KR101233117B1 - Extracting method of isoflavone aglycone from soybean isoflavone by microwave assisted acid hydrolysis - Google Patents

Abstract

The present invention relates to a method for efficiently extracting soybean isoflavone aglycone by acid hydrolysis using microwave treatment, and more particularly, soaking dried and pulverized soybean seed powder in an acid solution and irradiating with microwaves. And adding alcohol to the mixture of the hydrolyzed soybean seed powder and the acid solution and leaving it at room temperature to extract isoflavone aglycone, and filtering and separating the isoflavone aglycone. It is characterized by.
According to the present invention, microwave application can provide an acid hydrolysis method that is easier, less time consuming, and has higher reproducibility and reliability compared to the conventional method for extracting soy isoflavone aglycone using dry oven treatment. have.

Description

Extracting method of isoflavone aglycone from soybean isoflavone by microwave assisted acid hydrolysis}

The present invention relates to a method for the simple and easy hydrolysis and extraction of isoflavone glycosides from soybeans in the form of non-glycoside isoflavone aglycones.

Beans [ Glycine max (L.) Merr.] Isoflavones containing In in vitro and In Various human health including in vivo similar estrogen / anti-estrogen activity, antiproliferation, cell-cycle arrest and induction of cancer cell death, oxidation prevention, regulation of host immune system and cell signal change There have been a lot of research reports that it works. In addition, isoflavones have been reported to significantly affect the effects of lowering the risk of cardiovascular disease represented by soybeans or preventing mineral loss of bone in uterine-excised rats.

Such isoflavone extraction methods include classical soxhlet extraction, magnetic stirring, supercritical fluid extraction, pressurized liquid extraction, and solid phase extraction. And ultrasound assisted extraction, and isoflavones are usually extracted using a water-soluble organic solvent (methanol, ethanol or acetonitrile), followed by analysis using HPLC-UVD (UV detector). have.

In general, isoflavones contained in plants exist in the form of isoflavone glycosides in which aglycone and sugar are combined, and isoflavones contained in soybean are conjugated between aglycone and β-glucoside. Glycoside derivatives, ie glycosides. Therefore, in the case of extracting the isoflavones of soybeans by the above-described method, most of the isoflavone glycosides are extracted.

Currently, about 12 kinds of isoflavones contained in soybean are known. Nine of these components are known to exist in glycoside form, and the remaining three components are present in the form of aglycone in trace amounts, and nine glycoside derivatives are hydrolyzed. To the three aglycone forms. Thus, as an experimental method for the quantitative analysis of total isoflavones contained in food and soybeans, when it is analyzed with glycoside-type isoflavones, 12 kinds of separation and analysis require a long analysis time, and 12 kinds of quantitative standards Due to the many time and economic disadvantages, such as should be provided by using a hydrolysis method to convert the three types of aglycone is used a lot of methods for easy analysis.

In addition, since there is almost no standard for analysis of commercially available isoflavone glycoside derivatives, or even occasionally, trace amounts are extremely high, these factors have been a challenge for qualitative and quantitative analysis of isoflavones. In order to induce the conversion of glycoside derivatives to aglycone after hydrolysis in isoflavone analysis, separation and detection were carried out, and this hydrolysis method caused the use of quantization of free aglycone.

A method for hydrolysis of isoflavone glycosides has been reported by several researchers, among which acid hydrolysis by hydrochloric acid (HCl) using a dry oven is the most widely used method, and hydrolysis by other enzymes and alkalis Decomposition has also been reported.

However, the hydrolysis methods described above are relatively complicated, time-consuming, labor-intensive, and require various experimental facilities, and lack of reproducibility. In addition, acid hydrolysis method using a dry oven has been widely used for isoflavone analysis, but accurate acid hydrolysis time for the analysis of isoflavone content in soybean seeds, extraction time of isoflavone aglycone after acid hydrolysis, and Accurate information on the amount of extraction is not provided.

Therefore, it will be necessary for various researchers studying isoflavones to provide efficient hydrolysis techniques that are simple, efficient, economical and reliable.

The present invention has been made to solve the above problems, when using the microwave treatment in the acid hydrolysis of soybeans isoflavone aglycone in the form of a non-glycoside isoflavone aglycone in a short time can be converted and extracted in a simple and high efficiency It was confirmed that the present invention was completed.

Accordingly, an object of the present invention is to provide a novel method for converting and extracting isoflavones into isoflavone aglycone form from soybean by acid hydrolysis using microwave treatment.

As an example for achieving the above object, a method for extracting soybean isoflavone aglycone of the present invention is a step of immersing dried and pulverized soybean seed powder in an acid solution and hydrolyzing while irradiating microwaves, the hydrolyzed soybean seed The method includes extracting isoflavone aglycone by adding alcohol to the mixture of the powder and the acid solution and leaving it at room temperature, and separating and filtering the isoflavone aglycone.

Hereinafter, a method for extracting soybean isoflavone aglycone according to the present invention will be described in detail.

First, the dried and pulverized soybean seed powder is immersed in an acid solution and hydrolyzed while irradiating microwaves.

Bean seeds are prepared by drying and grinding, to which the acid solution is mixed. The acid solution may use various organic and inorganic acids including hydrochloric acid (HCl), and the concentration is preferably adjusted to 0.1 N to 6 N levels. Such acid solution is preferably added 5 to 30 times the dry weight of the beans so that the dried soybean powder is sufficiently immersed in the acid solution.

Microwaves, on the other hand, have been used as an alternative to classical methods for extracting organic compounds from plants and foods. This utilizes the principle of selective and rapid local heating of moisture in a sample by microwaves, which increases the intracellular pressure of the sample by local heating and rapidly transfers the compound from the cell into the extraction solvent. In addition, the use of closed vessels allows the extraction by microwaves to be performed at high temperatures, thus accelerating the transfer of the desired compound from the sample matrix. Thus, microwaves can reduce solvent content and improve extraction efficiency.

In the present invention, microwave irradiation is used for acid hydrolysis of soy-containing isoflavone glycosides. At this time, the microwave irradiation is performed for 45 to 55 minutes, preferably 50 minutes is good in terms of yield of soy isoflavone aglycone, hydrolysis using the microwave irradiation as described above is 45 at 95 to 105 ℃ conditions To 55 minutes, preferably at 100 ° C. for 50 minutes.

Next, alcohol is added to the mixture of the hydrolyzed soybean seed powder and the acid solution and left to stand at room temperature to extract isoflavone aglycone.

Alcohol is added to the mixture of the hydrolyzed soybean seed powder and the acid solution after the above-mentioned hydrolysis performed using microwave irradiation, and such alcohol may be a lower alcohol having 1 to 4 carbon atoms, preferably It is recommended to use ethanol. Such alcohol is preferably added 1 to 20 times the weight of the mixture of hydrolyzed bean seed powder and acid solution. Alcohol is added as above and left at room temperature, which is preferably performed for 2 to 3 hours, preferably 3 hours.

Through hydrolysis carried out using microwave irradiation as described above, soybean isoflavone glycoside is converted to isoflavone aglycone, which is a nonglycoside, and isoflavone nonglycoside isoflavone while leaving at room temperature for a certain time by adding alcohol. The flavone aglycone is completely dissolved in the alcoholic extraction solution and extracted.

Finally, the isoflavone aglycone extraction solution is filtered.

The alcohol was added to the mixture of the hydrolyzed soybean powder and the acid solution in the above process, and the mixture was left at room temperature for a certain time, and then the reaction solution was removed by filtration to remove the solids, followed by RP-HPLC. By soybean isoflavone aglycone can be obtained with high efficiency.

The present invention is an acid hydrolysis method using microwave treatment capable of extracting isoflavone aglycone in a shorter time in a shorter time compared to conventional dry oven treatment under proper acid hydrolysis conditions considering the isoflavone content in soybean. Was presented for the first time. The present invention performs soy isoflavone content measurement according to the three parameters of acid hydrolysis method, hydrolysis time and isoflavone extraction time, which is faster and more efficient than conventional methods when microwave treatment is introduced during acid hydrolysis. The present invention relates to providing a reliable acid hydrolysis method. In addition, it was confirmed that the optimal condition was to hydrolyze the beans using 1 N HCl for 50 minutes at 100 ℃ using microwave.

According to the present invention described above it can be seen that the acid hydrolysis method by microwave treatment is an attractive method that can be used to convert isoflavone glycosides in glycoside form into their aglycones.

According to the present invention, it is easier, faster, more efficient, less expensive, and more reliable acid hydrolysis method than conventional soybean isoflavone aglycone extraction methods such as dry oven treatment using microwaves. can do.

In addition, the present invention can be expected to be effective as a method of deglycosylation of isoflavones from soybean and soybean-derived foods.

In addition, isoflavones exist in the form of isoflavone glycoside, which is the most stable form in storage in plants, but is known to be metabolized into isoflavone aglycone after absorption in glycoside form in the human body. To this end, isoflavones ingested in the form of isoflavone glycosides are deglycosylated by the enzymatic reaction in the liver and converted into isoflavone aglycones. According to the present invention, isoflavones in the glycoside form are actually used and metabolized in the body. Phosphorus isoflavones can be easily converted to and separated from the aglycone form and can be ingested in the form of isoflavones aglycone, thereby reducing the burden on the liver.

In addition, the present invention is expected to have a great effect on various related industries, including the manufacture of drugs, foods, and the like, which can be ingested in the form of isoflavone aglycone together with the ease of producing isoflavone aglycone standard. .

In Figure 1 (A) shows the chemical structure of the 12 kinds of isoflavones contained in soybeans, (B) is the non-hydrolysis of Sinpalda bean, domestically grown soybean varieties (80% ethanol aqueous solution, 85 ℃, extracted for 3 hours) Soy isoflavone HPLC chromatogram of sample extract is shown [1; Diamond, 2; Glycidine, 3; Genistin, 4; Malonyldiazine, 5; Malonylzenistin, 6; Diazein, 7; Glycinein, 8; Genistein].
Figure 2 shows the aglyconylation of soy isoflavone glycosides with hydrolysis time in a 100 ° C. dry oven in HPLC chromatogram [A; 30 minutes, B; 60 minutes, C; 90 minutes, D; 120 min, E; 180 minutes, F; 240 minutes, 1; Diamond, 2; Glycidine, 3; Genistin, 4; Malonyldiazine, 5; Malonylzenistin, 6; Diazein, 7; Glycinein, 8; Genistein].
Figure 3 shows the change of the diazane, glycine, genistein and total isoflavone content with hydrolysis time in a 100 ℃ dry oven.
Figure 4 shows the HPLC chromatogram of soy isoflavones with microwave treatment and hydrolysis time [A; 10 minutes, B; 20 minutes, C; 30 minutes, D; 40 minutes, E; 50 minutes, F; 60 minutes, 1; Diamond, 2; Glycidine, 3; Genistin, 4; Malonyldiazine, 5; Malonylzenistin, 6; Diazein, 7; Glycinein, 8; Genistein].

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples.

Reference Example  1. Samples used, chemicals and utensils used

The soybean seeds were used as Shinpaldalkong from the Functional Crop Division of the National Institute of Crop Science under the Rural Development Administration. The bean seeds were dried for 24 hours at 40 ° C., ground using a micro mill (Culatty AG, Zurich, Swiss), sieved through a 1.0 mm screen sieve, sealed in a plastic bag, and stored at 4 ° C.

Methanol, ethanol, acetonitrile and hydrochloric acid (HCl) were purchased from Merck Co. Darmstadt, Germany. Among the isoflavone standards, daidzein, genistein and glycite were obtained from a prior researcher (Lee et al., 2008). Diadzin, genistin and glycytin were purchased from Apin Chemicals LTD.Oxon, UK. Malonyl glucosides and acetyl glucosides were purchased from Fujicco Co. Ltd., Kobe, Japan and Nakalai Tesque Inc. Kobe, Japan. Ultrapure water was prepared using a Milli-Q water purifier system from Millipore, Bedford, Mass., USA. The relative purity of isoflavone glucoside and aglycone was more than 98%, and the relative purity of malonyl glucoside and acetyl glucoside was more than 90%. Stock solutions of each isoflavone were prepared with methanol content of 80% (v / v) and used while stored at -20 ° C.

Ultraviolet-visible absorption spectra were measured using an Infinite M200 spectrophotometer (Tecan Austria GmbH, Untersbergstrasse 1A, Austria). HPLC analysis is an HPLC analyzer (Agilent 1200 series, Wilmington, DE), a quaternary pump, a photodiode array detector (Agilent 1200 series), Nova-Pac C18 guard column (Waters, Milford) YMC ODS AM 303 column (5 μm; 250 × 4.6 mm id, Waters, Milford, Mass.), Was used.

Comparative example  One. HPLC Analysis of Soy Isoflavones by

For analysis of soy isoflavones by HPLC, an HPLC analyzer (Agilent 1200 series HPLC system, Wilmington, DE) equipped with a photodiode array detector with an analytical wavelength set to 254 nm was used. Separation was performed using a YMC ODS AM 303 column (5 μm; 250 × 4.6 mm id, Waters, Milford, MA) equipped with a Nova-Pak C18 guard column (Waters, Milford, Mass.). The column temperature was maintained at 30 ° C., and the mobile phase was prepared with solvent A (0.1% (v / v) acetic acid in ultrapure water purified by milli-Q) and solvent B (0.1% (v / v) acetic acid in acetonitrile). Was done. Separation was carried out using the concentration gradient of the solvent B described below. That is, 10 minutes to 15 to 28%, 20 minutes to 28 to 35%, 10 minutes to 35 to 55%, and 10 minutes to 55 to 75%, followed by 10 minutes of isocratic elution. The flow rate was adjusted to 1.0 mL / min and the sample injection amount was 20 μL. The content of isoflavones was calculated by comparison with HPLC peak areas by an external standard calibration curve. The external standard calibration curve ( r = 0.999 ^** ) was prepared by injecting 20 μL of an 80% methanol solution containing 0.05 to 1 μg of isoflavone standard content.

Comparative example  2. Hydrolysis of Isoflavones by Dry Oven Treatment

To analyze the isoflavone-containing behavior of Shinpaldalkong, 50 mL of 80% ethanol aqueous solution was added to non-hydrolysis soybean seeds (control, 1.0 g) and reflux extractor at 85 ° C for 3 hours. Extracted using (Murphy, Barua, & Hauck, 2002). Also for acid hydrolysis by dry oven treatment, 1.0 g of soybean seed was administered to a 25 mL cap-reaction vial with 10 mL of 1N HCl solution. The mixture was heated in a 100 ° C. dry oven for each hydrolysis time (30-240 minutes) and then cooled at room temperature. After cooling, 15 mL of ethanol was added to each mixture and left for 3 hours to extract the isoflavones.

In order to determine the optimal isoflavone extraction time, the resultant obtained according to the extraction time (1, 3, 6, 9, and 12 hours) using an acid hydrolyzed soybean sample for 120 minutes was filtered with a filter paper and 0.45 μm membrane filter (Millex- Filtered using HN, Millipore, Bedford, Mass., USA, and the reaction solution was diluted with ethanol (1 mL of reaction solution: 1 mL of EtOH). The 20 μL reaction solution was injected into HPLC and analyzed.

Example . Acid Hydrolysis of Isoflavones by Microwave Treatment

Acid hydrolysis of isoflavones by microwave treatment was performed on microwave extractors (ETHOS TOUCH CONTROL, Milestone, Sorisole, Italy). Microwave acid hydrolysis of isoflavones was performed with magnetic stirring at 100 ° C. and 500 W, and four vessels (one for reference with three samples and temperature probes) were used at a time. Hydrolysis of soy isoflavones was performed during each hydrolysis time (10-60 minutes).

That is, 10 mL of 1N HCl solution containing 1 g of pulverized soybeans contained in a cap-reaction vial was placed in the microwave extractor for each hydrolysis time, and then hydrolyzed, and the mixture was cooled at room temperature. 15 mL of ethanol was added to each mixture and left for 3 hours to extract the isoflavones. This was filtered using a filter paper and a 0.45 μm membrane filter (Millex-HN, Millipore, Bedford, Mass., USA), and the reaction solution was diluted with ethanol (1 mL of reaction solution: 1 mL of EtOH). 20 μL of the reaction solution was injected into HPLC and analyzed.

Reference Example  2. Statistical Analysis

Statistical analysis was performed by the general linear model procedure (GLM) using SAS statistical programs (Version 9.1, 2002, SAS Cary, NC). Experimental design of the present specification was performed by randomized block design and was carried out in three iterations. Duncan's multiple range test (DMRT) was set at a probability level of 0.05.

Experimental Example  1. of soy isoflavones HPLC Chromatogram

Eight isoflavone peaks were obtained by HPLC chromatogram of soy isoflavone extract obtained by reflux extraction of soybean meal. The eight isoflavones include diadzin, glycidin, genistin, malonyldaidzin, malonylgenistin, diadonzegen, and glycidine ( glycitein) and genistein, and the base-line resolution was very good within 50 minutes of retention time in HPLC analysis (FIG. 1B).

In general, soybean seeds contain a total of 12 isoflavones as shown in A of FIG. 1 (Lee, Yan, Ahn, & Chung, 2003; Rostagno, Palma, & Barroso, 2004). Eight isoflavones were identified from the soybean extracts of the soybean seed. Differences in the composition and content of individual isoflavones according to genotype are well known in the previous studies (Lee, Yan, Ahn, & Chung, 2003; Lee et al., 2003).

Experimental Example  2. Determination of proper acid hydrolysis and extraction time by dry oven treatment

Soy isoflavones were obtained by acid hydrolysis with 1N HCl in a 100 ° C. dry oven for each hydrolysis time (30, 60, 90, 120, 180, and 240 minutes).

Each soy isoflavone HPLC chromatogram obtained according to hydrolysis time is shown in FIG. 2. At the end of each hour's hydrolysis, the isoflavone aglycone content also increased, and most of the diadazine, glycidine, genistin, malonyldiazine, and malonylgenistin hydrolyzed to aglycone at 120 minutes [ 2D]. In addition, isoflavone glucoside was completely converted to each aglycone at 180 minutes [E in FIG. 2]. Figures 3 and 4 show the change of the diazezein, glycinein, genistein and total isoflavone content with hydrolysis time in the 100 ℃ dry oven treatment.

The contents of diazeine, glycine and genistein were markedly different depending on the hydrolysis time. When the hydrolysis time increased up to 180 minutes, the contents of diazein and glycatein increased to reach the highest content and reached equilibrium after 180 minutes. Genistein, on the other hand, reached its highest content at 120 minutes [Fig. 3]. The content of genistein tended to decrease after reaching the highest content (after 120 minutes), probably due to the degradation of the genistein molecule with increasing hydrolysis time (Chiang, Shih, & Chu, 2001). . In addition, the total isoflavone content with hydrolysis time was significantly correlated with the genistein content.

The above results indicate that acid hydrolysis carried out in a 100 ° C. dry oven for 120 minutes is the best condition for obtaining maximum isoflavone aglycone from soybean seeds. To extract the maximum content of isoflavones from the acid hydrolyzed sample, alcohol is added to the acid hydrolyzed sample for 120 minutes to increase the isoflavone extraction time from 1 hour to 3, 6, 9 and 12 hours The results of the examination are shown in Table 1 below.

Extraction time (hours) Isoflavones content (㎍ / g) Diamond Jane Glycitane Genistein Total content One 508.7a ^a 173.3a 648.8c 1330.8b 3 484.3b 159.7a 700.2b 1334.3ab 6 500.5ab 173.6a 714.2ab 1388.3a 9 494.2ab 172.2a 692.4b 1358.8ab 12 496.9ab 167.7a 731.9a 1396.5a ^a Data with different letters in a same column differed significantly at p = 0.05.

 As shown in Table 1, most of the isoflavone aglycone present in the acid hydrolysis solution of the soybean was extracted almost 99% in 3 hours, the total isoflavones obtained the maximum content by 12 hours extraction. Statistical significance was not recognized between 3, 6, 9 and 12 hours extraction time for total isoflavone content, and thus it can be seen that extraction over 3 hours is not significant.

That is, in order to obtain the maximum isoflavone content in soybean seed, it is necessary to leave it for extraction for 3 hours at room temperature after completion of acid hydrolysis and hydrolysis for 120 minutes using a 100 ° C. dry oven treatment.

Experimental Example  3. Determination of Acid Hydrolysis and Extraction Time by Microwave Treatment

In the case of microwave irradiation, soybean samples were hydrolyzed with 1N HCl for 10 to 60 minutes in time to determine the proper hydrolysis time. The HPLC chromatogram of the result obtained by hydrolysis at each time using microwave irradiation is shown in FIG. 4. All isoflavone glucosides were fully hydrolyzed to their aglycone in 50 minutes [Fig. 4E]. The change of isoflavone aglycone content with hydrolysis time is shown in Table 2.

Hydrolysis time (minutes) Isoflavones content (㎍ / g) Diamond Jane Glycitane Genistein Total content 10 239.2d ^a 148.8c 420.3d 808.3e 20 384.3e 170.4ab 674.8c 1229.4d 30 406.1b 178.9a 681.6c 1266.5d 40 418.7b 167.0ab 724.4b 1313.0c 50 487.5a 167.8b 792.9a 1448.2a 60 485.5a 174.6ab 718.6b 1378.6b ^a Data with different letters in a same column differed significantly at p = 0.05.

As shown in Table 2, the content of each isoflavone aglycone increased with hydrolysis time. Diadzein and Genistein reached maximum content at 50 minutes, whereas the maximum content of glycine reached 30 minutes. In addition, the total isoflavone content showed a similar tendency to change pattern of diazein and genistein with different hydrolysis time. The total isoflavone content increased significantly during the hydrolysis up to 50 minutes, while the total content of isoflavones was decreased for the 60 minute hydrolysis than for the 50 minutes.

Microwaves, which have been used as an alternative to traditional methods for the extraction of organic compounds from plants and foods, can be found to improve extraction efficiency by reducing the amount of solvent used and extraction time.

Table 1 and 2 comparing the individual aglycone and total isoflavone contents obtained by the conventional acid hydrolysis method subjected to acid hydrolysis for 120 minutes during the dry oven treatment and the method of the present invention which was acid hydrolyzed for 50 minutes using microwave treatment. In consideration, there was no difference in the isoflavone aglycone and total isoflavone contents of the two methods.

These results suggest that at least 120 minutes of acid hydrolysis time is required to obtain maximum isoflavone aglycones by dry oven treatment, i.e. conventional conventional acid hydrolysis [FIGS. 2 and 3], but from microwave seed treatment by microwave treatment. This means that only 50 minutes of acid hydrolysis time is required to obtain the maximum isoflavone aglycone [FIG. 4 and Table 2]. Therefore, it can be seen that the acid hydrolysis method of isoflavone glycoside by microwave treatment can obtain similar results compared to the dry oven treatment with only a relatively small amount of time.

Based on the above results, it can be seen that acid hydrolysis using microwave treatment is a simpler and more reliable acid hydrolysis method for evaluating the content of soy isoflavones.

The present invention described above is not limited to the above-described embodiment and the accompanying drawings, and various substitutions, modifications, and changes are possible within the scope without departing from the technical spirit of the present invention. It will be evident to those who have knowledge of.

1, 1 is diaidzin, 2 is glycytin, 3 is genistin, 4 is malonyldaidzin, 5 is malonylgenistin, 6 is dia. Dezezein, 7 is glycite and 8 is genistein.
In Fig. 2, 1 is diazine, 2 is glycidine, 3 is zenithine, 4 is malonyl diazine, 5 is malonyl zenithine, 6 is diadzeze, 7 is glycitine and 8 is zensteine.
In Fig. 4, 1 is diadazine, 2 is glycidine, 3 is zenithine, 4 is malonyl diazine, 5 is malonyl zenithine, 6 is diadzein, 7 is glycitine and 8 is genistein.

Claims (6)

Soybean isoflavone glycosides are non-glycoside isoflavones by immersing the dried and pulverized soybean seed powder in an acid solution of 5 to 30 times the weight of soybeans and acid hydrolyzing under microwave irradiation for 45 to 55 minutes at 95-105 ° C. Converting to aglycone;
Alcohol was added to the mixture of the hydrolyzed bean seed powder and the acid solution and allowed to stand at room temperature to obtain diadzin, genistin, diadzein, genistein, glycidine, malonyldiadzin, malonylgenistin, and glycinein. Extracting eight isoflavone aglycones; And
Separating the isoflavone aglycones by filtration and diluting with ethanol;
Soy isoflavone aglycone extraction method comprising a.
delete delete delete The method according to claim 1,
The alcohol is a soybean isoflavone aglycone extraction method characterized in that the addition of 1 to 20 times the weight of the mixture of the hydrolyzed bean seed powder and the acid solution.
The method according to claim 1,
Extracting the soybean isoflavone aglycone, characterized in that the room temperature standing for 2 to 3 hours.

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