A simple method for determining the content of oleic acid in Alpinia oxyphylla

Abstract

The present invention provides a method for simply judging the content of oleanone in Alpinia oxyphylla kernels, and belongs to the technical field of traditional Chinese medicine detection. The method comprises the following steps: (1) preparing two kinds of Alpinia oxyphylla kernels as standard Alpinia oxyphylla kernels, and determining the oleanone content of the two kinds of Alpinia oxyphylla kernels; allowing an evaluation personnel to taste the two kinds of Alpinia oxyphylla kernels, and obtaining the bitterness value and standard deviation of the standard Alpinia oxyphylla kernels; (2) selecting Alpinia oxyphylla kernels with non-bitter outer arils as Alpinia oxyphylla kernels to be tested; (3) scoring the bitterness degree of the selected Alpinia oxyphylla kernels to be tested, and obtaining the bitterness value x; (4) judging the oleanone content y of the Alpinia oxyphylla kernels to be tested according to the relationship between the bitterness value of the standard Alpinia oxyphylla kernels and the oleanone content. The present invention proposes to use the bitterness degree as an indicator for quickly and roughly evaluating the oleanone content in Alpinia oxyphylla kernels. The method has extremely high operability, and extremely low manpower, material and time costs, and has obvious scientific significance and application value for the cultivation and quality control of Alpinia oxyphylla kernels.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for simply judging the content of oleocanthoidene in Alpinia oxyphylla seeds. Background Art

[0002] Alpinia oxyphylla Miq. is the fruit of Alpinia oxyphylla Miq., a plant of the ginger family. It has the effects of warming the spleen, stopping diarrhea, relieving saliva, warming the kidney, contracting urine and consolidating semen. It is commonly used for spleen and stomach deficiency, vomiting, diarrhea, cold pain in the abdomen, excessive salivation, kidney deficiency, enuresis, frequent urination, spermatorrhea, and leukorrhea. It can be widely used as a spice, Chinese medicine and raw material for extracting grapefruit ketone.

[0003] The main active ingredient of Alpinia oxyphylla is nootkatone, and the content of nootkatone represents the quality of Alpinia oxyphylla. However, in this field, the methods for determining the content of nootkatone in Alpinia oxyphylla are mainly chemical analysis and instrument detection methods, which include steps such as sample crushing, extraction, instrument detection and analysis. This method not only relies on the laboratory and the large-scale instruments and reagents therein, but also requires high technical requirements for personnel, and takes a lot of time. It is difficult to grade Alpinia oxyphylla in a short time, and it is also impossible to conduct direct and rapid evaluation in the fields, Chinese herbal medicine trading markets or pharmacies, which restricts the industrial development of Alpinia oxyphylla.

[0004] Based on this, we hope to find a method that is highly operable, simple, convenient, and can quickly determine the content range of oleic acid ketone in Alpinia oxyphylla kernels, so as to facilitate rapid grading of Alpinia oxyphylla kernels. Summary of the invention

[0005] The purpose of the present invention is to provide a method for simply judging the content of oleanone in Alpinia oxyphylla seeds, so as to facilitate rapid grading of Alpinia oxyphylla seeds.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for simply determining the content of oleic acid in Alpinia oxyphylla seeds, comprising the following steps:

[0008] (1) Prepare two kinds of Alpinia oxyphylla seeds as standard Alpinia oxyphylla seeds, and determine the content of y CK1 ±σ CK1 and CK2 ±σ CK2 , where y CK1 <y CK2 ; Let the testers taste the standard Alpinia oxyphylla kernels and find their bitterness value x CK1 、x CK2 and standard deviation SD1, SD2, the x CK1 and x CK2 Between 0 and 15; the bitterness value of the standard Alpinia oxyphylla kernel satisfies xCK1 +SD1<x CK2 -SD2;

[0009] (2) Selecting Alpinia oxyphylla kernels whose outer aril is not bitter as the Alpinia oxyphylla kernels to be tested;

[0010] (3) asking the testers to chew the Alpinia oxyphylla kernels to be tested, and then scoring the bitterness to obtain a bitterness value x, where x is between 0 and 15;

[0011] (4) the content of oleic acid in the Alpinia oxyphylla seeds to be tested is y,

[0012] When x≤x CK1 -SD1, y≤y CK1 -σ CK1 ;

[0013] When x CK1 -SD1<x<x CK1 +SD1, y CK1 -σ CK1 <y<y CK1 +σ CK1 ;

[0014] When x CK1 +SD1≤x≤x CK2 -SD2, y CK1 +σ CK1 ≤y≤y CK2 -σ CK2 ;

[0015] When x CK2 -SD2<x<x CK2 +SD2, y CK2 -σ CK2 <y<y CK2 +σ CK2 ;

[0016] When x ≥ x CK2 +SD2, y≥y CK2 +σ CK2 .

[0017] Preferably, the bitterness degree in step (3) is divided into no bitterness, slight bitterness, medium bitterness, and strong bitterness.

[0018] Preferably, after obtaining the bitterness score of each assessor, the average value is calculated to obtain the bitterness value x CK1 、x CK2 and x.

[0019] Preferably, the bitterness score is: 0 for no bitterness, 1-5 for slight bitterness, 6-10 for moderate bitterness, and 11-15 for strong bitterness.

[0020] Preferably, when determining the content of the grapefruit ketone, the number of the evaluators is no less than 8.

[0021] Preferably, the standard deviations SD1 and SD2 are integers after rounding.

[0022] The present invention finds that the outer layer of Alpinia oxyphylla kernel is aril through microstructural analysis, and flavor analysis shows that the outer aril has no bitterness but sweetness. After removing the aril, chewing the seeds has obvious bitterness. Therefore, the bitterness of the outer aril is used as a standard for whether the Alpinia oxyphylla kernel is artificially added with other substances or contaminated. The 1448 identified components are searched in the flavor database, and 54 bitter substances are screened, including the most important bioactive substance, oleanone. The present invention conducts differential metabolite analysis, flavor omics analysis and correlation analysis on peels and seeds at different developmental stages, and finds that the three differential metabolites enriched in dry seeds are the source of the bitterness of Alpinia oxyphylla kernel. Combined with the results of HPLC-DAD analysis, it is found that oleanone is the most important compound with an absolute advantage in content in Alpinia oxyphylla and is the main source of bitterness in Alpinia oxyphylla kernel. Based on this, the present invention proposes to use the bitterness degree alone as an indicator for quickly and roughly evaluating the content of oleic acid ketone in Alpinia oxyphylla kernel, and further provides a method for roughly judging the content of oleic acid ketone in Alpinia oxyphylla kernel by using the bitterness value. The method has extremely high operability, can greatly save manpower, material resources and time costs, and has obvious scientific significance and application value for the breeding of fine varieties of Alpinia oxyphylla kernel, optimization of cultivation technology, and quality control of medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the OPLS-DA analysis score diagram between different sample groups in Example 1.

[0024] Figure 2 This is the arrangement diagram of OPLS-DA analysis between different sample groups in Example 1.

[0025] Figure 3 This is a flavor wheel diagram comparing the relative content of DAM and its corresponding aroma among the sample groups in Example 1.

[0026] Figure 4 This is a Sankey diagram comparing the flavor changes corresponding to the relative content changes of DAM among the sample groups in Example 1.

[0027] Figure 5 These are the morphological pictures of the peel and seeds of different groups at different time periods in Example 2.

[0028] Figure 6 This is a diagram showing the development of the seed coat pigment layer and oil cells of different groups in Example 2.

[0029] Figure 7 This is a graph showing the accumulation of metabolites at different developmental stages in the pericarp and seed coat in Example 2.

[0030] Figure 8 This is a diagram showing the accumulation of essential oils in the pericarp and seed coat at different developmental stages in Example 2.

[0031] Fig. 9 This is a flavor analysis diagram of the mature Yiqi fruit in Example 3.

[0032] Fig.10 The following are the classification and counting results of 1448 metabolites in Example 3.

[0033] Fig.11 The PCA analysis results of 22 sweet smell substances, 45 sweet taste substances, 11 spicy smell substances, 17 spicy taste substances and 54 bitter taste substances in Example 4 are shown.

[0034] Fig.12 This is the HCA diagram of the relative content of bitter substances in different samples in Example 4.

[0035] Fig.13 4. The TIC diagrams of the nutmeg ketone standard and the RS sample in Example 4 (the upper diagram is the TIC diagram of the nutmeg ketone standard, and the lower diagram is the TIC diagram of the RS).

[0036] Fig.14 This is a graph showing the relationship between bitterness and the content of naringenone in Example 4.

[0037] Fig.15 The contents of different active compounds in the Yiqi fruit in Example 5 ((a) 34 representative compounds and (b) 8 most important active compounds).

[0038] Fig.16 for Figure 4 Enlarged view of panel f.

[0039] Fig.17 for Figure 4 Enlarged view of Figure g.

[0040] Fig.18 for Figure 4 Enlarged view of Figure j.

[0041] Fig.19 for Figure 4 Enlarged view of the middle k figure.

[0042] Fig. 20 for Figure 4 Enlarged view of Figure 1. DETAILED DESCRIPTION

[0043] In the present invention, Ep: seed coat epidermis; Hy: hypodermis; OL: oil cell layer; PL: pigment layer; PC: pigment cell; En: endocarp; Vb: vascular bundle; Oc: oil cell; CW: cell wall; O: osmiophilic substance.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] Sample collection and analysis methods

[0047] Qiongzhong No. 1 Alpinia oxyphylla was used as the material and cultivated in the Hainan Alpinia oxyphylla planting demonstration base. In this work, fresh samples and dry samples were prepared synchronously. For the collection of fresh samples, fruits were collected 1, 2, and 7 weeks after flowering (color measurements were performed on standardized lighting and white balance photos using ImageJ software). Then, the peel and seeds were immediately peeled off, and the samples were named YP (fresh skin picked 1 week after flowering), GP (fresh skin picked 2 weeks after flowering), RP (fresh skin picked 7 weeks after flowering), YS (fresh seeds picked 1 week after flowering), GS (fresh seeds picked 2 weeks after flowering), and RS (fresh seeds picked 7 weeks after flowering). For the preparation of dry samples, fresh skin and fresh seeds were picked 7 weeks after flowering and stored in an oven at 40°C with a relative humidity of 40-50% for one week. Accordingly, these samples were named DP (dried skin) and DS (dried seeds), respectively. Three biological replicates were prepared for each sample. The samples were fixed in FAA fixative (formalin: acetic acid: 70% ethanol = 5:5:90; V:V:V) for one day to prepare paraffin sections. Frozen sections and semi-thin sections were prepared at the same time.

[0048] An extensive targeted metabolomics analysis of non-volatile metabolites in Amomum tsao-ko was performed. The analysis method can be referred to “P. Li, G. Bai, J. He, et al., Chromosome-level genome assembly of Amomum tsao-ko provides insights into the biosynthesis of flavor compounds”.

[0049] The volatile metabolites in the fruit of Yiqi were analyzed. The analysis method can be found in "D. Zhang, D. Shen, Y. Cao, et al., Widely targeted metabolomic approach reveals dynamic changes in non-volatile and volatile metabolites of peanuts during roasting"

[0050] The data analysis method was as follows: after obtaining the bitterness score of each evaluator, statistical analysis was performed, the mean value was calculated, the bitterness value x was obtained, and difference analysis was performed. The comparison between two groups of samples was performed using the independent sample t test, and the Duncan's new multiple range test AONVA was used for three groups or more samples. For the samples of Alpinia oxyphylla with a difference p<0.05 between the groups, the bitterness and content were evaluated to be significantly different, and the difference was statistically significant. The corrected peak area was calculated as the relative content during metabolome analysis. Principal component analysis (PCA) was performed using MetWare Cloud (https: / / cloud.metware.cn), and the function parameter was set to scale=True. Hierarchical agglomerative cluster analysis (HCA) was performed using the TBtools v2.0 software suite. Three biological replicates were performed for each sample. The differential cumulative metabolites (DAMs) between groups were determined by VIP≥1, p<0.05, and log2FC (fold change) absolute value ≥1. VIP was derived by orthogonal partial least squares discriminant analysis (OPLS-DA) using the R package MetaboAnalystR, with score plots ( Figure 1 ) and Pareto diagrams ( Figure 2 ). To avoid overfitting, we performed 200 permutation tests and the results showed that Q 2 All of them were greater than 0.85, indicating that there were significant differences among the groups and the model for screening differential metabolites was reliable. 2 The y values ​​are all 1, indicating a good fit. Flavor analysis requires first retrieving the metabolites identified in the fruit from the flavor database, then annotating the corresponding flavor-related information to these metabolites (http: / / www.thegoodscentscompany.com; http: / / perflavory.com / ; http: / / foodflavorlab.cn / ; https: / / cosylab.iiitd.edu.in / flavordb / ), and drawing a flavor wheel based on the relationship between metabolites and flavors (e.g. Figure 3 ) and Sankey diagrams ( Figure 4 ), Figures 1 to 4 Group a is a comparison between YP and GP; Group b is a comparison between GP and RP; Group c is a comparison between YP and RP; Group d is a comparison between RP and DP; Group e is a comparison between YS and GS; Group f is a comparison between GS and RS; Group g is a comparison between YS and RS; Group h is a comparison between RS and DS; Group i is a comparison between YS and YP; Group j is a comparison between GS and GP; Group k is a comparison between RS and RP; Group l is a comparison between DS and DP. The enlarged image of group f in the Sankey diagram is Fig.16 , the enlarged image of group g is Fig.17 , the enlarged image of group j is Fig.18 , the enlarged picture of group k is Fig.19, the enlarged image of group l is Fig. 20 .

[0051] Example 2

[0052] Morphological observations showed that the fruit of the genus Artemisia chinensis expanded rapidly from 1 week after flowering (WA) to 2WA, and there was no obvious change thereafter; the outer layer of the seed was the aril, and the color of the inner seed changed from 2WA to 7WA, and at the same time, the aril liquefied. One week after harvesting (1WP), the peel and seeds dried and turned brown ( Figure 5 The oil cells in the seeds gradually increase in size from YS to RS, and the aril cells first swell and then collapse, which is consistent with the appearance of liquefied arils in RS. The pigment layer stains darker with Safranin O, indicating that the degree of lignification increases with seed development ( Figure 6 ).

[0053] One-step localization of essential oils using histochemical staining revealed that oil cells were dispersed throughout the pericarp and seed coat at all developmental stages ( Figure 7 The development of the hypodermis (Hy) and pigment layer (PL) cells begins at YS. The semi-thin section results show that dark osmiophilic substances are enriched in the hypodermis and pigment layer at the YS stage ( Figure 8 ). The diameter of oil cells in the pericarp and seeds differed significantly. There was no obvious osmiophilic substance in the aril, indicating that the accumulation of secondary metabolites, especially oil compounds including oleoyl ketone, in seeds was mainly concentrated in the seed coat rather than the aril.

[0054] Example 3

[0055] Sensory analysis and metabolomics analysis

[0056] The Institutional Review Board of the Institute of Tropical Crop Genetic Resources, Chinese Academy of Tropical Agricultural Sciences, approved the ethical review of this study (PZSYYSP-202307151, July 15, 2023) and approved the participation of human subjects in this study. Before participation, all subjects signed informed consent. Samples were stored in odorless beakers for evaluation. The score ranged from 0 to 15 points, with 0 for no bitterness, 1 to 5 for slight bitterness, 6 to 10 for moderate bitterness, and 11 to 15 for strong bitterness.

[0057] The results of human sensory flavor analysis showed that the ripe fruit exudes a spicy and sweet aroma, and the spiciness increases after being cut ( Fig. 9 a), the spicy taste mainly comes from the peel ( Fig. 9 b), the aril has a distinct sweet taste ( Fig. 9 c) The seeds without the aril have a distinct bitter taste ( Fig. 9 d).

[0058] The headspace injection-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) technology was used for volatile metabolomics detection and ultra-high performance liquid chromatography-electrospray ion source-tandem quadrupole composite linear ion trap-tandem mass spectrometry (UPLC-ESI-QTRAP-MS / MS) was used for extensive targeted metabolic detection, and metabolomics analysis of the samples was performed. A total of 1,448 metabolites were identified, and the metabolites were classified into 28 categories based on the metabolomics data, among which flavonoids, phenolic acids, lipids, and terpenes were the most abundant ( Fig.10 ), there were 768 common metabolites between the peel and seeds at different stages (accounting for 53% of the total number of metabolites).

[0059] Example 4

[0060] Flavor analysis

[0061] Based on the sensory analysis results, we cross-referenced the volatile metabolites in the flavor database (http: / / www.thegoodscentscompany.com; http: / / perflavory.com / ; http: / / foodflavorlab.cn / ; https: / / cosylab.iiitd.edu.in / flavordb / ), and annotated a total of 82 odor substances. We then analyzed the relative content of DAMs and their corresponding aromas (e.g. Figure 3 and Figure 4 The flavor transition from 1WA to 2WA is less and relatively simple ( Figure 3 a. Figure 3 e. Figure 4 a and Figure 4 e), while the flavor changes from 2WA to 7WA become more numerous and complex ( Figure 3 b. Figure 3 f. Figure 4 b and Figure 4 f). At each stage, the peel and seeds exhibit different flavors, with the peel having a richer aroma, including sweet, spicy, woody, green, herbal, floral, camphorous, balsamic, and pine ( Figure 3 i~ Figure 3 1. Figure 4 i~ Figure 4 l). One week after harvest (1WP), the accumulation of trans-geranic acid methyl ester (KMW0480), ethyl hexanoate (KMW0709), methyl 2-hydroxy-3-methylvalerate (XMW0152) and 2-butyl-5-ethylthiophene (XMW1459) in the peel increased significantly, and the fruity aroma was more obvious ( Figure 4 d). As the fruit develops and matures, the flavor gradually intensifies, with sweetness and spiciness being the most prominent characteristic smells of the fruit.

[0062] At the same time, PCA analysis was performed on the 22 sweet smell substances, 45 sweet taste substances, 11 spicy smell substances, 17 spicy taste substances and 54 bitter taste substances identified ( Fig.11 ), the results showed that in most cases the pericarp and seeds were clearly separated, and the separation intensified with development, only the inter-tissue separation of sweet substances decreased with development.

[0063] According to the HCA analysis of bitter substances ( Fig.12 ) The results showed that five bitter substances were significantly enriched in RS: oleanone, piperonal, butea, catechin and epicatechin. The bitter DAM analysis results between RS and RP also showed that compared with RP, RS was significantly enriched in the above five DAMs, among which oleanone had an absolute advantage in the content of Alpinia oxyphylla (such as Fig.13 The bitter taste of oleic acid ketone has a dose advantage in affecting the flavor of Alpinia oxyphylla. In DS, the levels of catechin and epicatechin decreased significantly, and only three differential metabolites were the source of the bitter taste of Alpinia oxyphylla.

[0064] According to the results of correlation analysis, bitterness and oleic acid content are significantly positively correlated ( Fig.14 ).

[0065] Example 5

[0066] Bioactive substances in yizhi fruit

[0067] 41 sesquiterpenes such as oleanone, 135 flavonoids such as chrysin, phenylpropanoids such as oxyphylla A, 17 gingerols and their derivatives such as 6-gingerol, and 43 diphenylheptanes represented by yakuchinone A and oxyphyllacinol were identified. 34 bioactive substances were selected for HCA (see Fig.15 a), the results showed that the samples were clustered into two groups, RS and DS ( Fig.15 a) clustered into the first cluster, and the rest clustered into the second cluster. This indicates that fresh and dried mature seeds have the most special accumulation characteristics of bioactive substances. In the second cluster, RP and DP were clustered into one class, while YP, GP, YS and GS were clustered into one class. The results showed that 34 bioactive metabolites were mainly present in mature tissues and differed significantly between mature peels and seeds. Mature seeds were enriched in sesquiterpenes, and mature peels were enriched in diphenylheptane compounds. On the other hand, immature tissues were mainly enriched in vitamin C (ascorbic acid) and dehydroascorbic acid. The eight substances of most concern (nopalxinone, OxyphyllaA, protocatechuic acid, chrysin, chrysin, cinnamaldehyde, 6-gingerol and curcumin) were further analyzed ( Fig.15b), the results showed that RS enriched naringenone, OxyphyllaA, protocatechuic acid and 6-gingerol. RP enriched chrysin, chrysin, cinnamaldehyde and curcumin. Drying treatment increased the contents of naringenone, OxyphyllaA, protocatechuic acid, 6-gingerol, chrysin, chrysin and curcumin in mature fruits, but reduced the content of cinnamaldehyde.

[0068] Flavour analysis involves both olfactory and gustatory sensory experiences, and results from human sensory studies have highlighted clear tissue specificity in the flavour of Alpinia oxyphylla: a sweet and spicy aroma in the peel, sweetness in the arils and bitterness in the seeds.

[0069] In the flavoromics analysis, it was found that the aroma of the fruit became sweeter and spicier with maturity, and postharvest processing can lead to richer flavor compounds in 1WP than in 7WA. At the same time, it was also observed that there was a significant difference in the flavor accumulation of the peel and seeds. The sweetness in the peel came from esters and aromatic hydrocarbons, while there were fewer sweet substances in mature seeds.

[0070] The present invention found five main bitter substances in RS, including piperonal, butein, catechin and epicatechin in addition to a large amount of naringenone, resulting in an overall bitter taste, among which naringenone is absolutely dominant and has a strong correlation with bitterness. The ratio of catechin and epicatechin gradually decreases from RS to DS, further supporting the conclusion that naringenone is the main source of bitterness in DS.

[0071] The accumulation of bioactive substances was also tissue-specific, with enrichment being particularly evident in mature tissues. Postharvest treatments increased the abundance of traditional bioactive compounds, but decreased the abundance of curcumol, epi-naringol, naringol, and osmoticone A. Some molecules with "neuroprotective properties" were increased in DS compared with other osmotic fruit tissues. The increase in naringolone, OxyphyllaA, and protocatechuic acid suggests that DS may have characteristics associated with osmotic fruit and is a "fruit that is beneficial to intelligence." Oil cells are generally the accumulation site for secondary metabolites, especially essential oils containing terpenoids. Histochemical localization showed that essential oil groups accumulate in seed coat oil cells and pigment layers, while metabolomics analysis showed that the sesquiterpene naringolone mainly accumulates in seeds. Therefore, it can be inferred that naringolone is concentrated in the seed coat, probably mainly in the seed coat oil cells. In general, the appearance of the fruit peel of osmotic fruit does not change significantly during fruit ripening, but metabolites accumulate in osmotic fruit during seed coloration and oil cell development.

[0072] The aroma characteristics of the fruit of the Chinese ginseng are sweet and spicy. The sweetness of the fruit of the Chinese ginseng comes from the disaccharides at the time of harvest, which are converted into monosaccharides after harvest. The spicy taste comes from dihydrocapsaicin, gingerol and their derivatives. The bioactive compounds are mainly found in mature tissues, and their concentrations increase after postharvest drying. The bitterness of the fruit of the Chinese ginseng is extremely significantly linearly positively correlated with the accumulation of oleic acid in DS oil cells. Compared with other components, the content of oleic acid has an absolute advantage in DS, and its bitterness has the greatest impact on the flavor of the kernel of the Chinese ginseng. Therefore, the degree of bitterness can be used as a reference for the content of oleic acid in DS.

[0073] Example 6

[0074] Two samples of Alpinia oxyphylla reference substances with known oleic acid content were prepared, namely CK1 and CK2. The oleic acid content in CK1 was 0.69±0.06 mg / g, and the oleic acid content in CK2 was 1.00±0.05 mg / g. Eight testers were asked to score the bitterness of CK1 and CK2, calculate the mean and standard deviation (SD), and grade them according to the content. The results are shown in Tables 1 and 2:

[0075] Table 1 Calculation of bitterness scores of Alpinia oxyphylla reference substances CK1 and CK2

[0076] Bitterness value x Standard Deviation (SD) The standard deviation (SD) of bitterness value is rounded CK1 8.5 1.195 1 CK2 13 0.926 1

[0077] Table 2 Grades of Alpinia oxyphylla

[0078] Bitterness Classification Definition Bitterness rating range Estimated content of naringenone (mg / g) grade <![CDATA[x≤x CK1 -SD1]]> x≤7.5 y≤0.63 5 <![CDATA[x CK1 -SD1<x<x CK1 +SD1]]> 7.5＜x＜9.5 0.63＜y＜0.75 4 <![CDATA[x CK1 +SD1≤x≤x CK2 -SD2]]> 9.5≤x≤12 0.75≤y≤0.95 3 <![CDATA[x CK2 -SD2<x<x CK2 +SD2]]> 12＜x＜14 0.95＜y＜1.05 2 <![CDATA[x≥x CK2 +SD2]]> ≥14 y≥1.05 1

[0079] Please ask the above 8 evaluation personnel to taste the Alpinia oxyphylla samples to be tested. If the outer aril tastes bitter, it means other substances have been artificially added or the medicinal materials are contaminated, and the evaluation cannot continue. The bitterness of the 9 Alpinia oxyphylla samples that passed the initial inspection will be evaluated, and the bitterness value will be calculated. The content of oleic acid in the Alpinia oxyphylla and the grade of the Alpinia oxyphylla will be roughly evaluated according to the bitterness value falling into the range in Table 2. The content of oleic acid in the Alpinia oxyphylla will be detected by instrument and the grades will be divided according to the oleic acid content. The difference between the grade inferred based on bitterness and the grade detected by instrument will be compared, and the accuracy will be calculated and it will be judged whether the inferred grade is correct.

[0080] (Accuracy% = |estimated grade - detected grade| / 5*100. Correctness% = number of times the estimated grade is correct / total number of estimates*100. The results show that the accuracy of the estimated grade of the Pu'er tree according to this method is 98%, and the correctness is 88% (Table 3)).

[0081] Table 3 Statistics on the accuracy of predicting the grade of Alpinia oxyphylla kernels based on bitterness

[0082]

[0083]

[0084] As can be seen from Table 3, the method proposed by the present invention for rapidly and roughly evaluating the grade of Alpinia oxyphylla using the bitterness degree has a correct rate of 88% and an accuracy rate of 98%. At the same time, the method of the present invention does not require professional technical operations such as sample processing, chemical analysis, instrument detection, and professional detection personnel, has extremely high operability, greatly reduces the cost of manpower, material resources, and time, and has obvious scientific significance and application value for rapid quality evaluation and control of Alpinia oxyphylla in field research, test fields, planting bases, and medicinal material trading markets.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simply determining the content of naringenone in Alpinia oxyphylla kernel, characterized in that: The following steps are involved: (1) Prepare two kinds of Alpinia oxyphylla kernels as standard Alpinia oxyphylla kernels, and determine the content of oleic acid in the standard Alpinia oxyphylla kernels as y CK1 ±σ CK1 and CK2 ±σ CK2 , where y CK1 <y CK2 ; Let the testers taste the standard Alpinia oxyphylla kernels and find their bitterness value x CK1 、x CK2 and standard deviation SD1, SD2, the x CK1 and x CK2 Between 0 and 15; the bitterness value of the standard Alpinia oxyphylla kernel satisfies x CK1 +SD1<x CK2 -SD2; (2) Select Alpinia oxyphylla kernels with non-bitter outer aril as the Alpinia oxyphylla kernels to be tested; (3) asking the testers to chew the Alpinia oxyphylla kernels to be tested, and then score the bitterness to obtain a bitterness value x, where x is between 0 and 15; (4) the content of oleic acid in the Alpinia oxyphylla kernel to be tested is y, When x≤x CK1 -SD1, y≤y CK1 -σ CK1 ; When x CK1 -SD1 < x < x CK1 +SD1, y CK1 -σ CK1 < y < y CK1 +σ CK1 ; When x CK1 +SD1 ≤ x ≤ x CK2 -SD2, then y CK1 +σ CK1 ≤ y ≤ y CK2 -σ CK2 ; When x CK2 -SD2 < x < x CK2 +SD2, then y CK2 -σ CK2 < y < y CK2 +σ CK2 ; When x ≥ x CK2 +SD2, y≥y CK2 +σ CK2 .

2. The method according to claim 1, characterized in that: The bitterness level in step (3) is divided into four categories: no bitterness, slight bitterness, moderate bitterness and strong bitterness.

3. The method according to claim 2, characterized in that: After obtaining the bitterness score of each evaluator, calculate the average value and obtain the bitterness value x CK1 、x CK2 and x.

4. The method according to claim 3, characterized in that: The bitterness score is: 0 for no bitterness, 1-5 for slight bitterness, 6-10 for moderate bitterness, and 11-15 for strong bitterness.

5. The method according to claim 4, characterized in that: When determining the content of the grapefruit ketone, the number of the evaluators is no less than 8.

6. The method according to claim 5, characterized in that The standard deviations SD1 and SD2 are rounded to integers.

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