula, A represents the pigment loss rate, A0 represents the absorbance of natural safflower yellow pigment before treatment, and A1 represents the absorbance of natural safflower yellow pigment after treatment.

[0010] The coloring components of natural safflower yellow pigment are mainly chalcone glycosides, and changes in their content can indirectly reflect the overall content level of natural safflower yellow pigment. Therefore, under experimental conditions with consistent sample mass and dilution factor, the stability of natural safflower yellow pigment can be assessed by measuring changes in absorbance. The degree of decrease in absorbance directly characterizes the pigment loss rate; the higher the value, the worse the stability of natural safflower yellow pigment and the higher the degree of pigment degradation.

[0011] The second technical solution of the present invention: The present invention also provides a method for preparing a highly stable natural safflower yellow pigment-β-cyclodextrin inclusion complex, comprising the following steps: β-Cyclodextrin was dissolved in water to form a saturated β-cyclodextrin solution. Natural safflower yellow pigment was added to the saturated β-cyclodextrin solution. After stirring and freeze-drying, the natural safflower yellow pigment-β-cyclodextrin was obtained.

[0012] Furthermore, the stirring speed is 500 rpm, the time is 0.5 to 2.5 h, and the temperature is 30 to 70 ℃.

[0013] More preferably, the stirring time is 1.0 h and the temperature is 50 ℃.

[0014] Furthermore, the freeze-drying process is as follows: first, pre-freeze at -50℃ for 6 hours, then desorb and dry at -20 to -10℃ under a vacuum of 15Pa for 24 hours, and then raise the temperature to 25℃ and continue drying for 9 hours.

[0015] Beneficial effects: The freeze-drying method described above can promote the stable formation of inclusion structures: the pre-freezing stage allows the solution to quickly form uniform ice crystals, which helps to fix the inclusion structure between β-cyclodextrin and safflower yellow pigment; the subsequent desorption drying at low temperature and low pressure can slowly remove moisture without disturbing the already formed inclusion complex, which is beneficial to maintaining intermolecular forces and improving inclusion efficiency and stability.

[0016] Improve product porosity and resolubility: Freeze-dried products have a porous and loose structure with a large specific surface area, which not only facilitates storage and transportation, but also allows for rapid resolubility in water during use, thus improving application convenience.

[0017] Furthermore, the mass ratio of the β-cyclodextrin to water is 24:100.

[0018] The beneficial effects of this invention compared to the prior art are as follows: This invention studies a natural safflower yellow pigment-β-cyclodextrin inclusion complex, in which β-cyclodextrin is the host and natural safflower yellow pigment is the guest. By optimizing three key process parameters—the host-guest molecular ratio, stirring temperature, and stirring time—an inclusion complex with low pigment loss rate and high photostability was successfully prepared, improving the protective effect on natural safflower yellow pigment. This provides solid data support and theoretical basis for expanding the practical application of this natural pigment, and offers an effective stability solution for its application in food, pharmaceuticals, and other fields. Furthermore, the natural safflower yellow pigment-β-cyclodextrin inclusion complex constructed in this invention acts as a molecular barrier, shielding the pigment molecules from direct attack by external factors such as light radiation, thereby slowing down the degradation rate of natural safflower yellow pigment. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a bar chart showing the effect of different host-guest molecular ratios on pigment loss rate in the single-factor experimental analysis of this invention. Figure 2This is a bar chart showing the effect of different stirring temperatures on pigment loss rate in the single-factor experimental analysis of this invention; Figure 3 This is a bar chart showing the effect of different stirring times on pigment loss rate in the single-factor experimental analysis of this invention; Figure 4 The response surface plot shows the effect of the interaction between the host-guest molecular ratio, stirring temperature, and stirring time on the pigment loss rate in the model verification experiment of this invention. Here, a is the interaction between the host-guest molecular ratio and stirring temperature, b is the interaction between the host-guest molecular ratio and stirring time, and c is the interaction between stirring temperature and stirring time. Figure 5 The image shows the UV-Vis spectrum of the natural safflower yellow pigment-β-cyclodextrin inclusion complex in the present invention, including the natural safflower yellow pigment raw material, β-cyclodextrin, and the natural safflower yellow pigment-β-cyclodextrin inclusion solution. Figure 6 These are microscopic images of β-cyclodextrin, natural safflower yellow pigment raw material, a physical mixture of β-cyclodextrin and natural safflower yellow pigment raw material with a host-guest molecular ratio of 1:1, and the natural safflower yellow pigment-β-cyclodextrin inclusion complex in the microscopic examination of the natural safflower yellow pigment-β-cyclodextrin inclusion complex of the present invention. Among them, a is a microscopic image of natural safflower yellow pigment raw material, b is a microscopic image of β-cyclodextrin, c is a microscopic image of the physical mixture of β-cyclodextrin and natural safflower yellow pigment raw material with a host-guest molecular ratio of 1:1, and d is a microscopic image of the natural safflower yellow pigment-β-cyclodextrin inclusion complex. Figure 7 This is a bar chart showing the loss rate of the inclusion complex and pigment raw material under different light conditions during the photostability test of the natural safflower yellow pigment-β-cyclodextrin inclusion complex of this invention. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The natural safflower yellow pigment used in the following experiments of this invention was purchased from Zhuhai Yafuxingyuan Food Industry Co., Ltd. β-Cyclodextrin was purchased from Jiangsu Aogu Biotechnology Co., Ltd.

[0027] The instruments used in the following experiments of this invention are: UV-1800 ultraviolet-visible spectrophotometer (Shimadzu Corporation, Japan), C-MAG HS 7 thermostatic magnetic stirrer (IKA Corporation, Germany), FDU-2100 vacuum freeze dryer (Tokyo Rika K.K., Japan), KBW400 light irradiator (Binder Corporation, Germany) and DM4B microscope (Leica Corporation, Germany).

[0028] Each of the following experiments in this invention was repeated at least 3 times, and the results were expressed as mean ± standard deviation. The data are expressed as mean ± standard deviation (±s). SPSS 22.0 statistical software was used for data analysis (significance level set to 0.05). Design Expert 8.0.6 software was used for response surface analysis, and Origin 8.0 software was used for plotting.

[0029] 1. Single-factor screening test of natural safflower yellow pigment-β-cyclodextrin inclusion complex (1) Test methods To screen the optimal single-factor conditions for the preparation of natural safflower yellow pigment-β-cyclodextrin inclusion complex, the pigment loss rate after 5 hours of strong light irradiation of the prepared inclusion complex was used as the indicator. Specifically, after the inclusion complex was prepared, it was diluted to a concentration of 15 μg / mL and subjected to a full-wavelength scan to determine the maximum absorption wavelength. The absorbance value A0 was measured at this wavelength, and then the mixture was placed in a light irradiator for 5 hours before measuring the absorbance value A1. The absorbance was calculated using the formula A = (A0 - A1) × A0. -1 The pigment loss rate was calculated as (A × 100%, where A0 represents the absorbance value of natural safflower yellow pigment before treatment, and A1 represents the absorbance value of natural safflower yellow pigment after treatment). The host-guest molecular ratio, stirring temperature, and stirring time were selected as single-factor experiments. In each experimental level, two factors were fixed, and only the third single-factor was changed. The relatively fixed host-guest molecular ratio, stirring temperature, and stirring time were 1:1, 40℃, and 1 h, respectively. The single-factor experimental levels are shown in Table 1.

[0030] Table 1

[0031] (2) Single-factor experimental analysis 1) Effect of host-guest molecular ratio on pigment loss rate β-cyclodextrin and natural safflower yellow pigment were weighed at host-guest ratios of 1:0.5, 1:1, 1:1.5, 1:2, and 1:2.5. The β-cyclodextrin was dissolved in 100 g of water to form a saturated β-cyclodextrin solution. Natural safflower yellow pigment was then added to the saturated β-cyclodextrin solution, and the mixture was stirred at 500 rpm and 40 °C for 1 h to obtain a natural safflower yellow pigment-β-cyclodextrin inclusion complex. The effect of different host-guest ratios on the loss rate of natural safflower yellow pigment was investigated, and the results are as follows: Figure 1 As shown. By Figure 1It is observed that as the host-guest molecular ratio increases from 1:2.5 to 1:0.5, the pigment loss rate gradually decreases. The lowest pigment loss rate (4.87%) is achieved when the host-guest molecular ratio is 1:1. Subsequently, the pigment loss rate increases with further increases in the host-guest molecular ratio. This is because the formation of inclusion complexes between the guest molecule, natural safflower yellow pigment, and the host molecule, β-cyclodextrin, is a dynamic equilibrium. From a host-guest molecular ratio of 1:2.5 to 1:1, the system contains an excess of the guest molecule, natural safflower yellow pigment, while the host molecule, β-cyclodextrin, is relatively insufficient. As the relative proportion of β-cyclodextrin increases, i.e., the host-guest molecular ratio approaches 1:1, more pigment molecules have the opportunity to enter the cavity of the β-cyclodextrin, forming stable inclusion complexes. The formation of these inclusion complexes protects the pigment molecules, making them less susceptible to degradation or loss during subsequent processing. Therefore, as the proportion of the host molecule increases, the inclusion and protection of the pigments strengthens, resulting in a gradual decrease in the pigment loss rate. The optimal inclusion effect is achieved near the theoretical ratio of 1:1, at which point the pigment loss rate is lowest. When the host-guest molecular ratio continues to increase from 1:1 to 1:0.5, there is an excess of host molecules in the system that are not bound to the pigment. The high concentration of β-cyclodextrin will change the viscosity and other physicochemical properties of the entire solution, which is not conducive to the inclusion reaction and thus indirectly leads to an increase in pigment loss rate. Therefore, considering both inclusion efficiency and pigment stability, 1:1 is chosen as the host-guest molecular ratio.

[0032] 2) Effect of stirring temperature on pigment loss rate Weigh out β-cyclodextrin and natural safflower yellow pigment with a host-guest molecular ratio of 1:1. Dissolve β-cyclodextrin in 100 g of water to form a saturated β-cyclodextrin solution. Add natural safflower yellow pigment to the saturated β-cyclodextrin solution. Stir at 500 rpm for 1 h at temperature gradients of 30 ℃, 40 ℃, 50 ℃, 60 ℃, and 70 ℃ to obtain a natural safflower yellow pigment-β-cyclodextrin inclusion complex. Investigate the effect of different stirring temperatures on the loss rate of natural safflower yellow pigment. The results are as follows: Figure 2 As shown. By Figure 2 It was found that the pigment loss rate first decreased and then increased with increasing stirring temperature, with a significant effect (P < 0.05). The pigment loss rate reached its minimum of 4.82% at 40 ℃. This is because as the temperature increases, the movement of guest molecules intensifies, which is conducive to diffusion into the β-cyclodextrin lumen and the formation of inclusion complexes. However, as the temperature continues to increase, natural safflower yellow pigment, as a bioactive molecule, is quite sensitive to high temperatures. When the stirring temperature is too high, some free pigment molecules, and even some already included pigment molecules, undergo degradation or oxidation reactions due to the increased temperature, leading to structural damage and inactivation, thus directly manifesting as an increase in the pigment loss rate. Therefore, considering both inclusion efficiency and pigment stability, 40 ℃ was selected as the optimal stirring temperature.

[0033] 3) Effect of stirring time on pigment loss rate Weigh out β-cyclodextrin and natural safflower yellow pigment with a host-guest ratio of 1:1. Dissolve β-cyclodextrin in 100 g of water to form a saturated β-cyclodextrin solution. Add natural safflower yellow pigment to the saturated β-cyclodextrin solution and stir at 500 rpm and 40℃ for 0.5 h, 1.0 h, 1.5 h, 2.0 h, and 2.5 h to obtain a natural safflower yellow pigment-β-cyclodextrin inclusion complex. Investigate the effect of different stirring times on the loss rate of natural safflower yellow pigment. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that the pigment loss rate first decreases and then increases with the extension of stirring time. When the stirring time is 1.0 h, the pigment loss rate is 4.89%, which is significantly lower than other levels (P<0.05). This is because in the initial stage of the inclusion reaction, with the extension of stirring time, the contact between β-cyclodextrin and natural safflower yellow pigment molecules is more sufficient, and the inclusion reaction gradually tends to equilibrium, forming a more stable inclusion complex, thereby reducing the free state of the pigment and reducing its loss in subsequent processing. However, when the stirring time exceeds 1.0 h, excessive mechanical stirring can cause some of the formed inclusion complex structures to be destroyed or cause the degradation of pigment molecules, thus increasing the pigment loss rate again. In addition, long-term stirring will also accelerate the contact between oxygen and pigment, thereby triggering an oxidation reaction and leading to a decrease in pigment content. Therefore, considering both inclusion efficiency and pigment stability, 1.0 h was selected as the optimal stirring time.

[0034] 2. Response surface methodology (1) Response surface optimization of the preparation process of natural safflower yellow pigment-β-cyclodextrin inclusion complex To determine the optimal preparation conditions for the natural safflower yellow pigment-β-cyclodextrin inclusion complex, a three-factor, three-level response surface methodology was used to optimize the results based on single-factor experiments. The response surface methodology factor levels are shown in Table 2, where 0, 1, and -1 represent coded values, with 0 representing the intermediate level, 1 representing the high level, and -1 representing the low level.

[0035] Table 2

[0036] (2) Response surface verification test The optimal preparation process of the inclusion complex was obtained through response surface methodology optimization. Verification experiments were conducted, and the actual values ​​were compared and analyzed with the theoretical values ​​to verify the feasibility of the model.

[0037] 1) Establishment and testing of regression models A three-factor, three-level Box-Benhnken central composite experiment was conducted to investigate the effects of host-guest ratio (A), stirring temperature (B), and stirring time (C) on pigment loss rate (Y). The experimental results are shown in Table 3. In Table 3, 0, 1, and -1 in the host-guest ratio represent coded values, which correspond to those in Table 2.

[0038] Table 3

[0039] A multiple regression analysis was performed on the data in Table 3, and the resulting regression equation is as follows:

[0040] The regression determination coefficient (R²) of the model was calculated. 2 The R-value is as high as 0.9940, indicating that the model can explain up to 99.40% of the variation in response values, with only about 0.60% of the total variation remaining unexplained. Generally speaking, R... 2 The closer the value is to 1, the better the model fits the experimental data. In response surface methodology, R0... 2 A value greater than 0.90 is considered a good model fit, and the R-value of this model is... 2 The value is much higher than this standard, proving that the regression model has extremely strong explanatory power. The model's corrected coefficient of determination (Adj R²) 2 The value is 0.9864. (Adj R) 2 The number of independent variables was adjusted to more objectively measure the model's goodness of fit. In this model, R0 2 With Adj R 2 The difference between the two values ​​is extremely small (0.9940 - 0.9864 = 0.0076), and both are at very high levels, indicating that the model is reasonably constructed, there is no overfitting, and it has high reliability. The model's coefficient of variation (CV) is 0.41%. The lower the CV value, the better the repeatability of the experiment and the smaller the dispersion of the data. Generally, a CV value below 10% is considered to indicate good reproducibility. The CV value of this model is far below 1%, which is at an extremely low level, proving that the experimental data has high stability and reliability. The model's signal-to-noise ratio (SNR) is 36.168. According to statistical requirements, the SNR threshold is 4. The SNR of this model far exceeds this threshold, indicating that the model can be used for prediction and optimization analysis. The results of the analysis of variance for the regression equation of pigment loss rate are shown in Table 4. The lack-of-fit term was not significant (P=0.8137>0.05), indicating that the established response surface model can well describe the influence of each factor on the response value, and its prediction results are reliable. This equation can be used to accurately predict and analyze the pigment loss rate. The model equation P=0.0095, indicating that the model has excellent significance. 2 The effects of item A, B, and B are extremely significant. 2Items A and B have a significant impact, while items C and related items do not reach a significant level, indicating that their impact on pigment loss rate is relatively weak. The F-value shows that the order of influence of the three factors on pigment loss rate is: host-guest molecular ratio > stirring temperature > stirring time. This result suggests that in actual process optimization, the host-guest molecular ratio should be adjusted first, followed by the stirring temperature, while the stirring time can be adjusted within a wide range without significantly affecting pigment stability.

[0041] Table 4

[0042]

[0043] 2) Response Surface Analysis The effect of the interaction between the host-guest molecular ratio, stirring temperature, and stirring time on the pigment loss rate is as follows: Figure 4 As shown in Table 4, the significance test results indicate that the interaction between the host-guest ratio and stirring temperature (AB) has a significant effect on the pigment loss rate, while the interactions between the host-guest ratio and stirring time (AC) and stirring temperature and stirring time (BC) are not significant. This suggests that there is a significant synergistic effect between the host-guest ratio and stirring temperature influencing the pigment loss rate, while the combination of stirring time and the other two factors has virtually no coupling effect on the response value. The significant interaction (AB) in... Figure 4 In response surface a, the response surface exhibits significant curvature, with corresponding contour lines displaying an elliptical distribution. This indicates that the combined changes in the host-guest ratio (A) and stirring temperature (B) have a nonlinear synergistic effect on the pigment loss rate. In contrast, the interactions between the host-guest ratio and stirring time (AC), and between stirring temperature and stirring time (BC), are more pronounced in the response surface. Figure 4 The relatively flat curved surfaces and approximately circular contour lines in b and 4c further confirm the statistical result that the interaction between AC and BC is not significant.

[0044] 3) Model validation experiment The optimal conditions for the three factors were obtained through response surface regression equation: host-guest molecular ratio of 1:1, stirring temperature of 50℃, and stirring time of 1 h. Under these conditions, the model predicted a pigment loss rate of 5.07%, and the actual pigment loss rate was 5.09%, which is close to the theoretical value. The RSD was 0.31%, proving that the regression model is reliable.

[0045] 3. Analysis and detection of inclusion compounds To confirm whether an inclusion complex has successfully formed between the host and guest molecules, commonly used verification methods include microscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy, and ultraviolet-visible spectrophotometry. This invention first uses ultraviolet-visible spectrophotometry to determine the position of the maximum absorption peak of the natural safflower yellow pigment-β-cyclodextrin inclusion complex, and then combines this with microscopic observation to characterize the prepared inclusion complex, thereby verifying the inclusion complex formed between natural safflower yellow pigment and β-cyclodextrin.

[0046] Weigh out β-cyclodextrin and natural safflower yellow pigment with a host-guest molecular ratio of 1:1. Dissolve β-cyclodextrin in 100 g of water to form a saturated β-cyclodextrin solution. Add natural safflower yellow pigment to the saturated β-cyclodextrin solution and stir at a constant temperature of 500 rpm and 50 ℃ for 1 h to obtain a natural safflower yellow pigment-β-cyclodextrin inclusion complex. After freeze-drying, obtain the natural safflower yellow pigment-β-cyclodextrin inclusion complex.

[0047] 1) UV-Vis spectroscopy detection of natural safflower yellow pigment-β-cyclodextrin inclusion complex Equal amounts of natural safflower yellow pigment, β-cyclodextrin, and the aforementioned natural safflower yellow pigment-β-cyclodextrin inclusion complex were taken, diluted with water to 15 μg / mL, and scanned at wavelengths of 300-600 nm using water as a blank. The results are as follows. Figure 5 As shown.

[0048] Depend on Figure 5It was observed that the host molecule, β-cyclodextrin, did not exhibit any significant characteristic absorption peak in the 300-600 nm scanning range, which is consistent with its molecular structure. β-cyclodextrin is a cyclic oligosaccharide composed of glucose units, and it itself lacks chromophores in this wavelength range, thus exhibiting no UV-Vis absorption. The guest molecule, natural safflower yellow pigment, showed a maximum absorption peak at 403 nm, determined by the conjugated chalcone glycoside structure in its molecular structure, which is a characteristic absorption of this compound. When natural safflower yellow pigment formed an inclusion complex with β-cyclodextrin, its maximum absorption wavelength shifted from 403 nm to 397 nm, exhibiting a significant blue shift, which usually indicates a decrease in the polarity of the microenvironment in which the chromophore molecule resides. The inner wall of the cavity of β-cyclodextrin is a hydrophobic environment composed of carbon-hydrogen bonds. When natural safflower yellow pigment molecules are encapsulated within the hydrophobic cavity of β-cyclodextrin, its chromophore shifts from the external polar aqueous environment to the internal nonpolar cavity. This significant reduction in the polarity of the microenvironment leads to an increase in the energy required for electronic transitions, resulting in a "blue shift" in the absorption peak position on the spectrum. This change in spectral behavior is strong evidence of inclusion interaction between the host and guest components. The blue shift caused by inclusion interaction suggests that the chromophore of the natural safflower yellow pigment molecule is protected by the β-cyclodextrin cavity.

[0049] 2) Microscopic detection of natural safflower yellow pigment-β-cyclodextrin inclusion complex β-cyclodextrin, natural safflower yellow pigment raw material, a physical mixture of β-cyclodextrin and natural safflower yellow pigment raw material with a host-guest molecular ratio of 1:1, and natural safflower yellow pigment-β-cyclodextrin inclusion complex powder were respectively placed on glass slides and observed under a 10×10 magnification microscope. The results are as follows: Figure 6 As shown.

[0050] pass Figure 6 The microscopic morphology of each sample group can be clearly observed. Natural safflower yellow pigment powder is orange-yellow, irregular in shape, rough in surface, and lacks obvious crystalline structure. β-cyclodextrin consists of colorless, transparent, or translucent irregular particles, typically characterized by a distinct hollow cavity structure. In the physical mixture sample, the orange-yellow natural safflower yellow pigment particles and the hollow β-cyclodextrin particles are mixed together, but each maintains its original morphology, exhibiting a loose and independent distribution, with no obvious structural fusion observed. However, in the natural safflower yellow pigment-β-cyclodextrin inclusion complex sample of this invention, the hollow regions of β-cyclodextrin are filled with orange-yellow material, forming a complex in both color and structure, with a morphology significantly different from the simple coexistence state of the two in the physical mixture. These microstructural characteristics indicate that natural safflower yellow pigment has been embedded in the cavities of β-cyclodextrin, forming an inclusion complex.

[0051] 3) Photostability test of natural safflower yellow pigment-β-cyclodextrin inclusion complex The prepared natural safflower yellow pigment-β-cyclodextrin inclusion complex was diluted with pure water to 15 μg / mL, and its absorbance was measured at 397 nm. Four aliquots of the inclusion solution, approximately 10 mL each, were placed in a light irradiator, a clean bench, a laboratory bench, and a darkroom, respectively, and subjected to strong light irradiation, ultraviolet irradiation, natural light, and light protection. After 7 days of light exposure, the absorbance of each sample was measured again, and the pigment loss rate was calculated using the formula. Simultaneously, an unincluded natural safflower yellow pigment raw material solution was used as a control, treated using the same method, and its absorbance was measured at 403 nm to compare the pigment loss.

[0052] like Figure 7 ( As shown in the figure (with highly significant differences), both natural safflower yellow pigment and its natural safflower yellow pigment-β-cyclodextrin inclusion complex exhibited varying degrees of pigment loss under different light conditions. Overall, the high-light treatment group showed the most severe loss, while the darkroom treatment group showed the least loss. Compared to the raw pigment, the natural safflower yellow pigment-β-cyclodextrin inclusion complex demonstrated higher stability under all light conditions. Under the most destructive high-light conditions, after 7 days of irradiation, the loss rate of the raw pigment reached 60.72%, while the loss rate of the natural safflower yellow pigment-β-cyclodextrin inclusion complex was only 38.47%, a significant reduction of 22.25%. Statistical analysis showed that, except for no significant difference under dark conditions, the pigment loss rates of the natural safflower yellow pigment-β-cyclodextrin inclusion complex and the pigment raw material were significantly different under strong light, ultraviolet light, and indoor light conditions (p<0.01). This indicates that the inclusion technology can effectively inhibit the degradation of natural safflower yellow pigment by light, especially strong light. This may be because the β-cyclodextrin inclusion material encapsulates the pigment molecules in its hydrophobic cavity, forming a physical barrier that effectively reduces the degradation of the pigment by external factors such as light, heat, and oxygen. The natural safflower yellow pigment-β-cyclodextrin inclusion complex has better stability than the free pigment, which is of great significance for extending its shelf life as a food additive or pharmaceutical excipient.

[0053] In summary, this invention optimized the preparation process of the natural safflower yellow pigment-β-cyclodextrin inclusion complex using response surface methodology, determining the optimal process conditions as follows: host-guest molecular ratio of 1:1, inclusion temperature of 50 °C, and stirring time of 1 h. The optimized model verified a high degree of agreement between experimental and predicted values, demonstrating the reliability of this model in guiding and predicting inclusion processes.

[0054] Ultraviolet-visible absorption spectroscopy and microscopic observation provided direct evidence for the formation of the inclusion complex. The blue shift of the characteristic absorption peak of safflower yellow pigment after inclusion indicates that its chromophore is embedded in the hydrophobic cavity of β-cyclodextrin, reducing the polarity of the molecular microenvironment and confirming the occurrence of host-guest inclusion at the molecular level.

[0055] Compared to the raw pigment, the natural safflower yellow pigment-β-cyclodextrin inclusion complex exhibits significantly enhanced stability under various light conditions, especially under strong light irradiation, where its pigment retention rate is greatly improved. This demonstrates that the β-cyclodextrin inclusion technology effectively constructs a molecular barrier, shielding the pigment molecules from direct attack by external factors such as light radiation, thereby slowing down its degradation rate.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A natural saffron yellow pigment-β-cyclodextrin inclusion complex having high stability, characterized in that, The natural saffron yellow pigment-beta-cyclodextrin inclusion compound comprises beta-cyclodextrin and natural saffron yellow pigment with a molecular ratio of 1:(0.5-1.5).

2. The natural curcumin-β-cyclodextrin inclusion compound having high stability according to claim 1, characterized by, The molecular ratio of the beta-cyclodextrin and the natural saffron yellow pigment is 1:

1.

3. The natural saffron yellow dye-β-cyclodextrin inclusion complex having high stability according to claim 1, characterized in that, The pigment loss rate of the natural saffron yellow pigment-beta-cyclodextrin inclusion compound is 4.82-5.47%.

4. A method for preparing natural saffron yellow pigment-β-cyclodextrin inclusion complex having high stability according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: The beta-cyclodextrin is dissolved in water to form a beta-cyclodextrin saturated solution, and the natural saffron yellow pigment is added to the beta-cyclodextrin saturated solution, and the natural saffron yellow pigment-beta-cyclodextrin is obtained after stirring and freeze-drying.

5. The preparation method according to claim 4, characterized in that, The stirring speed is 500 rpm, the stirring time is 0.5-2.5 h, and the stirring temperature is 30-70 ℃.

6. The preparation method according to claim 4, characterized in that, The freeze-drying process is as follows: pre-freezing at-50 ℃ for 6 h, desorption drying at-20 to-10 ℃ and a vacuum degree of 15 Pa for 24 h, and then continuing drying at 25 ℃ for 9 h.

7. The preparation method according to claim 4, characterized in that, The mass ratio of the beta-cyclodextrin and water is 24:100.

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