TWI834477B - Extract of chenopodium formosanum koidz., preperation method thereof and use for anti-oxidation, anti-cytotoxicity and anti-inflammation - Google Patents

Abstract

The invention provides a Taiwanese quinoa sprout extract and a preparation method thereof. The Taiwanese quinoa sprout extract is prepared through germination, fermentation and extraction processes in sequence. The invention also provides a use of Taiwanese quinoa sprout extract for antioxidant, anti-cytotoxicity or anti-inflammation.

Description

Taiwanese quinoa sprout extract and its preparation method and use for antioxidant, anti-cytotoxicity or anti-inflammation

The present invention relates to a Taiwanese quinoa sprout extract and its preparation method. In particular, it is a preparation method including germination, fermentation and extraction processes to prepare the Taiwanese quinoa sprout extract and its use for antioxidant, anti-cytotoxicity or anti-inflammation. .

With the development of social economy, human productivity has improved, living standards have also improved day by day, and people's demand for healthy food has become increasingly urgent. Modern people's long-term irregular lifestyle and abnormal diet have led to a decline in body functions. Moreover, due to industrial development, pollution has increased, and the suspended particles produced have caused damage to the human respiratory system, especially suspended particles with a particle size of ≦2.5 microns (μm). Known as "fine suspended particulates (PM2.5)", they can pass through the alveoli and enter the human circulatory system and are not easily discharged, posing a major threat to health.

Taiwanese Chenopodium (scientific name: Chenopodium formosanum Koidz.) is a native Taiwanese plant of the Chenopodium genus of the Amaranthaceae family, Chenopodium subfamily. Traditionally called red pigweed, it was renamed Taiwanese pigweed in December 2008. It is a traditional crop cultivated by Taiwanese aborigines for more than a hundred years. It is an annual herb with strong plant growth and excellent drought tolerance, and the grains are Called "the ruby of the culinary world." In Taiwan, red quinoa is a traditional food crop of the aborigines. It is often cooked with rice, glutinous rice or taro to become rice dumplings, bamboo tube rice, or used to brew millet wine. Non-aboriginals have also begun to cook it with flour. , into baking and meal dishes, or combined with millet food, such as red quinoa millet porridge, red quinoa millet wine, red quinoa millet donuts. Currently, it is cultivated by aborigines in Majiajia, Taitung and Hualien areas of Pingtung County. The shell of Taiwanese quinoa contains slightly toxic "saponin", which makes Taiwanese quinoa bitter and tastes bad when eaten in the shell, making it difficult to process and eat. It also limits the intake of Taiwanese quinoa. Therefore, there are many Taiwanese quinoas after harvesting. The reddish-brown shell will be removed, but Taiwanese quinoa shells are rich in many nutrients and active ingredients. If eaten with the shell, it should be beneficial to health.

In addition to the above dietary uses, whether Taiwanese quinoa has any effect on human health still needs further research. Previous literature has pointed out that the active ingredients of Taiwanese quinoa are affected by factors such as different harvest periods, weather conditions, and extraction methods. Internal changes, resulting in changes in composition. Therefore, there is still a need for further analysis and research on the effectiveness of the active ingredients of Taiwanese quinoa under what cultivation or extraction conditions.

The present invention therefore proposes an application method using Taiwanese quinoa as a material, including the extracted Taiwanese quinoa sprout extract and its preparation method, and further confirms that it has antioxidant, anti-cytotoxic and anti-inflammatory effects.

According to one embodiment of the present invention, the present invention provides a method for preparing a Taiwanese quinoa bud extract, which includes first subjecting a Taiwanese quinoa bud to a germination process to form a Taiwanese quinoa bud, and then subjecting the Taiwanese quinoa bud to an inoculation process. A fermentation process is performed to obtain a fermented Taiwanese quinoa sprout. Finally, the fermented Taiwanese quinoa sprout is subjected to an extraction process to obtain a Taiwanese quinoa sprout extract. The Taiwanese quinoa sprout extract is obtained by taking a final water layer in the extraction process.

According to another embodiment of the present invention, a Taiwanese quinoa sprout extract is provided. The active ingredients of the Taiwanese quinoa sprout extract include: pyroglutamic acid, gluconic acid, uridine, malic acid, xanthine nucleoside, 3-hydroxy -3-Methylglutaric acid, acetyl-L-carnitine, pantothenic acid and 4-hydroxyproline.

According to another embodiment of the present invention, a use of Taiwanese quinoa sprout extract for preparing a composition with antioxidant, anti-cytotoxicity or anti-inflammatory properties is provided.

In order that those with ordinary knowledge in the technical field to which the present invention belongs can further understand the present invention, the preferred embodiments of the present invention will be listed in the following description, and together with the drawings, the content of the present invention and the effects to be achieved will be described in detail. .

The present invention first provides a preparation method of Taiwanese quinoa sprout extract, which includes the following steps: (A) subjecting a Taiwanese quinoa sprout to a germination process to become a Taiwanese quinoa sprout; (B) subjecting the Taiwanese quinoa sprout to an inoculation fermentation process A fermented Taiwanese quinoa sprout is obtained; (C) the fermented Taiwanese quinoa sprout is subjected to an extraction process to obtain a Taiwanese quinoa sprout extract.

In step (A), the Taiwanese quinoa can be Taiwanese quinoa of any grain color, such as red quinoa, red orange quinoa, orange quinoa, red purple quinoa, black quinoa, and white quinoa, but is not limited thereto. In a preferred embodiment, unhulled Taiwanese quinoa is used to retain the nutrients in the shell to the maximum extent. In one embodiment of the present invention, the germination process includes a sterilization step, a cleaning step and a water sprinkling step. The sterilization step can use any chlorine-containing sterilization solution that does not harm Taiwanese quinoa, such as sodium hypochlorite; the cleaning step uses water or other similar solutions to remove the aforementioned sterilization solution by washing or soaking, and then drain the Taiwanese quinoa; sprinkle The water step is preferably carried out in a light-proof environment, and water is sprinkled on the surface at certain intervals to promote the germination of Taiwanese pigweed. In a preferred embodiment of the present invention, Taiwanese quinoa sprouts are selected after the 4th day of germination to obtain the maximum extract content, and the Taiwanese quinoa sprouts after 4 days of germination are stored in a refrigerator at -80°C.

In step (B), Taiwanese quinoa sprouts are subjected to an inoculation fermentation process to obtain fermented Taiwanese quinoa sprouts. In a preferred embodiment of the present invention, solid-state fermentation is used, which may be flat plate fermentation or bioreactor fermentation. The plate fermentation process includes cultivating the Taiwanese Chenopodium buds on a substrate, applying an auxiliary liquid, and then inoculating the Taiwanese Chenopodium buds with a spore suspension to allow fermentation and culture. The culture medium can be a matrix containing different amounts of free water and can provide a certain nitrogen source. In one embodiment, it is, for example, potato dextrose agar (PDA). The auxiliary liquid includes auxiliary substances that can assist fermentation, such as sucrose and buffer. In one embodiment, the sucrose concentration is 0.1~5%, and the buffer is 0.1~5% KH ₂ PO ₄ . The spore suspension is made from spores of filamentous fungus, which is characterized by producing a higher concentration of protease, such as Aspergillus (such as Aspergillus awamari), Rhizopus ( For example, Rhizopus microspores var. oligosporus), etc., but are not limited to this. In one embodiment, the amount of inoculated bacteria is 1% to 10% (v/w). If a bioreactor is used, the inoculation and fermentation process includes placing the Taiwanese Chenopodium buds into a bioreactor, applying an auxiliary liquid, and then inoculating the Taiwanese Chenopodium buds with a spore suspension to allow fermentation and culture. For example, the bioreactor has a holding tank for placing the aforementioned culture medium, and the holding tank has functions of adjusting temperature and air flow. Please refer to Figure 1, which is a schematic diagram of an embodiment of a bioreactor of the present invention. As shown in Figure 1, the bioreactor has a tank, and the stirring shaft in the tank can be connected to a drive motor to adjust the stirring speed or set the stirring time. The bioreactor is also equipped with a controller that can sense the temperature of the holding tank through a thermal sensor and adjust the temperature of the holding tank in real time through an external water bath. Temperature; in one embodiment, the controller also has an air valve control (flow meter) to control the air inlet volume and air outlet volume of the accommodation tank. In a preferred embodiment of the present invention, the fermentation temperature is maintained at 32~38°C, and the air flow is controlled at 1~3 LPM (liter of air volume/min).

In step (C), Taiwanese quinoa sprouts are fermented through an extraction process to obtain a Taiwanese quinoa sprouts extract. In one embodiment, the extraction process sequentially includes a rough extraction step, a concentration step and a layered extraction step. In one embodiment, the crude extraction step includes adding an alcohol (such as ethanol) to the freeze-dried sample of step (B) and extracting it more than twice to obtain a crude extract. In one embodiment, the concentration step is, for example, a vacuum concentration step, which includes depressurizing the crude extract to volatilize the alcohol to a predetermined volume, and then adding water to dissolve it to obtain a concentrated aqueous layer. In one embodiment, the layered extraction step includes adding an alkane to the aforementioned concentrated aqueous layer for extraction, and then extracting a first aqueous layer; then adding an ester to the first aqueous layer for extraction, and then extracting a second aqueous layer. ; Finally, an alcohol is added to the second water layer for extraction, and a final water layer is obtained. The final water layer obtained is the Taiwanese quinoa sprout extract of the present invention. In one embodiment, the alkane is, for example, n-pentane ( n -pentane), n-hexane ( n -hexane), n-heptane ( n -heptane), preferably n-hexane, but is not limited thereto; ester The alcohol is, for example, ethyl acetate; the alcohol is, for example, propan-1-ol or butanol, preferably butanol. The number of extractions mentioned above is more than 2 times, preferably 3 times. The preferred extraction ratios mentioned above are all 1:1 (v/v).

The Taiwanese quinoa sprout extract obtained by the present invention has antioxidant and anti-inflammatory effects, and can inhibit the body toxicity caused by PM2.5. The effective dose used is 10-100 ppm, preferably 20-75 ppm. In one embodiment, the active ingredients of the Taiwanese quinoa sprout extract include: pyroglutamic acid, gluconic acid, uridine, malic acid, xanthine nucleoside, 3-hydroxy-3-methylglutarate, acetyl L-carnitine, pantothenic acid and 4-hydroxyproline. In one embodiment, at the effective extraction dosage (for example, 25 ppm) of the aforementioned Taiwanese quinoa sprout extract, the concentrations of the active ingredients are as follows: pyroglutamic acid is 20-200 ppb, preferably 50-150 ppb, Gluconic acid is 10-100 ppb, preferably 20-80 ppb, uridine is 1-50 ppb, preferably 5-35 ppb, malic acid is 1-50 ppb, preferably 3-15 ppb, xanthine Nucleosides are 0.1-10 ppb, preferably 0.5-5 ppb, 3-hydroxy-3-methylglutaric acid is 0.1-10 ppb, preferably 0.5-2 ppb, acetyl L-carnitine is 0.01-5 ppb, preferably 0.1-1 ppb, pantothenic acid is 0.01-1 ppb, preferably 0.05-0.5 ppb, and 4-hydroxyproline is 0.01-1 ppb, preferably 0.05-0.5 ppb. Relevant embodiments and experimental data will be presented below, and diagrams will be used to assist in explaining the efficacy of the Taiwanese quinoa sprout extract of the present invention.

Preparation method of Taiwan Quinoa sprout extract

Step (A): Germination: Use unhulled Taiwanese quinoa as raw material, soak in 1.25% sodium hypochlorite aqueous solution for 10 minutes for surface sterilization, then rinse with clean water for 30 minutes to remove impurities and sodium hypochlorite, and soak in water at room temperature for 19 hours. Drain, lay flat in a plastic tray to avoid light, sprinkle water on the surface every 24 hours, harvest after the 4th day of germination and store in -80°C refrigerator.

Step (B): The inoculation fermentation process can use two implementation methods: "plate fermentation" or "bioreactor fermentation".

1. Plate fermentation method: Use the plate to ferment the Taiwanese quinoa sprouts on the 4th day of germination. First, perform matrix pretreatment. Add 1.3% sucrose and 0.3% KH ₂ PO ₄ to the Taiwanese quinoa sprouts on the 4th day of germination. Use a medical spoon. After mixing thoroughly, sterilize the kettle with moist heat at 121°C for 20 minutes, then lower the temperature to below 35°C on a sterile operating table for inoculation. Sterilized and cooled germinated Taiwanese pigweed was filled into sterile Petri dishes (100×15 mm) under aseptic operation. Each Petri dish was filled with 20 g wet weight sample and inoculated with 1 mL of 10 ⁶ spores/mL oligospores. Rhizopus microspores var. oligosporus ( BCRC 31996) spore suspension, the inoculation amount is 5% (v/w), mix well with a spatula, ferment and culture at 35℃, relative humidity (RH) 80% From 0 to 10 days, the fermented bean sprouts will be taken for the subsequent extraction process on the 5th day.

2. Bioreactor fermentation method: Use the bioreactor shown in Figure 1 to ferment the above-mentioned Taiwanese quinoa sprouts on the 4th day of germination. Place 1.5 kg of germinated Taiwanese quinoa into the holding tank of the bioreactor, and Add 1.3% sucrose and 0.3% KH ₂ PO ₄ and stir evenly. Then, the entire holding tank is completely covered with a sterilization bag and placed into a sterilization kettle. After sterilization with moist heat at 121°C for 40 minutes, it is cooled for 10 hours. After the storage tank and controller are assembled, inoculate 75 mL of 10 ⁶ spores/mL spore suspension at the feeding port with an inoculation amount of 5% (v/w). After stirring evenly with the stirring shaft, set it at the fermentation temperature. Allow for static fermentation at 35°C and a ventilation rate of 2 LPM (liter of air volume/min). Turn the stirring shaft to turn the fermentation substrate once a day to increase yield.

Step (C): Extraction process:

1. Crude extraction step First, add 10-15 times (weight) of alcohol (1:15 = freeze-dried sample: alcohol). After stirring for 0.5-1 hour, use suction filtration to separate the freeze-dried sample for the second extraction. Repeat the previous steps 3 times to complete 3 extractions.

2. Concentration step Concentrate the alcohol extract under reduced pressure until it is completely dry (the round-bottom bottle must be washed and weighed first). After concentration under reduced pressure and weighing again, the weight of the concentrate can be obtained. Take 50-150 mL of secondary water and re-dissolve (ultrasonic vibration can be used to assist the re-dissolution, 50 mL each time. If it can be re-dissolved, there is no need to add water). Aliquot into centrifuge tubes, centrifuge at 5000 rpm for 10 minutes, and take the supernatant (i.e. concentrated aqueous layer) for subsequent processes.

3. Layered extraction step Alkane extraction: Prepare a separatory funnel, pour the above supernatant (the volume of the solution should not exceed three-quarters of the volume of the separatory funnel), and then add an equal volume of n-hexane (for example: 100 mL of supernatant, then n-hexane Add 100 mL of alkane). After tightening the lid, shake the separatory funnel vigorously to mix the two immiscible solutions (during this period, you can open the stopcock to deflate as needed, about once every five shakes). Place the separatory funnel on a rack, open the lid and let it sit to allow the layers to separate. The denser water layer back to the solution is at the bottom, and the n-hexane layer is at the top. Let stand for about 10 minutes. Discharge the water layer back into the beaker and shake the separatory funnel slightly to allow the n-hexane layer to settle. Then collect the n-hexane layer into the serum bottle. Repeat three layered extractions with n-hexane, which is n-hexane division (hereinafter referred to as Hex group or HEX group in the experiment).

Ester extraction: Pour the water layer discharged into the beaker (i.e. the first water layer) back into the separatory funnel, add an equal volume of ethyl acetate for extraction, secure the lid and shake the separatory funnel vigorously to make the two immiscible. Mix the solution (during this period, you can open the stopcock to deflate as needed, about once every five shakes). Place the separatory funnel on a rack, open the lid and let it sit to allow the layers to separate. The denser water layer is at the bottom and the ethyl acetate layer is at the top. Let stand for about 10 minutes. Drain the aqueous layer back into the beaker and shake the separatory funnel slightly to allow the ethyl acetate layer to settle. Collect the ethyl acetate layer into a serum bottle. Repeat three layered extractions with ethyl acetate, which is the ethyl acetate partition (hereinafter referred to as EA group in the experiment).

Alcohol extraction: Pour the water layer returned to the solution (i.e. the second water layer) discharged into the beaker back into the separatory funnel, and add an equal volume of butanol for extraction. After tightening the lid, shake the separatory funnel vigorously to mix the two immiscible solutions (during this period, you can open the stopcock to deflate as needed, about once every five shakes). Place the separatory funnel on a stand, open the lid and let it sit. Make layers. The denser water layer is at the bottom, and the butanol layer is at the top. Let stand for about 10 minutes. Drain the water layer back into the beaker and shake the separatory funnel slightly to allow the butanol layer to settle. Collect the butanol layer into a serum bottle. Repeat three layered extractions with butanol, which is the butanol division (hereinafter referred to as the BU group or BuOH group in the experiment).

The remaining water layer solution was also collected into the serum bottle to divide the water into layers (i.e. the final water layer, referred to as the WA group or Water group in the following experiments). Concentrate each divided layer under reduced pressure. When about 20 mL remains, transfer the solution to a brown bottle (the brown bottle needs to be dried first and weighed as W1). Then concentrate each divided layer to dryness under reduced pressure, and then dry the brown bottle and weigh it as W2, and you can know the solid weight (=W2-W1).

Experiment 1: The influence of Taiwanese quinoa sprout extracts produced by different germination days in the germination process of step (A). Please refer to Figure 2, which shows the Taiwanese quinoa in the germination process of the present invention according to the germination of Taiwanese quinoa. After different days, they are divided into 8 groups (G0 is the 0th day of germination (that is, it has not yet germinated), G1 is the 1st day after germination, G2 is the second day after germination, and so on) and then goes through step (B) After the inoculation fermentation process (take the fermented Taiwanese quinoa sprouts on the 5th day) and the extraction process in step (C) (take the final water layer WA group), analyze the free radical scavenging effect of the Taiwanese quinoa sprout extract produced in each group. As shown in Figure 2 (A), it shows the germination status of different days groups. It can be observed from the picture that as the number of days increases, the germination status of Taiwanese pigweed is good and consistent, and there is no situation of being too dense or too sparse. It shows that the experimental model is healthy and normal; in the picture (B), the horizontal axis is the germination time (days) from day 0 to day 7 (G0~G7), and the vertical axis is the germination rate (%). , it can be seen from the figure that as the number of days evolves, all germination occurs from about the 2nd to the 3rd day, indicating that the cultivation environment of this embodiment is suitable for Taiwanese pigweed germination; in the figure (C), the horizontal axis is the 0th day of germination. From day to day 7 (G0~G7), the vertical axis is the sprout length (unit: mm), among which statistically G7, G6, G5, G4, and G3 are the groups with the highest sprout length (a , b), G2 and G1 are the second highest groups (represented by c, d), and G0 is the control group (represented by e). The figure shows that as the days progress, the bud length also increases in equal proportion; in (D ) In the figure, the horizontal axis is the germination days from day 0 to day 7 (G0~G7), and the vertical axis is the free radical scavenging efficiency of DPPH (α,α-Diphenyl-β-picrylhydrazyl). The experimental procedure is to mix the ethanol solution of DPPH Prepare a concentration of 0.1 mM and mix it with the sample evenly (1:1, v/v) and let it stand for 30 minutes in a dark environment to make the final concentration 0.2~7 mg Taiwanese Chenopodium bud extract/mL. After a light-proof reaction After about 30 minutes, measure the absorbance value at 517 nm with a spectrophotometer (SpectraMax ^® ), where the DPPH free radical scavenging rate (%) = (1-absorbance value of the experimental group/absorbance value of the control group), and additionally configure 0.01~0.1 mg/mL of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as the standard curve of the reaction in the same experiment, and the unit sample was converted by interpolation method to have the same DPPH radical scavenging ability. The Trolox equivalent concentration (mg/mL) is expressed by the total antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC) (mg TE/g). In terms of DPPH free radical scavenging efficiency, statistically G7, G6, G5, and G4 are the highest groups (represented by a, ab, b), G3 and G2 are the second highest groups (represented by c, d), and G1 is The poor group (represented by e), G0 is the control group (represented by f). From the comparison of the above values, it can be seen that as the number of days evolves, a comparable free radical scavenging effect has been achieved in G3, while the free radical scavenging effect in G4~G7 is approximately the same; in the figure (E), the horizontal axis is the 0th day of germination. From day to day 7 (G0~G7), the vertical axis is the free radical scavenging efficiency of ABTS (Azino-bis. (3-ethylbenzthiaoline)-6-sulfonic acid). The experimental conditions are as follows: prepare 7 mM ABTS solution and 2.45 mM potassium persulfate (K ₂ S ₈ O ₂ ) solution, mix the two solutions in equal volumes (1:1 ratio) in advance and store in a light-proof environment for 12 hours, the ABTS free radical solution was diluted with distilled water before use until the absorbance value at 734 nm was 0.7±0.02 as the reaction solution. Take 20 μL of the diluted sample extract and 180 μL of the ABTS free radical reaction solution and add it to 96 well. After mixing and reacting for 6 minutes, measure the absorbance value at the wavelength of 734 nm. ABTS clearance rate (%) = (1-absorbance value of the experimental group) /absorbance value of the control group), and additionally configure 0.01~0.1 mg/mL Trolox as the standard curve of the reaction in the same experiment, and convert the Trolox equivalent concentration (mg/mL) of the unit sample with the same ABTS free radical scavenging ability, Expressed as total antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC) (mg TE/g). Statistically, G7, G6, G5, and G4 are the highest groups (represented by a and b), G3, G2, and G1 are the second highest groups (represented by c and d), and G0 is the control group (represented by e). It can be seen from this figure that as the days progress, a comparable free radical scavenging effect has been achieved in G3, while the free radical scavenging effect in G4~G7 is approximately the same, which is consistent with the aforementioned DPPH data. As can be seen from Figure 2 (A) ~ (E), in the germination process of step (A) of the present invention, the free radical scavenging effect after the 4th day (G4 ~ G7) is roughly the same. Therefore, the industrialization process of the product should be paid attention to. In terms of time efficiency, choosing Taiwanese quinoa sprouts on the 4th day after germination can achieve the fastest results without sacrificing quality.

Experiment 2: The influence of Taiwanese quinoa sprout extract produced by different fermentation days in the inoculation fermentation process of step (B) . Please refer to Figure 3, which shows the Taiwanese quinoa of the present invention after the germination process of step (A) (take the 4-day-old Taiwanese quinoa buds), and then go through step (B) of the inoculation and fermentation process, which is divided into 11 groups according to the number of days of inoculation and fermentation of Taiwanese quinoa buds (PF0 is the 0th day of inoculation and fermentation (that is, it has not yet been inoculated and fermented). ), PF1 is the first day of inoculated fermentation, PF2 is the second day after inoculated fermentation, and so on), followed by step (C) extraction process (take the final water layer WA group), and finally analyze the products produced by each group Free radical scavenging effect of Taiwanese quinoa sprout extract. In Figure 3 (A), it shows the fermentation status of different groups of days. Observing the picture, we can see that at PF0, Taiwanese quinoa buds are evenly distributed on the culture plate, and as the number of days increases, Taiwanese quinoa buds are evenly distributed on the culture plate. After the Chenopodium quinoa buds were implanted into the spore liquid, they covered the entire culture medium at approximately PF2, and evenly and densely interfered with the Taiwan Chenopodium buds for interactive fermentation. The picture shows that the fermentation state of the inoculated bacteria is good; in (B), the horizontal axis is the fermentation state of the inoculated bacteria. The bacterial fermentation days are from day 0 to day 10 (PF0~PF10). The vertical axis is the DPPH free radical scavenging efficiency (the experimental procedure is the same as the aforementioned Figure 2 (D)). In terms of DPPH free radical scavenging efficiency, statistically speaking, PF2 , PF3 is the highest group (expressed by a, ab), PF4, PF5, and PF6 are the second highest groups (expressed by bc), PF1 and PF7 are the second best groups (expressed by cd), PF8, PF9, and PF10 is the unsatisfactory group (expressed by d), and PF0 is the control group (expressed by d). From the above data, it can be seen that in the early stage of inoculation fermentation, due to insufficient fermentation time, good Taiwanese quinoa sprout extract DPPH free radical scavenging performance cannot be achieved. After a certain period of fermentation (PF2~PF6), a considerable free radical scavenging effect can be achieved, but after a long time (PF7~PF10), the free radical scavenging ability decreases; in the picture (C), the horizontal axis represents the inoculated fermentation. From day 0 to day 10 (PF0~PF10), the vertical axis is ABTS free radical scavenging efficiency. In terms of ABTS free radical scavenging efficiency, statistically speaking, PF3, PF2, PF5, PF4, PF6, and PF7 are the highest groups ( Indicated by a, ab, b), PF9, PF8, and PF10 are the second highest group (indicated by c, cd), PF1 is the poor group (indicated by de), and PF0 is the control group (indicated by e). From From the above data, it can be seen that in the early stage of inoculation fermentation, due to insufficient fermentation time, the ABTS free radical scavenging effect of Taiwan Chenopodium quinoa bud extract cannot be achieved. However, after a certain period of fermentation (PF2~PF7), it can achieve a comparable free radical scavenging effect. However, After a long time (PF8~PF10), the free radical scavenging ability decreases, which is consistent with the aforementioned data of DPPH. It can be seen from Figure 3 (A) ~ (C) that in the inoculation fermentation process of step (B) of the present invention, the free radical scavenging effect is particularly good from about the 2nd to the 7th day, and in the industrialized process of the product Paying attention to time efficiency, taking comprehensive considerations into fermenting Taiwanese quinoa sprouts on the 5th day after inoculation and fermentation can achieve the fastest effect without sacrificing quality.

Experiment 3: Antioxidant efficacy test of Taiwanese quinoa sprout extract. The TEAC method was used to test the antioxidant capacity (DPPH and ABTS) of four groups of samples. The DPPH antioxidant experimental process is as follows: Prepare an ethanol solution of DPPH with a concentration of 0.1mM and mix it evenly with the sample (1:1, v/v), then let it stand in a dark environment for 30 minutes to make the final concentration 0.2~7 mg. Taiwan Chenopodium bud extract/mL, after reacting in the dark for about 30 minutes, measure the absorbance at 517 nm with a spectrophotometer ( ^SpectraMax® ), where the DPPH free radical scavenging rate (%) = (1-absorbance value of the experimental group/control The absorbance value of the group), and additionally configure 0.01~0.1 mg/mL Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) in the same experiment as the standard curve of the reaction, and within Interpolation is used to convert the Trolox equivalent concentration (mg/mL) of the unit sample with the same DPPH free radical scavenging ability, expressed as total antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC) (mg TE/g). The ABTS antioxidant experimental steps are as follows: prepare 7 mM ABTS (Azino-bis. (3-ethylbenzthiaoline)-6-sulfonic acid) solution and 2.45 mM potassium persulfate (potassium persulfate, K ₂ S ₈ O ₂ ) solution, and combine the two Equal volumes of the solution (1:1 ratio) were mixed in advance for 12 hours. The ABTS free radical solution was diluted with distilled water before use until the absorbance value at 734 nm was 0.7±0.02 as the reaction solution. Add 20 μL of diluted sample extract and 180 μL of ABTS free radical reaction solution to 96 well, mix and react for 6 minutes, then measure the absorbance value at 734 nm wavelength, and additionally prepare 0.01~0.1 mg/mL Trolox for the same experiment. As the standard curve of the reaction, the Trolox equivalent concentration (mg/mL) of the unit sample with the same ABTS free radical scavenging ability is converted and expressed as total antioxidant capacity (Trolox equivalent antioxidant capacity, TEAC) (mg TE/g). The results in Table 1 below show that in the DPPH antioxidant experiment, the "BU" group had the best antioxidant capacity (represented by a), the "EA" group was second (represented by b), and the "WA" group was third (represented by Indicated by c), the "HEX" group is last (indicated by d). In the ABST antioxidant experiment, the "WA" group had the best antioxidant capacity (represented by a), followed by the "BU" group (represented by b), the "HEX" group third (represented by c), and the "EA" group. ”The last one in the group (indicated by d). Table 1 Antioxidant test of Taiwanese quinoa sprout extract layered DPPH (mg TE/mg extract) TEAC _ABTS (mg TE/mg extract) WA 2.07± ^0.02c 15.06±2.35 ^a BU 58.47± ^4.77a 12.23± ^0.07b HEX 8.20±0.06 ^days 11.03±0.49 ^c EA 35.46±7.18 ^b 14.45±0.19 ^days

Experiment 4: Safety test of Taiwanese quinoa sprout extract. This experiment used CCK-8 (Dojindo laboratories, Kumamoto, Japan) to test eight groups of Taiwanese quinoa sprout extract (“Hex”, The safety of "EA", "BuOH" and "Water"). The experimental steps are as follows: During the experiment, take 100 μL of cell suspension and seed it into a 96-well plate at a cell concentration of 10 ⁵ cells/mL. Cultivate it in a 37°C, 5% CO ₂ incubator for 24 hours. After that, remove the culture medium and use the cells. Stain with 10% CCK-8 solution and incubate for 1 hour. Absorbance was measured at 450 nm using an Elisa Reader (MULTISKAN GO, Thermo Fisher Scientific, Waltham, MA, USA) and cell viability was calculated using the following formula: Cell viability = (OD ₄₅₀ sample/OD ₄₅₀ control) x 100%.

Please refer to Figure 4, which shows a bar chart of the toxicity and dose test of the Taiwanese quinoa sprout extract of the present invention, in which the horizontal axis represents each group and the vertical axis represents cell viability (unit: %). As shown in Figure 4, among the eight groups of Taiwanese quinoa sprout extracts, compared with the survival rate (100%) of the control group (Control, CTRL group), the "Water" group has the best survival rate at a concentration of 25 ppm. The cell survival rate (expressed as a) was followed by the concentration of 50 ppm (expressed as ab), and the other groups were not as good as the above two groups. It is obvious that the "Water" group has the best safety.

Experiment 5: Inhibition of ROS production and restoration of cell viability induced by PM2.5 using Taiwanese quinoa bud extract. PM2.5 was purchased from Sigma-Aldrich (now a subsidiary of Merck) with the number CRM558. The ingredients are shown in Table 2. . Table 2 Composition Classification concentration Quartz STOT RE 2; H373 ≧90, ≦100% Baseoil Carc. 1B; H350 ≧0.1, ≦1% Fuel oil NO.2 Carc. 2; H351 ≧0.1, ≦1%

The PM2.5 used in the experiment was dissolved in PBS at a concentration of 10,000 μg/mL and prepared as a stock solution. When processing cells, first use ultrasonic shock for 30 minutes, and then conduct experiments with the cell culture medium diluted as the target concentration. During the experiment, MHS cells (a type of alveolar cells) were inoculated into a 96-well plate at 1×10 ⁴ /well and cultured in a 37°C constant-temperature incubator for 24 hours. After the cells were attached, the medium was removed and 25 ppm Taiwanese After treatment for 24 hours in the "WA", "BU", "HEX" and "EA" groups of Chenopodium bud extracts, the culture medium was removed and replaced with the general culture medium.

Please refer to Figure 5, which shows the test chart of the Taiwan Chenopodium quinoa sprout extract of the present invention for inhibiting PM2.5-induced ROS production and restoring cell viability. Picture (A) is a cell microscope picture, and the left part is a general microscope. Figure, the right part is a fluorescence microscope image of reactive oxygen species (ROS); Figure (B) is a bar chart of values calculated based on the number of cells and fluorescence intensity in Figure (A), which are grouped as follows : Control group (Control, CTRL group), 0 μg/mL PM2.5 (PBS only, VEH group), 200 μg/mL PM2.5 (200PM group), 200 μg/mL PM2.5+0.5 mM N-acetyl cysteine (NAC) (200PM+NAC group), 200 μg/mL PM2.5+25 ppm "BU" extract (200PM+bu25 group), 200 μg /mL of PM2.5+25 ppm "EA" extract (200PM+EA25 group), 200 μg/mL of PM2.5+25 ppm "HEX" extract (200PM+hex25), 200 μg/mL of PM2.5+25 ppm "WA" extract (200PM+Water25); (C) is a bar graph of the calculated values based on the quantification of the number of cells in (A) (CTRL is 100%), The groups are as follows: control group (Control, CTRL group), 0 μg/mL PM2.5 (PBS only, VEH group), 200 μg/mL PM2.5 (PM group), 200 μg/mL PM2. 5+0.5 mM N-acetyl cysteine (PM+NAC group), 200 μg/mL PM2.5+25 ppm "BU" extract (BU group), 200 μg/mL PM2.5+ 25 ppm "EA" extract (EA group), 200 μg/mL PM2.5+25 ppm "HEX" extract (HEX group), 200 μg/mL PM2.5+25 ppm "WA" Extracts (Group WA).

As shown in Figure 5(B), in terms of ROS generation, the "200PM+Water25", "200PM+EA25" and "200PM+bu25" groups all have lower ROS generation, which is close to the control group (CTRL group), "200PM+hex25" has a higher ROS generation amount, which is close to only joining the PM2.5 group (200PM group). As shown in Figure 5(C), in terms of cell survival rate, the "WA" group is closest to the control group (CTRL group), and the other groups are not ideal. It can be seen that the "WA" group can significantly reduce the ROS damage caused by PM2.5 and improve the cell survival rate, which has a significant effect on reducing the damage caused by PM2.5 to cells.

Experiment 6: The anti-inflammatory effect of the " WA " group of Taiwanese quinoa sprout extract. This experiment further studies the regulation of inflammation/anti-inflammatory related pathway (NF-κB pathway) proteins of the "WA" group of Taiwanese quinoa sprout extract. . Please refer to Figure 6, which is a schematic diagram showing the expression of NF-κB protein in MHS cells induced by PM2.5 in the "WA" group of Taiwanese quinoa bud extract of the present invention. The upper part is NF-κB p65 and Western blot diagram of Ser536 phosphorylated NF-κB p65 protein. The lower half shows the phosphorylation ratio (p-NF-κB p65/NF-κB p65) converted according to the Western blot diagram. As shown in Figure 6, after treatment with PM2.5 (PM group), the NF-κB phosphorylation ratio of MHS cells increased significantly compared with the control group (CTRL group), and after PM2.5 plus Taiwan After simultaneous treatment with the "WA" group of Chenopodium quinoa bud extract (PM+WA group), the NF-κB phosphorylation ratio of MHS cells was significantly reduced compared to the aforementioned PM 2.5 treatment (PM group), and was even close to that of the control group ( CTRL group), representing the "WA" group of the Taiwanese quinoa sprout extract of the present invention, which can effectively inhibit the phosphorylation of NF-κB.

Please also refer to Figure 7, which is a schematic diagram showing the expression of inflammatory genes and anti-inflammatory genes after PM2.5 induces MHS cells in the "WA" group of Taiwanese quinoa sprout extract of the present invention, in which the horizontal axis IL1B (Interleukin -1beta, interleukin 1β), IL6 (Interleukin-6, interleukin 6), TNFa (Tumor Necrosis Factor, tumor necrosis factor) have the effect of promoting inflammation, IL1Ra (Interleukin 1 receptor antagonist, interleukin 1 receptor Antagonist) and IL10 (leukin 10) have the effect of inhibiting inflammation, and the vertical axis represents the mRNA expression amount of the above proteins (compared to the CTRL ratio in the control group). As shown in Figure 7, after treatment with PM 2.5 (PM group), the mRNA expression levels of the inflammatory proteins IL1B, IL6 and TNFa in MHS cells increased significantly compared with the control group (CTRL group), and the anti-inflammatory protein IL1Ra The mRNA expression levels of IL10 and IL10 decreased; however, after simultaneous treatment with PM2.5 and the "WA" group of Taiwanese quinoa sprout extract (WA group), the mRNA expression levels of inflammatory proteins IL1B, IL6, and TNFa were reduced. It can increase the expression of mRNA of the anti-inflammatory protein IL10. This experiment can prove that the "WA" group of the Taiwanese quinoa sprout extract of the present invention can effectively inhibit the adverse inflammatory consequences caused by PM2.5, and therefore has anti-inflammatory effects.

Experiment 7: Composition analysis of the " WA " group of Taiwanese quinoa sprout extract. The present invention conducts composition analysis on the "WA" group of Taiwanese quinoa sprout extract, and can obtain the composition and proportion table as shown in Table 3 (effective at 25 ppm measured at the extraction dose). Table 3 Identification of ingredients in the "WA" group of Taiwanese quinoa sprout extract No. Element chemical formula molecular weight Concentration(ppb) 1 L-Pyroglutamic Acid C ₅ H ₇ NO ₃ 129.04 95.57 ± 30.39 2 Gluconic Acid C ₆ H ₁₂ O ₇ 196.16 46.93 ± 18.23 3 Uridine C ₉ H ₁₂ N ₂ O ₆ 244.20 18.62 ± 6.83 4 Malic acid (L-(-)-Malic Acid) C ₄ H ₆ O ₅ 134.08 9.27 ± 3.27 5 Xanthosine C ₁₀ H ₁₂ N ₄ O ₆ 284.22 2.78 ± 0.96 6 3-Hydroxy-3-methylglutaric Acid C ₆ H ₁₀ O ₅ 162.14 1.19 ± 0.26 7 Acetyl-L-carnitine C ₉ H ₁₇ NO ₄ 203.23 0.48 ± 0.22 8 Pantothenic Acid C ₉ H ₁₇ NO ₅ 219.23 0.20 ± 0.05 9 4-Hydroxyproline (4-Oxoproline) C ₅ H ₇ NO ₃ 129.11 0.20 ± 0.06

As can be seen from the above table, pyroglutamic acid, gluconic acid, uridine, malic acid, xanthine nucleoside, 3-hydroxy-3-methylglutaric acid, acetyl-L-carnitine, pantothenic acid and 4-hydroxyproline , are all effective anti-inflammatory substances. It is obvious that the "WA" group of the Taiwanese quinoa sprout extract of the present invention does have anti-inflammatory potential.

In summary, the "WA" group of Taiwanese quinoa sprout extract of the present invention is not only prepared through a special preparation method, but also has been experimentally proven to have the effects of antioxidant, resisting cytotoxicity and cell inflammation caused by PM2.5, and It can be used to prepare drugs for treating inflammatory reactions caused by PM2.5.

without

Figure 1 is a schematic diagram of an embodiment of a bioreactor of the present invention. Figure 2 shows the Taiwanese quinoa in the germination process of step (A) of the present invention, which is divided into 8 groups according to different days after germination of Taiwanese quinoa, and then undergoes the inoculation fermentation process of step (B) and the step (C). ), the status of Taiwanese quinoa sprout extract produced by each group after the extraction process. Figure 3 shows the Taiwanese quinoa of the present invention after the germination process in step (A), and then the inoculation and fermentation process in step (B). The Taiwanese quinoa buds are divided into 11 groups according to the number of days of inoculation and fermentation. The status of the Taiwanese quinoa sprout extract produced by each group after the subsequent extraction process in step (C). Figure 4 shows a bar chart of the toxicity and dose tests of the Taiwanese quinoa sprout extract of the present invention. Figure 5 shows a test chart of the inhibition of PM2.5-induced ROS production and restoration of cell viability by the Taiwanese quinoa sprout extract of the present invention. Figure 6 shows a schematic diagram of the expression of NF-κB protein in MHS cells induced by PM2.5 in the "WA" group of Taiwanese quinoa sprout extract of the present invention. Figure 7 shows a schematic diagram of the expression of inflammatory genes and anti-inflammatory genes after PM2.5 induces MHS cells in the "WA" group of Taiwanese quinoa sprout extract of the present invention.

Claims (14)

A method for preparing a Taiwanese quinoa sprout extract, including: subjecting a Taiwanese quinoa sprout to a germination process to obtain a Taiwanese quinoa sprout; subjecting the Taiwanese quinoa bud to an inoculation fermentation process to obtain a fermented Taiwanese quinoa sprout; and fermenting the Taiwanese quinoa sprout Taiwanese quinoa sprouts are obtained through an extraction process, wherein the extraction process includes: a rough extraction step; a concentration step to obtain a concentrated aqueous layer; and a layered extraction step, wherein the Taiwanese quinoa sprouts extract is It is obtained by taking a final water layer in this layered extraction process. According to the preparation method described in item 1 of the patent application, the Taiwanese quinoa is unhulled Taiwanese quinoa. According to the preparation method described in item 1 of the patent application, the Taiwanese quinoa sprouts are Taiwanese quinoa sprouts that have germinated for more than 4 days. For the preparation method described in item 1 of the patent application, the inoculation amount of the inoculation fermentation process is 1~10% (v/w). For the preparation method described in item 1 of the patent application, the inoculation fermentation process uses plate fermentation or bioreactor fermentation. The preparation method described in item 1 of the patent application, wherein the inoculation fermentation process uses a spore suspension of filamentous fungus. According to the preparation method described in item 1 of the patent application, the fermentation days of the fermented Taiwanese quinoa sprouts are 2 to 7 days. As for the preparation method described in item 1 of the patent application, the layered extraction step includes: After adding an alkane to the concentrated aqueous layer for extraction, a first aqueous layer is taken; after adding an ester to the first aqueous layer for extraction, a second aqueous layer is taken; and an alcohol is added to the second aqueous layer. After extraction, the final aqueous layer is obtained. A Taiwanese quinoa sprout extract, wherein the active ingredients of the Taiwanese quinoa sprout extract include: pyroglutamic acid, gluconic acid, uridine, malic acid, xanthine nucleoside, 3-hydroxy-3-methylglutarate, Acetyl L-carnitine, pantothenic acid and 4-hydroxyproline. The Taiwanese quinoa sprout extract described in item 1 of the patent application is prepared by using the preparation method described in the patent application scope 1. For the Taiwanese quinoa sprout extract described in item 10 of the patent application, the extraction process includes: a rough extraction step; a concentration step to obtain a concentrated aqueous layer; and a layered extraction step. For the Taiwanese quinoa sprout extract described in item 11 of the patent application, the layered extraction step includes: adding an alkane to the concentrated aqueous layer for extraction, and then taking a first aqueous layer; adding an ester to the third aqueous layer. After the first water layer is extracted, a second water layer is taken; and after an alcohol is added to the second water layer for extraction, the final water layer is taken. A use of Taiwanese quinoa sprout extract for preparing a composition with antioxidant, anti-cytotoxicity or anti-inflammatory properties, wherein the active ingredients of the Taiwanese quinoa sprout extract include: pyroglutamic acid, gluconic acid, uridine, apple Acid, xanthine nucleoside, 3-hydroxy-3-methylglutaric acid, acetyl-L-carnitine, pantothenic acid, and 4-hydroxyproline. For the use described in item 13 of the patent application, the effective dose of the Taiwanese quinoa sprout extract is 10-100 ppm.