TWI752555B - Encapsulated microparticle for promoting chlorophyll stability and the manufacturing method for the same - Google Patents

Abstract

The invention provides an encapsulated microparticle for promoting chlorophyll stability, which includes: a polycaprolactone shell and a chlorophyll core, the chlorophyll core encapsulated within the polycaprolactone shell. The present invention also provides a method for the same, which includes the steps of: emulsifying a dispersed phase composition to form monodispersed and uniform chlorophyll-polycaprolactone emulsions, the dispersed phase composition comprising chlorophyll and polycaprolactone; and solidifying the chlorophyll-polycaprolactone emulsions to form chlorophyll-polycaprolactone microparticles, wherein in each microparticle, the polycaprolactone encapsulates the chlorophyll.

Description

Coated microparticles capable of improving chlorophyll stability and method for producing the same

The present invention relates to the coating technology of chlorophyll, and particularly relates to a coating particle which can improve the stability of chlorophyll and a manufacturing method thereof.

Colorants are widely used in the food industry to ensure uniform color and the desired appearance of food products. Although some synthetic colors are FDA-approved and approved for use in food, some artificial colors may cause harm to human health, such as: hyperactivity, irritability, sleep disturbance, aggression, and Symptoms such as oversensitivity. Therefore, the ability to manufacture colorants from natural materials has become a growing concern for health-conscious consumers. Chlorophyll is a natural yellow-green pigment that can be easily obtained from plants and algae. The uniqueness of chlorophyll makes it have potential applications in many fields of science and industrial technology. Chlorophyll has been approved as a coloring additive for health food, and can also be used in the cosmetic and pharmaceutical industries. Clinically, chlorophyll is also a part of photodynamic therapy, which can assist in the localization of malignant tumors and sentinel lymph nodes. Chlorophyll is one of the most popular natural colorants in the food industry due to its health benefits such as low toxicity and reduced cancer rates. Chlorophyll has been used as the main pigment in the manufacture of oils, waxes and canned liquids such as deodorants. It has also been used in conjunction with liposomes to deliver biologically active ingredients to drug target sites. Chlorophyll is often used as a photosensitizer in photodynamic therapy of cancer. Compared with traditional cancer therapy, the advantage of using chlorophyll as a photosensitizer is that chlorophyll can only irradiate cancer tissue, so it can avoid irreversible damage to cells caused by reactive oxygen species produced by general photosensitizers during the irradiation process. Through chlorophyll, cancer cells can be specifically treated.

Chlorophyll will degrade and change color under the action of heat, oxygen, light, and acids, and the color change is mainly due to the structure of the final and intermediate products of the degradation and the biochemistry of the porphyrin cracking reaction. The instability of chlorophyll limits its use in the food industry as a natural pigment and in photodynamic therapy. In order to prolong the storage time of chlorophyll and preserve the pigment of chlorophyll, some methods involving cyanine killing, alkalizing agent, copper complex reaction, glycerol and low temperature storage have been proposed in the prior art. Generally speaking, cyanidation is a heating operation for fruits and vegetables, and the purpose of cyanidation is to inactivate enzymes, maintain the color of fruits and vegetables, and remove trapped air. However, tissue damaged during the cyanocyanin process can cause some degradation of chlorophyll. The original intention of alkalization treatment is to avoid the acidic environment, and it is mostly carried out together with the cyanine killing operation to improve the stability of the green pigment. If chlorophyll is placed in an acidic environment, the chlorophyll molecules will be converted into fucoidins due to the influence of pH, thus changing from green to olive green. In the above reaction, the chlorophyll molecule becomes the corresponding fucoidin due to the substitution of the magnesium ion in the porphyrin ring by the hydrogen ion. At present, it is known that pH value affects the stability of chlorophyll, and color is one of the most important characteristics of vegetable products. Therefore, previous technologies have conducted various studies on the color change or degradation of chlorophyll in the kinetic mode of first-order reaction. Unfortunately, most methods require that the pH value must be close to neutral to reduce unfavorable chemical reactions, and because the alkalizing agent cannot continuously neutralize the acidic substances in the internal tissue during long-term storage, the alkalizing treatment The effect is not ideal. In order to solve the problem of stability, another document proposes to use chlorophyll derivatives to produce green copper complexes with a color similar to natural chlorophyll. However, the general public prefers natural chlorophyll and does not like artificial colors. Other literatures also propose chlorophyll coating, as well as coating with polymers such as liposomes and glycerol. However, the high purchase cost of liposomes and glycerol is not conducive to commercialization.

In order to improve the stability of chlorophyll, the present invention proposes a novel polymer coating technology; specifically, this technology, for example, uses polycaprolactone (PCL) to coat chlorophyll. PCL is favored because of its biodegradability and its versatility as a drug delivery agent, while being cost-effective, tough, and biocompatible. For example, delivery of water-soluble vitamins with hydrophobic PCL polymeric nanofibers can prolong vitamin delivery time in transdermal patches. Based on the above properties, PCL polymers may be used as drug carriers to protect sensitive drug molecules. In the present invention, susceptible chlorophyll molecules are protected with PCL polymers. In addition, the inventors have extensive experience in how to form uniform monodisperse microparticles using the principles of droplet microfluidics. The present inventors hope to combine the biodegradability of PCL polymers and the reliable production technology of droplet microfluidics to manufacture PCL microparticles (Chl-PCL microparticles) with chlorophyll-coated inside.

Therefore, the present invention provides a coated particle which can improve the stability of chlorophyll, which comprises: a polycaprolactone shell and a chlorophyll inner core, and the chlorophyll core is coated by the polycaprolactone shell.

In a preferred example, the average diameter of the coated particles is 30 to 70 μm, and preferably 35.7 to 68.1 μm.

In addition, the present invention further provides a method for producing coated microparticles that can improve the stability of chlorophyll, which comprises: emulsifying a dispersed phase, wherein the dispersed phase contains chlorophyll and polycaprolactone, so as to form a plurality of monodisperse and uniform chlorophyll - polycaprolactone emulsions; and curing these chlorophyll-polycaprolactone emulsions to form a plurality of chlorophyll-polycaprolactone microparticles, wherein in each chlorophyll-polycaprolactone microparticle, the polycaprolactone is coated in Outside of chlorophyll.

In a preferred embodiment, the emulsification step includes: providing a microfluidic device, the microfluidic device has a middle channel, a central inlet connected to one end of the middle channel, a central outlet connected to the other end of the middle channel, and two connections. The side inlets on the opposite sides of the middle channel; the dispersed phase is injected from the central inlet, and a continuous phase is injected from these side inlets, so that the dispersed phase and the continuous phase flow to the middle channel to form these monodisperse and uniform chlorophyll- polycaprolactone emulsions; and collecting these chlorophyll-polycaprolactone emulsions from a central outlet.

In a preferred embodiment, the continuous phase contains polyvinyl alcohol.

In a preferred example, the injection flow rate of the dispersed phase is 0.2 to 0.5 mL/h, and preferably 0.3 to 0.5 mL/h.

In a preferred embodiment, the injection flow rate of the continuous phase is 0.1 to 0.4 mL/min, and preferably 0.2 to 0.4 mL/min.

In a preferred embodiment, the side inlets are symmetrically connected to two opposite sides of the middle channel.

In a preferred embodiment, the temperature used in the curing step is 35 to 40°C, preferably 37°C.

In a preferred embodiment, the curing step is performed for 20 to 28 hours, and preferably 24 hours.

In a preferred example, the average diameter of the particles is 30 to 70 μm, and preferably 35.7 to 68.1 μm.

According to the present invention, a polymer coating technology is proposed to improve the stability of chlorophyll, and this microfluidic emulsification technology can make chlorophyll-polycaprolactone particles with uniform size distribution. The experimental results showed that the chlorophyll-polycaprolactone microparticles had lower reaction constants than chlorophyll (k value 0.00693h ^-1 vs k value 0.05511h ^-1 ). In addition, the method proposed in the present invention has the following advantages: (1) the size of the chlorophyll-polycaprolactone particles can be controlled during the manufacturing process; (2) the chlorophyll-polycaprolactone has a high coating rate; (3) the chlorophyll-polycaprolactone The ester particles are in a solid state, which is convenient for storage and transportation.

In order to make the above-mentioned and/or other purposes, effects and features of the present invention more obvious and easy to understand, preferred embodiments are given below, and are described in detail below:

<Experimental Example 1: Materials>

PCL and polyvinyl alcohol (PVA, 88%-89% hydrolyzed) were purchased from Sigma. Silkworm sand is obtained from Chinese pharmacies. 95% ethanol was purchased from Echo Chemical Co., Ltd. The reference chlorophyll b reference with purity higher than 99% was purchased from Sigma.

<Experimental Example 2: Chlorophyll Extract>

First, 1 kg of silkworm sand was infiltrated with 10 L of 99% ethanol for 1 week. Afterwards, the resulting solution was filtered and concentrated on a rotary evaporator to a volume of 1 L. Chlorophyll in the concentrate was quantified by high performance liquid chromatography.

<Experimental Example 3: Arrangement of Microfluidic Chips>

A microfluidic fluid focusing wafer with cross-shaped channels was fabricated from a phenolic resin substrate (length x width x depth = 270 mm x 210 mm x 1 mm) with an engraving machine via micromachining. This microfluidic chip has three layers, and the three layers have the same size (length x width x depth = 86mm x 44mm x 1mm). The top cover layer (including three reagent inlets and 20 fixing bolt holes), the middle layer (including cross-shaped channels and 20 bolt holes) and the bottom layer (including one product outlet and 20 bolt holes) are made of 20 M4 bolts. (Pitch 0.5mm, diameter 4mm, tightened with a torque of 2mN) to form a microfluidic chip. The bolt heads are respectively located at the entrance and exit of the top surface and the bottom surface of the integrated chip to balance the bonding force on the chip. Each inlet and outlet is provided with pads to assist in securing the polytetrafluoroethylene (PTFE) tubing for injection and collection of solutions. Disassembled microfluidic chips are easy to fabricate, mount, organize, and program.

<Experimental Example 4: Synthesis of Chl-PCL Microparticles>

A dispersed phase consisting of chlorophyll (from silkworm sand) and PCL (1% wt/v) was prepared in chloroform. A continuous phase consisting of an aqueous solution of PVA (1% wt/v) was also prepared. The continuous and dispersed phases were connected to the microfluidic device using a PTFE tube (0.76 mm inner diameter, 1.22 mm outer diameter) attached to a syringe, where the syringe was controlled by three digits controlled by the syringe pump. As shown in Figure 1, the dispersed phase is injected into the middle channel (2) from the central inlet (1), and the continuous phase is injected from the two side inlets (3). The two-phase flow rate changes the shear force within the cruciform channel, resulting in the formation of a monodisperse homogeneous Chl-PCL emulsion. The flow rate of the dispersed phase is controlled between 0.2 and 0.5 mL/h, and the flow rate of the continuous phase is controlled between 0.1 and 0.4 mL/min. The Chl-PCL emulsion was dropped onto a 45 mL petri dish through the central outlet (4), and then cured at 37°C for 24 hours. The spheroids were collected by centrifugation, then washed twice with 30 mL of distilled water to remove excess PVA reagent, followed by observation of Chl-PCL microparticles.

<Experimental Example 5: Characteristic Analysis>

Chl-PCL emulsions and Chl-PCL microparticles were observed through optical microscopy. The average diameter of emulsion or microparticles (expressed as mean ± standard deviation (SD)) was measured using photographs, and about 100 independent samples were randomly selected to reduce sampling bias. The micromorphology and physical characteristics of the composite particles were analyzed by scanning electron microscopy and ultraviolet-visible spectrophotometry (UV-VIS), respectively.

<Experimental Example 6: Stability Test of Chl-PCL Microparticles>

The chlorophyll in the silkworm sand was extracted with ethanol, and then the chlorophyll was transferred into a sealed glass ampoule. Chl-PCL microparticles were dissolved in ethanol and sealed in glass ampoules. These ampoules were placed in a white light environment at room temperature to prevent chlorophyll from being degraded by oxidation reaction, and then spectrophotometrically measured at different exposure times (0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours), and then the degradation rate of chlorophyll under white light was obtained.

<Analysis Example 1: Form>

Compared to other oil-water mixing systems, microfluidic devices are a competitive approach because they allow controllable polydispersity of the particle size distribution through the emulsification chamber. In the hydrofocusing design of oil-water emulsions, the shear force is caused by the relative strength between the oil and water flowing simultaneously. When oil and water pass through the cross-shaped channel, the droplets are elongated by shearing force, forming emulsion particles. In this paper, the PCL or Chl-PCL emulsion is formed by subjecting the PCL or Chl-PCL liquid to the PVA aqueous solution under the action of shearing force. The PCL or Chl-PCL solution of the dispersed phase is from the central inlet channel, while the PVA solution of the continuous phase is from the channels on both sides of the microfluidic device. 2A to 2H are optical microscopy images of emulsions and microparticles used in a microfluidic device, where the flow rates of the continuous and dispersed phases were fixed at 0.2 mL/min and 0.5 mL/h, respectively. 2A and 2B show the spherical structure and 240 μm diameter of the PCL emulsion. The Chl-PCL emulsions with core-shell spherical structures in Figures 2C and 2D are relatively different, and have a diameter of 274.7 μm. The diameter of the PCL emulsion loaded with chlorophyll was slightly larger than that of the PCL emulsion without chlorophyll. After 24 hours of curing, the Chl-PCL emulsion was converted into microparticles (68.1 μm in diameter, as shown in Figures 2E to 2H) with a shrinkage of 70% to 80%. Next, SEM images of PCL particles and Chl-PCL particles were obtained. Figure 3A shows an SEM image of PCL particles, while Figure 3B shows an SEM image of Chl-PCL particles. The relatively good particle size distribution and relatively consistent morphology in the images are due to a continuous phase flow rate of 0.4 mL/min and a flow rate of 0.3 mL/min. h is the separated phase flow rate. It can be seen from FIG. 3A that the surface of the PCL particles has excellent uniformity and smooth sphericity, with an average diameter of 33 μm. Holes can be observed in the SEM image of the Chl-PCL particles in Figure 3B, and the average diameter of the spherical particles is 35.7 μm. In conclusion, the PCL particles with chlorophyll are slightly larger than the PCL particles without chlorophyll.

<Analysis Example 2: Detection of Chl-PCL Microparticle Formation>

Figure 4 shows the absorption spectra of chlorophyll, PCL particles and Chl-PCL particles. From this figure, it can be seen that chlorophyll has two main absorption peaks at about 420 and 660 nm, respectively. These two broad peak bands show that chlorophyll is mixed with other substances. PCL particles have no clear peaks in the visible spectrum (approximately 400 to 700 nm). Chl-PCL particles have two main absorption peaks at about 420 and 660 nm, respectively. Chl-PCL microparticles exhibited an absorption spectrum similar to that of chlorophyll. According to FIGS. 2A to 2H and FIG. 4 , it was confirmed that PCL successfully encapsulated chlorophyll therein.

Chlorophyll is a useful green chelating compound, which appears green under visible light irradiation, but fluoresces under ultraviolet light, and the fluorescence appears in the red band. In order to evaluate the fluorescence distribution of PCL before and after curing, Chl-PCL emulsion and Chl-PCL microparticles were used. Figures 5A and 5B are optical microscopic images and fluorescence microscopic images of Chl-PCL emulsions, and Figures 5C and 5D are optical microscopic images and fluorescence microscopic images of Chl-PCL microparticles. The results showed that the fluorescence of all spherical particles was uniformly distributed (see Figures 5B and 5D). The uniform fluorescence distribution showed that the chlorophyll in each Chl-PCL sphere was monodisperse.

<Analysis example 3: PCL coating improves the stability of chlorophyll>

Noichinda, Bodhipadma, Mahamontri, Narongruk, and Ketsa in 2007 found that light accelerates the decay of chlorophyll content. In this study, in order to observe the decay of chlorophyll content, the experiment must be carried out at low temperature. According to the method proposed by Chen and Huang et al. in 1998, when measuring the color decay rate of chlorophyll, in addition to the chlorophyll content, the absorption rate measured by spectrophotometry at wavelengths of 630, 647, and 664 nm must also be considered, etc. physical parameters. The stability of Chl-PCL particles is measured by the absorption decay rate under white light irradiation, which has been described by the modified first-order reaction kinetics proposed by Steet and Tong et al. in 1996. The first-order reaction based on chlorophyll concentration can be expressed as ln(C/C ₀ )=-kt, where C ₀ is the initial concentration of chlorophyll, C is the concentration of chlorophyll at time point t, and k is the reaction rate constant. Figure 6 shows the degradation of chlorophyll and Chl-PCL complexes under white light irradiation for 24 hours at room temperature, which is consistent with the first-order reaction mode. The first-order degradation curves of the coated and uncoated chlorophyll during the illumination period were linear regression curves, and the correlation coefficients between the natural logarithm of the concentration and the illumination time were r ² =0.7623 and r ² =0.9965, respectively. The results showed that the degradation of chlorophyll conformed to the first-order reaction mode, and the uncoated chlorophyll degraded faster (can be known from the corresponding k value of 0.05511h ^-1 ).

Coating chlorophyll with polymer is not the first time. For example, in 2012, Fan et al. encapsulated chlorophyll in nanoscale liposomes to improve the water solubility of chlorophyll, but the focus was not on the stability of chlorophyll. It is an innovation of this paper to improve the stability of chlorophyll by polymer coating technology. PCL polymers are often used as drug carriers to protect sensitive drug molecules. In this paper, PCL polymer is used for physical coating of chlorophyll. PCL polymers can isolate chlorophyll molecules from the air, thereby reducing the rate of chlorophyll oxidation. In addition, the PCL polymer exhibits an absorption band in the wavelength range of 260 to 340 nm in the UV-VIS spectrum, which means that the PCL polymer has the ability to reflect and/or scatter ultraviolet light, and can protect the photosensitive substance. After chlorophyll is coated with PCL polymer, the composition of PCL can make chlorophyll unaffected by UV radiation, thus improving chlorophyll stability.

However, the above are only preferred embodiments of the present invention, but cannot limit the scope of implementation of the present invention; therefore, any simple equivalent changes and modifications made according to the scope of the patent application of the present invention and the contents of the description of the invention, All still fall within the scope of the patent of the present invention.

(1): Central entrance

(2): Middle channel

(3): Side entrance

(4): Central Exit

Figure 1 is a schematic diagram illustrating the preparation of Chl-PCL microparticles of the present invention. Figures 2A to 2H are optical microscopic images, wherein Figures 2A and 2B present PCL emulsions at different magnifications, Figures 2C and 2D present Chl-PCL emulsions at different magnifications, and Figures 2E and 2H present Chl-PCL microparticles at different magnifications . 3A and 3B are SEM images, wherein FIG. 3A shows the sample surface of PCL particles, and FIG. 3B shows the sample surface of Chl-PCL particles. FIG. 4 is an absorption spectrum diagram illustrating the composition of chlorophyll, PCL particles and Chl-PCL particles. Figures 5A to 5D are photographs, wherein Figures 5A and 5B are optical microscopic images of Chl-PCL emulsions and corresponding fluorescence microscopic images, and Figures 5C and 5D are optical microscopic images of Chl-PCL particles and corresponding fluorescent microscopic images. Light microscopy images. FIG. 6 is a graph showing the results of degradation analysis, illustrating the degradation of chlorophyll and Chl-PCL microparticles under irradiation with white light for 24 hours at room temperature.

(1): Central entrance

(2): Middle channel

(3): Side entrance

(4): Central Exit

Claims (7)

A coated particle capable of improving the stability of chlorophyll, comprising: a polycaprolactone shell; and a chlorophyll inner core, which is coated by the polycaprolactone shell, wherein the manufacturing method of the coated particle comprises the steps: Emulsifying a dispersed phase containing chlorophyll and polycaprolactone to form a plurality of monodisperse and uniform chlorophyll-polycaprolactone emulsions; and curing the chlorophyll-polycaprolactone emulsions to form a plurality of Chlorophyll-polycaprolactone particles, in each chlorophyll-polycaprolactone particle, the polycaprolactone is coated on the chlorophyll; wherein, the emulsifying step includes: providing a microfluidic device, the microfluidic The control device has a middle channel, a central inlet connected to one end of the middle channel, a central outlet connected to the other end of the middle channel, and two side inlets connected to opposite sides of the middle channel; inject the dispersion from the central inlet phase, and inject a continuous phase from the side inlets, so that the dispersed phase and the continuous phase flow to the middle channel to form the monodisperse and uniform chlorophyll-polycaprolactone emulsion; and collected from the central outlet These chlorophyll-polycaprolactone emulsions, wherein the injection flow rate of the dispersed phase is 0.2 to 0.5 mL/h, and the injection flow rate of the continuous phase is 0.1 to 0.4 mL/min. A method for manufacturing coated microparticles capable of improving chlorophyll stability, comprising: emulsifying a dispersed phase containing chlorophyll and polycaprolactone to form a plurality of monodisperse and uniform chlorophyll-polycaprolactone emulsion; and curing the chlorophyll-polycaprolactone emulsions to form a plurality of chlorophyll-polycaprolactone particles, wherein in each chlorophyll-polycaprolactone particle, the polycaprolactone coats the chlorophyll ; Wherein, the emulsifying step includes: providing a microfluidic device, the microfluidic device has a middle channel, a central inlet connecting one end of the middle channel, a central outlet connecting the other end of the middle channel, and two connecting the middle channel Side inlets on opposite sides of the middle channel; inject the dispersed phase from the central inlet, and inject a continuous phase from the side inlets, so that the dispersed phase and the continuous phase flow to the middle channel to form the monodisperse and uniform chlorophyll-polycaprolactone emulsion; and collecting the chlorophyll-polycaprolactone emulsion from the central outlet, wherein the injection flow rate of the dispersed phase is 0.2 to 0.5 mL/h, and the injection flow rate of the continuous phase is 0.2 to 0.5 mL/h. The rate is 0.1 to 0.4 mL/min. The production method according to claim 2, wherein the continuous phase contains polyvinyl alcohol. The manufacturing method of claim 2, wherein the side inlets are symmetrically connected to opposite sides of the middle channel. The manufacturing method as claimed in claim 2, wherein the temperature used in the curing step is 35 to 40° C., and the time is 20 to 28 hours. The manufacturing method of claim 2, wherein the particles have an average diameter of 30 to 70 μm. The manufacturing method of claim 2, wherein the continuous phase contains polyvinyl alcohol, the temperature used in the curing step is 35 to 40° C., the curing step is performed for 20 to 28 hours, and the average of the particles is The diameter is 30 to 70 μm.

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