KR102624473B1 - A Manufacturing method of sliced bellflower sugared - Google Patents

Abstract

The present invention relates to a method for producing bellflower sugar segments that are effective in improving the respiratory system and a composite composition of sugar needle solution for producing the sugar segments.

Description

A manufacturing method of sliced bellflower sugared that is effective in improving the respiratory system

The present invention relates to a method of manufacturing balloon flower segments that can be stored for a long time and are effective in improving the respiratory system.

Currently, fine dust is a substance floating in the atmosphere containing very complex components such as sulfur oxides, nitrogen oxides, lead, ozone, and carbon monoxide, and is known to be mostly generated from automobile exhaust gases and road dust.

The size and composition of fine dust are very complex and diverse. PM10 is called fine dust with a particle size of 10 mm or less, and PM2.5 is called ultrafine dust with a particle size of 2.5 mm or less. The health hazard depends on the particle size, surface area, and chemical composition. It has many and varied impacts.

The impact of fine dust on the respiratory system is mainly caused by causing an inflammatory response in the bronchi, which causes or worsens asthma, chronic bronchitis, and airway obstruction.

In addition, fine dust can cause respiratory infections by interfering with the inactivation or removal of bacteria in lung tissue. Recently, fine dust has become an important risk factor for cardiovascular diseases such as myocardial infarction, stroke, abnormal heart rate, and sudden death. It is being accepted.

Therefore, exposure to such fine dust for a certain period of time is reported to be related not only to the occurrence of respiratory and cardiovascular diseases, but also to an increase in mortality. By age group, preschool children and those in their 70s or older, who have physically weak immune systems, are reported to be associated with fine dust exposure. It was found that the impact of dust and yellow dust was greater.

As concerns about respiratory health due to fine dust have recently increased rapidly, many products to protect respiratory health, such as industrial products, pharmaceuticals, and health functional foods, are being released.

However, among the health functional foods approved and distributed in Korea, there are no products that are effective in or have the functionality to prevent respiratory diseases caused by fine dust or yellow dust.

In addition, because the clear mechanism of how fine dust causes respiratory diseases has not been identified, there has been no development of related biopharmaceuticals yet.

Therefore, as a complementary method, research on functional foods that can reduce the toxicity of fine dust in vivo is needed.

In response to these demands, the demand for foods that help with respiratory health based on folk remedies and oriental medicine is increasing in addition to traditional food items such as dangjeolpyeon. In particular, in oriental medicine, bellflower root is used as an expectorant, bronchitis, tonsillitis, sore throat, and other respiratory diseases. It has been used for treatment purposes.

In addition, bellflower root components such as triterpene saponins, inulin, and sterol exhibit pharmacological effects, and natural platycodin is reported to be the main pharmacological ingredient. Bellflower root as a food is effective in treating adult diseases such as asthma, pulmonary tuberculosis, hypertension, diabetes, and inflammation. Most of the products are grown for folk remedies to be used as sedatives and painkillers and consumed as a simple side dish, or extracted from bellflower root alone or mixed with pears.

However, these things are difficult to consume easily.

Therefore, sugar-pickled bellflower root was recently released to make it easier to eat, but its effect on improving respiratory system has not been verified.

Therefore, the present invention uses bellflower root as the main raw material and prepares a sugar needle solution with a complex composition of Cheonmundong and yellow chili extract concentrate, which has been reported to be effective in respiratory health, to identify the respiratory improvement effect and add respiratory health functionality as well as palatability.

For this purpose, finely chopped and dried Cheonmundong and Hwangchil were extracted with 10 times the weight of 50% ethanol at 70°C three times for 24 hours, filtered and concentrated, and the freeze-dried extract was mixed with Cheonmundong and Hwangchil in a 5:5 ratio by weight. and mixed with red ginseng extract (10% by weight), honey (30% by weight), and Hwangchil + Cheonmundong extract blended in a 5:5 ratio (60% by weight) to prepare a sugar needle solution.

The final sugar steeping solution was prepared by mixing the sugar steeping solution and water (sterilized water) in a 1:1 ratio, then the final sugar steeping solution and dried bellflower slices were mixed in a 2:1 ratio, and then the dried bellflower slices were added to the final sugar steeping liquid. It was sufficiently immersed.

After immersion, the first sugar needle was heated at 70°C for 6 hours, the first sugar needle was aged at room temperature for 12 hours, and then the second sugar needle was heated at 70°C for 6 hours after aging.

After the sugar needle process was completed, it was dried at room temperature for 6 hours, and the sugar needle liquid on the bellflower was naturally drained to remove it, then dried at 45°C for 24 hours. After drying, the moisture content was confirmed to be less than 12% and then vacuum-packed.

Therefore, the bronchial and lung inflammation inhibition activity was high, and especially when Cheonmundong and Hwangchil were mixed in a 5:5 ratio, the highest inflammation inhibition activity was shown.

1 is a block diagram showing the manufacturing process of the present invention.

Hereinafter, the present invention will be described in detail based on an embodiment as follows.

As shown in Figure 1, the manufacturing method of the present invention consists of the following process sequence.
In the first step, the dried Cheonmundong and Hwangchil were finely chopped and extracted with 10 times the weight of 50% ethanol at 70°C three times for 24 hours, filtered, concentrated, and freeze-dried. This is a process to obtain a powdered extract.

In the second process, each powdered extract obtained in the first process was mixed with Cheonmundong and Hwangchil in a 5:5 ratio by weight to obtain 60% by weight of the powdered mixture of Hwangchil + Cheonmundong extract and 10% by weight of powdered red ginseng extract. This is a process of producing a sugar needle solution, that is, a composite composition, by mixing 30% by weight of gel-like honey.

The third process is a process of preparing the final sugar needle solution by mixing the sugar needle solution (composite composition) obtained in the second process and water (sterilized water) in a 1:1 ratio.

As for the 4th process. This is a process of mixing the final sugar-impregnated liquid obtained in the third process and dried bellflower slices in a 2:1 ratio by volume, and then sufficiently immersing the dried bellflower slices in the final sugar-impregnated liquid.
In the present invention, sufficiently immersing means soaking the dried bellflower slices for a certain period of time while preventing them from floating in the final sugar soaking liquid so that the dried bellflower slices are sufficiently penetrated with the final sugar soaking liquid.

The fifth process is a process of first sugaring by heating at 70°C for 6 hours in a state that has been sufficiently immersed in the fourth process.
This is to maintain uniform penetration while the final saccharide solution is also warmed as bellflower root dried at a certain temperature expands.
The sixth process is a process of maturing in the sugared state at room temperature for 12 hours after the first sugaring according to the fifth process.
Even at this time, it is best to keep it in a non-floating state if possible.
In the 7th process, the product aged in the 6th process is heated to a temperature of 70°C for 6 hours to perform secondary sugaring.
This is to ensure that the final sugar solution is sugared to a uniform consistency on the dried balloon flower.

The 8th process is a process of drying at room temperature for 6 hours after the sugar needle process is completed by the above process, removing the sugar needle liquid on the bellflower root by naturally flowing it, and then drying it at 45°C for 24 hours.
In the 9th process, the product dried in the 8th process is vacuum-packed after checking whether the moisture content is maintained at 12% or less.

The following are the experimental results of exaggeration obtained by each of these processes.

Finely chop the Cheonmundong and Hwangchil dried in the first process, add 50% ethanol 10 times the weight of the raw material, extract three times for 24 hours at 70℃, centrifuge, filter, concentrate the filtrate, and then freeze. By drying, extracts of Cheonmundong and Hwangchil powder were prepared.

Afterwards, to check the total polyphenol content, 10 ml of distilled water was added to 1 ml of each extract, then 2 ml of Folin-Ciocalteu phenol reagent was added, mixed, and reacted at room temperature for 5 minutes.

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2 mL of 20% sodium carbonate was added to the reaction mixture, mixed, reacted at room temperature for 1 hour, and absorbance was measured at 725 nm.

At this time, tannic acid was used as an indicator substance.

Afterwards, for antioxidant activity, the method used by Yoshida et al. (1989) was slightly modified to measure the scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical.

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DPPH radical scavenging ability was measured by dispensing 0.2mM 2,2-diphenyl-2-picrylhydrazl (DPPH) solution into 0.5mL (10.0μ) of the extract, leaving it for 30 minutes, and measuring the absorbance (GENESYS 10 UV) at 517 nm. .

The control group was measured in the same way by adding a solvent instead of the sample.

The positive control group was tested with 10.0 μL of Vitamin C.

Additionally, to correct the absorbance value for the color of the sample itself, methanol was added instead of 0.2 nm DPPH and the absorbance was measured.

At this time, the antioxidant activity of the extract was expressed as a percentage based on the obtained value, and the closer the converted value was to 100%, the higher the antioxidant activity was expressed.

To measure ABTS radical scavenging activity, a modified method of Roberta et al. was used.

To prepare the ABTS radical solution, 7mM ABTS and 2.45mM potassium sulfate were added to distilled water to produce ABTS cations for 16 hours at room temperature.

At this time, it was diluted and prepared so that the absorbance value at 734 nm was 0.7 or less.

50ul of the sample at each concentration was added to 950ul of the ABTS solution and reacted for 10 minutes, and then the absorbance was measured at 734nm.

As a positive control, BHA was used instead of the material in the examples.

The free radical scavenging effect of each sample was expressed as SC ₅₀ by calculating the concentration that scavenges 50% of the radicals.

At this time, the negative control was the absorbance response of the solution without any sample treatment.

For cell culture, to determine changes in cell viability by extracts of Cheonmun-dong and Hwangchil powder, MH-S lung macrophages (ATCC, CRL 2019) were cultured in RPMI 1640 medium containing 10% FBS, and then the cells were 90-100% dead. When cultured, it was passaged and used.

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In addition, the cell survival rate caused by LPS and fine dust of powdered Cheonmundong and Hwangchil and the killing effect of NO analysis complex composition on lung macrophages were tested using MTT assay.

MH-S cells were grown at a density of 1×10 ⁵ cells/well in RPMI1640 medium containing 10% fetal bovine serum (FBS; Gibco), 100 units/ml penicillin, and 100 ug/ml streptomycin. After culturing in a well culture plate at 37°C in a 5% CO ₂ environment for 1 day, the medium was replaced with RPMI1640 medium without serum, and complex compositions were added at different concentrations and cultured for 24 hours.

20ul of MTT solution (5mg/ml; Sigma, USA) was added per well, and after reaction at 37°C for 4 hours, the MTT solution was removed, and 100ul of DMSO was added per well.

After dissolving the formazan derivative at room temperature, the absorbance was measured at 490 nm.

For NO production, the supernatant was taken and the absorbance was measured at 540 nm using Griess reagent (0.2% N-(1-naphthyl)ethylene diamine solution: 0.2% sulfanilamide solution=1:1).

The NO production inhibitory activity was evaluated by comparing with the group treated with LPS only.

In addition, in order to obtain the anti-inflammatory effect and optimal composition ratio of Cheonmundong and Hwangchil extracts by PM, after culturing MH-S lung macrophages, Cheonmundong and Hwangchil extracts were mixed at a concentration of 0 to 100 ug/ml, and then treated with PM for 24 hours. An inflammatory response was induced.

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After 24 hours, cell survival rate and NO production were measured to establish the optimal composition ratio for developing the sugar syrup recipe.

In addition, 50Brix Cheonmundong and Hwangchil extracts were prepared to prepare the optimal bellflower syrup based on the optimal composite composition ratio in order to establish a sugar syrup recipe and prepare sugar-pickled bellflower root samples for animal testing.

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Four types of sugar needle solution were prepared by varying the contents of Cheonmundong and Hwangchil extract (Cheonmundong extract:Hwangchil extract = 7:3, 5:5, 3:7, 10:0), and then adding red ginseng extract (10% by weight). , honey (30% by weight), and Hwangchil + Cheonmundong extract mixture (60% by weight).

The final sugar syrup was prepared by mixing the four types of sugar syrup and water (sterilized water) in a ratio of 1:1, then mixing the sugar syrup and dried bellflower slices in a ratio of 2:1 by volume, and then thoroughly sacrificing the dried bellflower slices. immersed.

After immersion, the first sugar needle was heated at 70°C for 6 hours, the first sugar needle was aged at room temperature for 12 hours, and then the second sugar needle was heated at 70°C for 6 hours after aging.

After the sugar needle process was completed, it was dried at room temperature for 6 hours, and the sugar needle liquid on the balloon flower was naturally drained and removed, then dried at 45°C for 24 hours. After drying, the moisture content was confirmed to be less than 12%, and then vacuum-packed for storage and animal use. It was used in the experiment.

In addition, to measure the total number of bronchoalveolar lavage (BAL) cells, six Balb/c male mice were selected per group, and all groups except the normal group were treated with PM inhaled into the airway every day for 14 days. Injected twice.

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And extracts with different ratios of Cheonmundong and Hwangchil were fed at 500 mg/Kg once a day.

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Cheonmundong: Hwangchil ratio control group 0 PM 0 Experimental group 1 10:0 Experimental group 2 7:3 Experimental group 2 5:5 Experimental group 3 3:7

After completion of the experiment, an autopsy was performed and BAL fluid was recovered.

In addition, to measure the expression level of inflammatory factor mRNA in lung tissue, total RNA was isolated from the extracted lung tissue using Easy-Blue reagent (Intron Biotechnology Inc.).

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Total RNA was quantified using the NANO drop 1000 spectrophotometer system (Thermo), and reverse transcription was performed to synthesize cDNA.

Specifically, it contains 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl2, 10mM DTT (Invitrogen), 1U/ulRNasin (Invitrogen), 1mM each dNTP, 100ng of oligo(dT)20 and 200U of MMLV reverse transcriptase (Invitrogen). It was synthesized from 1ug of total RNA in 20ul of solution.

Quantitative real-time PCR was performed on the synthesized cDNA transcript using the StepOnePlus Real-time PCR system (Applied Biosystems) using SYBR green master mix (Applied Biosystems, USA) and the primers shown in <Table 7> below.

GAPDH was used for standardization of each sample.

GAPDH was used for standardization of each sample.

<Primers used for RT-PCR>

target gene Primer standing MUC5AC forward 5'-AGAATATCTTTCAGGACCCCTGCT-3' reverse 5'-ACACCAGTGCTGAGCATACTTTT-3' CCR5 forward 5'-ATTCTCCACACCCTGTTTCG-3' reverse 5'-AAGGTGGTCAGGAGGAGGAC-3' GAPDH forward 5’-TGATTCTACCCACGGCAAGT-3’ reverse 5'-AGCATCACCCCATTTGATGT-3'

In addition, to measure the expression of inflammatory factors in BAL fluid, the levels of TNF-a, MIP2, and RMFLRH CXCL-1 in BAL fluid were measured by ELISA.

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TNF-a antibody (MTA00B, R&D systems, Minneapolis, USA), MIP2 antibody (MM200, R&D systems, Minneapolis, USA), and CXCL-1 antibody (MKC00B, R&D systems, Minneapolis, USA) were diluted with buffer solution and used in 96 wells. After coating, the cells were incubated at 4°C for 16 hours.

After washing each well three times with a buffer solution, 100 mL of 10-fold diluted serum was dispensed.

After being left at room temperature for one hour, washed twice, treated with 100 mL of Avidin-HRP-conjugated antibody (DY007, R&D systems, Minneapolis, USA), left at room temperature for 1 hour, and washed again.

100 mL of TMB substrate (DY007, R&D systems, Minneapolis, USA) was dispensed, left in the dark for 30 minutes, treated with 50 mL stop solution (DY007, R&D systems, Minneapolis, USA), and absorbance was measured at 450 nm.

The results of the above experiment are as follows.

1. Change in extract content depending on solvent

As a result of measuring the yield of the extract after extraction at 50°C using water and 50% ethanol and freeze-drying, it was found that the content increased by about 50 to 55% in 50% ethanol.

In addition, the total polyphenol content was also found to be significantly higher in the ethanol extract than in the water extract, so a 50% ethanol extract was used as the sample.

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<Table 1> Change in extract content depending on solvent Cheonmundong Extract (AC) Hwangchil Extract (DM) water extract 12.5% 11.2% 50% ethanol extract 31.2% 23.5%

<Table 2> Total polyphenol content (mg/g) Cheonmundong Extract (AC) Hwangchil Extract (DM) water extract 52.2 43.6 50% ethanol extract 153.4 221.33

2. Antioxidant analysis

The antioxidant activity of Cheonmundong and Hwangchil powder extracts extracted with 50% ethanol as a solvent was measured using DPPH and ABTS radical scavenging activities.

The DPPH and ABTS radical scavenging ability was confirmed in a similar range to that of extracts derived from other natural products. The DPPH radical scavenging ability of the Cheonmundong extract was found to be 65.4 (SC50), which is about 1.6 times higher than that of the Hwangchil extract, and the ABTS radical scavenging ability was found to be 65.4 (SC50), which is about 1.6 times higher than that of the Hwangchil extract. It was found to be 9.35 (SC50), which is 3.4 times higher than the Cheonmundong extract.

<Table 3> Antioxidant activity Cheonmundong Extract (AC) Hwangchil Extract (DM) DPPH (SC50) 65.4 108.32 ABTS (SC50) 32.1 9.35

3. Anti-inflammatory effect of Cheonmundong extract and Hwangchil extract against fine dust

Cheonmundong and Hwangchil extracts were mixed with MH-S lung macrophages at a concentration of 0 to 100 ug/ml, treated with LPS, and the cell viability and NO production were checked. As a result, compared to treatment alone, 50 ug/ml of Cheonmundong and Hwangchil extracts were used. When treated ml at a time or mixed at a concentration of 30 ug/ml Cheonmundong and 70 ug/ml Hwangchil, the cell survival rate reduced by LPS was increased and the increased NO production was reduced (Figure 1).

In other words, when Cheonmundong and Hwangchil extracts were mixed in a ratio of 5:5 or 3:7, the anti-inflammatory effect was significantly higher than when treated alone.

This result was the same even when PM was treated instead of LPS (Figure 2).

< Figure 1 > Anti-inflammatory effect of LPS in mixed treatment of Cheonmundong and Hwangchil extracts

< Figure 2 > Anti-inflammatory effect of PM from mixed treatment of Cheonmundong and Hwangchil extracts

4. Total bronchoalveolar lavage fluid (BAL) cell count measurement

The effect on the total number of bronchoalveolar lavage fluid cells was tested using the method described in the following literature.

(Schines et al., Toxicol Appl Pharmacol. 195(1), 1-11 (2004) and Smith et al., Toxicol Sci, 93(2), 390-399 (2006)) Total bronchoalveolar lavage fluid cell count for each sample The results of measuring the impact are shown in Table 4.

The total number of BAL cells decreased compared to the induced group, confirming that all samples contributed to the reduction of inflammation levels. In particular, the sample mixed with Cheonmundong and Hwangchil extracts in a 5:5 ratio showed the lowest bronchial inflammation inhibitory activity.

<Table 4>Effect on total bronchoalveolar lavage fluid cell count Total BAL cells
(´10 ⁵ cells/ml) inhibition rate
(Based on trigger group) Jeongsang-gun 33.3 ± 4.7 Yuval-gun 127.8± 5.2 Bellflower Jeonggwa 101.1±6.2 22 Cheonmundong 72.1± 3.3 44 Hwangchil 92.3± 3.7 29 Cheonmundong: Hwangchil (7:3) 62.4± 4.2 52 Cheonmundong: Hwangchil (5:5) 42.5± 3.7 68 Cheonmundong: Hwangchil (3:7) 69.7± 3.2 47

5. Effect on inflammatory factor mRNA expression in the lungs

Table 5 shows the effect on inflammatory factor mRNA expression in the lung.

Inflammatory factors (MUC5AC, CCR5) in each sample administration group decreased compared to the induced group, and, consistent with the total bronchoalveolar lavage fluid cell count results, the highest inflammatory factor inhibition rate was shown in the sample mixed with Cheonmundong and Hwangchil extract in a 5:5 ratio. It was.

<Table 5>Effect on total bronchial alveolar lavage fluid cell count MUC5AC CCR5 amount of expression inhibition rate
(Based on trigger group) amount of expression inhibition rate
(Based on trigger group) Jeongsang-gun One One Yuval-gun 6.32 ± 0.9 8.2 ± 0.9 Bellflower Jeonggwa 5.82± 1.2 8 7.12 ± 0.8 14 Cheonmundong 4.24± 0.7 33 6.08± 0.7 26 Hwangchil 5.1± 0.8 19 7.32 ± 0.6 11 Cheonmundong: Hwangchil (7:3) 3.92± 0.7 38 5.24± 0.1 37 Cheonmundong: Hwangchil (5:5) 2.72± 0.6 67 4.17± 0.6 50 Cheonmundong: Hwangchil (3:7) 4.49± 0.7 29 6.47± 0.3 22

6. Measurement of inflammatory factor expression in bronchoalveolar lavage fluid

To determine the effect on the expression level of inflammatory factors in bronchoalveolar lavage fluid for each sample, an experiment was performed using the method described in the literature as follows (Brandt EB et al., J. Allergy Clin. Immunol. 135(5): 1194-1204 (2013)).

Table 6 shows the results of measuring the expression of inflammatory factors such as IL-17A, TNF-a, and MIP2 in bronchoalveolar lavage fluid.

Inflammatory factors in each sample administration group were all reduced compared to the induced group.

Consistent with the previous results, it was confirmed that the inhibition rate of inflammatory factor expression in bronchoalveolar lavage fluid was highest in samples mixed with Cheonmundong and Hwangchil extracts in a 5:5 ratio.

In other words, compared to the single extract, when the mixture of Cheonmundong and Hwangchil was used as a sugar needle solution, the bronchial and lung inflammation inhibition activity was higher. In particular, when Cheonmundong and Hwangchil were mixed at a ratio of 5:5, the highest anti-inflammatory activity was shown.

<Table 6>Measurement of expression of inflammatory factors in bronchoalveolar lavage fluid Concentration (pg/ml) / Inhibition rate based on induced group (%) IL-17A TNF-a MIP2 Jeongsang-gun 4.4 42.6 89.4 Yuval-gun 15.2 97.2 184.7 Bellflower Jeonggwa 13.1 / 14 89.1 / 9 179.1 / 4 Cheonmundong 8.9 / 42 77.4 / 21 164.2 / 12 Hwangchil 9.4 / 39 85.1 / 13 174.1 / 6 Cheonmundong: Hwangchil (7:3) 8.1 / 47 69.1 / 29 164.2 / 12 Cheonmundong: Hwangchil (5:5) 7.2 / 53 55.2 / 44 151.2 / 19 Cheonmundong: Hwangchil (3:7) 8.7 / 43 73.1 / 25 159.2 / 14

Claims (2)

Each dried Cheonmundong and Hwangchil were finely chopped and extracted repeatedly with 10 times the weight of 50% ethanol at 70°C three times for 24 hours, filtered, concentrated, and freeze-dried to obtain each powdered extract, as described above. The obtained powdered Cheonmundong and Hwangchil were mixed in a ratio of 5:5 by weight to obtain a Hwangchil + Cheonmundong extract blend, and in this state, the total weight was set to 100% by weight, and 60% by weight of the Hwangchil + Cheonmundong extract blend and red ginseng powder extract were added. Prepare sugar needle solution by mixing 10% by weight and 30% by weight of honey,
The final sugar needle solution was prepared by mixing the sugar needle solution prepared as above with sterilized water in a 1:1 ratio by weight,
The dried bellflower slices were mixed in the final sugar steeping solution at a volume ratio of 2:1, the dried bellflower slices were completely immersed so that they did not float in the final sugar steeping solution, and then heated at 70°C for 6 hours to make the first sugar needle. After the first sugar needle, the sugar was aged at room temperature for 12 hours in a non-floating state, and then heated at 70°C for 6 hours in a non-floating state for the second sugar needle.
After the first and second sugar needle processes, the sugar needle liquid on the balloon flower is naturally drained and removed while drying at room temperature for 6 hours.
A method of manufacturing balloon flower segments that are effective in improving the respiratory system by drying them at 45°C for 24 hours and then vacuum-packing them while maintaining a moisture content of 12% or less. delete

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