A compound and its application in the identification of Camellia ptilophylla varieties

Abstract

The present invention discloses a compound and its application in the identification of Camellia ptilophylla varieties. Its structural formula is shown in formula (I), #imgabs0#. It has been identified and named 4-amino-5-hydroxypentanoic acid. It is a new natural compound discovered in the new shoots of Camellia ptilophylla and is also a newly discovered amino acid. It has not been detected in other tea varieties, but can be detected in all populations of Camellia ptilophylla. By detecting this substance in the new shoot tissue or in the tea leaves made from the new shoots as raw materials, effective identification of Camellia ptilophylla tea products and seedlings can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of tea tree varieties and tea product identification. Specifically, it relates to a compound and its application in the identification of Camellia ptilophylla varieties. Background Art

[0002] China's tea tree resources are widely distributed and rich in types. Most of the tea tree germplasms distributed in the wild are distributed in the form of communities. Through years of natural and artificial selection, rich variations have occurred, forming multiple species and varieties. Therefore, tea plants are a huge family. Drinking tea can refresh the mind because there is caffeine in tea leaves. However, some people, including pregnant women, infants, those with poor sleep, and hypoglycemic patients, etc., should not drink too much caffeine.

[0003] Camellia ptilophylla is a rare tea tree germplasm resource discovered in Huizhou, Guangdong. Its buds and leaves are used to produce various teas such as green tea, white tea, and black tea. Camellia ptilophylla is famous for containing little or no caffeine in its new shoots. Due to its low caffeine characteristic, the products developed from it also have low caffeine characteristics. Therefore, developing tea products from Camellia ptilophylla has good application value and economic value.

[0004] Regarding the identification of Camellia ptilophylla tea products and Camellia ptilophylla seedlings, on the one hand, it can effectively standardize the purity of the seed source at the planting end, and on the other hand, it can trace the origin on Camellia ptilophylla products to ensure the reliability of tea products.

[0005] Traditional methods can detect the caffeine content to identify whether it is a Camellia ptilophylla seedling or product. However, since there are other tea tree germplasms with low caffeine characteristics. Therefore, detecting by caffeine content cannot fully prove that the tea tree seedlings or tea products are Camellia ptilophylla seedlings or Camellia ptilophylla tea products. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art, and provide a compound and its application in the identification of Camellia ptilophylla tea. The present invention detects 4-amino-5-hydroxypentanoic acid in the new shoots of seedlings or the tea made from new shoots, so as to detect Camellia ptilophylla seedlings or Camellia ptilophylla tea products.

[0007] The first object of the present invention is to provide a compound.

[0008] The second object of the present invention is to provide the application of the said compound in the identification of Camellia ptilophylla varieties.

[0009] The third object of the present invention is to provide a method for identifying Camellia ptilophylla.

[0010] The fourth object of the present invention is to provide a detection method for a compound with a structural formula shown in formula (I).

[0011] The fifth object of the present invention is to provide the use of the detection reagent of the said compound in the preparation of a kit for identifying Camellia ptilophylla Chang.

[0012] The sixth object of the present invention is to provide a kit for detecting and identifying Camellia ptilophylla Chang,

[0013] In order to achieve the above object, the present invention is realized by the following scheme: by detecting 4-amino-5-hydroxypentanoic acid in the new shoots of seedlings or the tea leaves made from the new shoots, so as to achieve the purpose of identifying the seedlings or the tea of Camellia ptilophylla Chang.

[0014] The present invention discovers through research that 4-amino-5-hydroxypentanoic acid is a new natural compound found in the new shoots of Camellia ptilophylla Chang, and it is also a newly discovered amino acid. It has not been detected in other tea species, but can be detected in all populations of Camellia ptilophylla Chang. Therefore, detecting it can identify the seedlings and the tea of Camellia ptilophylla Chang.

[0015] The present invention claims to protect a compound, the structural formula of which is shown in formula (I),

[0016]

[0017] It also claims to protect the use of the said compound in the variety identification of Camellia ptilophylla Chang.

[0018] Preferably, the use of the compound in the variety identification of the product and / or seedlings of Camellia ptilophylla Chang.

[0019] It also claims to protect a method for identifying Camellia ptilophylla Chang, by detecting the said compound in a sample to be tested:

[0020] The sample to be tested contains the said compound, and the sample to be tested is Camellia ptilophylla Chang.

[0021] Preferably, the sample to be tested is a new shoot.

[0022] Preferably, HPLC analysis is carried out on the DNFB derivatization product of the water extract of the sample to be tested.

[0023] It also claims to protect a detection method for a compound with a structural formula shown in formula (I), by carrying out HPLC analysis on the DNFB derivatization product of the water extract of the sample to be tested.

[0024] More preferably, the preparation method of the water extract of the sample to be tested is: the sample to be tested is mixed with boiling water, and fully extracted under boiling state, and the solid matter is removed to obtain the water extract.

[0025] Further preferably, the sample to be tested is powder, and the volume ratio of the sample to be tested to the water extract is 1 g: 80 ml to 120 ml; the extraction time is 30 min to 50 min.

[0026] Further preferably, the sample to be tested is powder, the volume ratio of the sample to be tested to the water extract is 1 g: 100 ml; the extraction time is 45 min.

[0027] Further preferably, the sample to be tested is the steamed green tea sample, the volume ratio of the sample to be tested to the water extract is 1 kg: 3 L to 5 L; the extraction time is 1 h to 3 h.

[0028] Further preferably, the sample to be tested is the steamed green tea sample, the volume ratio of the sample to be tested to the water extract is 1 kg: 4 L; the extraction time is 2 h.

[0029] Preferably, the derivatization method is as follows: a mixture of the water extract of the sample to be tested, the acetonitrile solution of DNFB and the NaHCO3 buffer solution is subjected to a derivatization reaction in the dark at 50-70 °C to obtain a derivatized product.

[0030] More preferably, the derivatization reaction is continued in the dark at 60 °C for not less than 1 hour.

[0031] More preferably, the mixture is a mixture of the water extract of the sample to be tested, the acetonitrile solution of 8-12 mL / L DNFB and the NaHCO3 buffer solution of 0.4-0.6 M pH 8.5-9.5 with a volume ratio of 1-10: 1-10: 1-1.

[0032] Most preferably, the mixture is a mixture of the water extract of the sample to be tested, the acetonitrile solution of 10 mL / L DNFB and the NaHCO3 buffer solution of 0.5 M pH 9.0 with a volume ratio of 10: 10: 1.

[0033] More preferably, after the derivatization reaction, purification is carried out and then HPLC analysis is carried out.

[0034] Most preferably, purification is carried out through a 0.45 μm filter head.

[0035] Preferably, the chromatographic column is Eclipse Plus C18, and the mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is the NaAc buffer solution containing N, N-dimethylformamide, and mobile phase B is the aqueous acetonitrile solution; gradient elution is carried out.

[0036] More preferably, the column temperature of the chromatographic column is 26-30 °C.

[0037] Most preferably, the column temperature of the chromatographic column is 28 °C.

[0038] More preferably, the detection wavelength is 300-420 nm.

[0039] Most preferably, the detection wavelength is 360 nm.

[0040] More preferably, mobile phase A is a 0.04 - 0.06 M NaAc buffer solution containing 8 - 12 mL / L of N,N - dimethylformamide with a pH of 6.0 - 7.0.

[0041] Even more preferably, mobile phase A is a 0.05 M NaAc buffer solution containing 10 mL / L of N,N - dimethylformamide with a pH of 6.5.

[0042] More preferably, mobile phase B is an aqueous acetonitrile solution with a V / V ratio of 1 - 2:1 - 2.

[0043] Even more preferably, mobile phase B is an aqueous acetonitrile solution with a V / V ratio of 1:1.

[0044] As a specific embodiment of the present invention, the specific method for HPLC analysis is as follows:

[0045] The chromatographic column is Eclipse Plus C18, and the mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is a 0.05 M NaAc buffer solution containing 10 mL / L of N,N - dimethylformamide with a pH of 6.5, and mobile phase B is an aqueous acetonitrile solution with a V / V ratio of 1:1;

[0046] Calculated by volume ratio, the gradient elution program is as follows:

[0047] 0 - 6 minutes: 100% mobile phase A, 0% mobile phase B;

[0048] 6 - 12 minutes: 90% mobile phase A, 10% mobile phase B;

[0049] 12 - 18 minutes: 85% mobile phase A, 15% mobile phase B;

[0050] 18 - 33 minutes: 80% mobile phase A, 20% mobile phase B;

[0051] 33 - 42 minutes: 75% mobile phase A, 25% mobile phase B;

[0052] 42 - 50 minutes: 70% mobile phase A, 30% mobile phase B;

[0053] 50 - 55 minutes: 50% mobile phase A, 50% mobile phase B;

[0054] 55 - 60 minutes: 20% mobile phase A, 80% mobile phase B;

[0055] 60 - 65 minutes: 0% mobile phase A, 100% mobile phase B;

[0056] 65 to 70 minutes: mobile phase A 0%, mobile phase B 100%;

[0057] 70 to 80 minutes: mobile phase A 100%, mobile phase B 0%;

[0058] The column temperature of the chromatographic column is 28°C, the detection wavelength is 360 nm, the flow rate of the mobile phase is 1.0 mL / min, the sample injection volume is 5 μL, and a characteristic peak appears at 42 minutes, indicating the presence of a compound with a structural formula as shown in formula (I).

[0059] The present invention also claims the use of the detection reagent for the compound in the preparation of a kit for identifying Camellia ptilophylla Chang.

[0060] The present invention also claims a kit for detecting and identifying Camellia ptilophylla Chang, containing the detection reagent for the compound.

[0061] Preferably, the detection reagent is a derivatization reagent and / or an HPLC analysis reagent.

[0062] More preferably, the derivatization reagent is an acetonitrile solution of DNFB and a NaHCO3 buffer solution

[0063] Still more preferably, the acetonitrile solution of DNFB is an 8 - 12 mL / L acetonitrile solution of DNFB.

[0064] Further preferably, the acetonitrile solution of DNFB is a 10 mL / L acetonitrile solution of DNFB.

[0065] Still more preferably, the NaHCO3 buffer solution is a 0.4 - 0.6 M NaHCO3 buffer solution with a pH of 8.5 - 9.5.

[0066] Further preferably, the NaHCO3 buffer solution is a 0.5 M NaHCO3 buffer solution with a pH of 9.0.

[0067] More preferably, the HPLC analysis reagent contains a mobile phase, and the mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is a NaAc buffer solution containing N,N - dimethylformamide, and mobile phase B is an aqueous acetonitrile solution.

[0068] More preferably, mobile phase A is a 0.04 - 0.06 M NaAc buffer solution containing 8 - 12 mL / L of N,N - dimethylformamide with a pH of 6.0 - 7.0.

[0069] Still more preferably, mobile phase A is a 0.05 M NaAc buffer solution containing 10 mL / L of N,N - dimethylformamide with a pH of 6.5.

[0070] More preferably, mobile phase B is an aqueous acetonitrile solution with a V / V ratio of 1 to 2:1 to 2.

[0071] Even more preferably, mobile phase B is an aqueous acetonitrile solution with a V / V ratio of 1:1.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] 4-Amino-5-hydroxypentanoic acid is a new natural compound discovered in the new shoots of Camellia ptilophylla, and it is also a newly discovered amino acid. It has not been detected in other tea species, but can be detected in all Camellia ptilophylla populations. In the new shoot tissue or in tea leaves made from new shoots, by detecting this substance, the tea products and seedlings of Camellia ptilophylla can be effectively identified. Description of the Drawings

[0074] Figure 1 Amino acid components of different tea types in Camellia ptilophylla populations and Yinghong No. 9; Ala is alanine, Arg is arginine, Asp is aspartic acid, Cys-cys is cysteine, Glu is glutamic acid, Gly is glycine, His is histidine, Ile is isoleucine, Leu is leucine, Lys is lysine, Met is methionine, Phe is phenylalanine, Ser is serine, The is theanine, Thr is threonine, Tyr is tyrosine, Val is valine.

[0075] Figure 2 Total amino acids of different tea types in Camellia ptilophylla populations and Yinghong No. 9.

[0076] Figure 3 Amino acid components in the new shoots and roots of Camellia ptilophylla; (A) 35 amino acid components in fresh leaves of Camellia ptilophylla and other tea resources; (B) Detection chromatogram of theanine in the roots of Camellia ptilophylla A35; (C) Detection chromatogram of theanine in the roots of Camellia oleifera; (D) Detection chromatogram of theanine in the roots of Yinghong No. 9; (E) Theanine standard.

[0077] Figure 4 Amino acid map characteristics of different tissue parts of Camellia ptilophylla No. 1 and Yinghong No. 9; (A) Sampling standards for different tissue parts, 1-9 represent bud, the first leaf, the second leaf, the upper part of the stem, the lower part of the stem, mature leaf, root system, flower bud and fruit respectively; (B) Amino acid determination chromatograms of Camellia ptilophylla No. 1 and Yinghong No. 9, a1-a9, b1-b9 represent different tea tree tissues of Camellia ptilophylla No. 1 and Yinghong No. 9 respectively; the black arrow represents the unknown amino acid in Camellia ptilophylla, and the red arrow indicates theanine in the tea tree.

[0078] Figure 5Amino acid contents in different tissues of three Camellia ptilophylla strains and three tea cultivars in spring; A to S represent the contents of total amino acids (Taa), theanine (The), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), serine (Ser), phenylalanine (Phe), threonine (Thr), lysine (Lys), alanine (Ala), isoleucine (Ile), tyrosine (Tyr), valine (Val), methionine (Met), histidine (His), cysteine (Cys-cys), glycine (Gly), leucine (Leu) and unknown amino acids in different tissues of six cultivars; CTN1 is Camellia ptilophylla No. 1, CTN2 is Camellia ptilophylla No. 2, CTN3 is Camellia ptilophylla No. 3, ZJ is Zijuan, YH9 is Yinghong No. 9, YH12 is Hongyan No. 12; Bud is the bud, First leaf is the first leaf of the new shoot, Second leaf is the second leaf of the new shoot, Mature leaf is the old leaf on the semi-lignified stem, stem1 is the upper section of the tender stem, stem2 is the lower section of the tender stem, Root is the fibrous root.

[0079] Figure 6 Amino acid contents in different tissues of three Camellia ptilophylla strains and three tea cultivars in summer; A to S represent the contents of total amino acids (Taa), theanine (The), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), serine (Ser), phenylalanine (Phe), threonine (Thr), lysine (Lys), alanine (Ala), isoleucine (Ile), tyrosine (Tyr), valine (Val), methionine (Met), histidine (His), cysteine (Cys-cys), glycine (Gly), leucine (Leu) and unknown amino acids in different tissues of six cultivars; CTN1 is Camellia ptilophylla No. 1, CTN2 is Camellia ptilophylla No. 2, CTN3 is Camellia ptilophylla No. 3, ZJ is Zijuan, YH9 is Yinghong No. 9, YH12 is Hongyan No. 12; Bud is the bud, First leaf is the first leaf of the new shoot, Second leaf is the second leaf of the new shoot, Mature leaf is the old leaf on the semi-lignified stem, stem1 is the upper section of the tender stem, stem2 is the lower section of the tender stem, Root is the fibrous root.

[0080] Figure 7Contents of different amino acids in different tissue parts of three Camellia ptilophylla strains and three tea cultivars in autumn; A to S represent the contents of total amino acids (Taa), theanine (The), aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), serine (Ser), phenylalanine (Phe), threonine (Thr), lysine (Lys), alanine (Ala), isoleucine (Ile), tyrosine (Tyr), valine (Val), methionine (Met), histidine (His), cysteine (Cys-cys), glycine (Gly), leucine (Leu) and unknown amino acids in different tissue parts of 6 cultivars; CTN1 is Camellia ptilophylla No. 1, CTN2 is Camellia ptilophylla No. 2, CTN3 is Camellia ptilophylla No. 3, ZJ is Zijuan, YH9 is Yinghong No. 9, YH12 is Hongyan No. 12; Bud is bud, First leaf is the first leaf of the new shoot, Second leaf is the second leaf of the new shoot, Mature leaf is the old leaf on the semi-lignified stem, stem1 is the upper section of the tender stem, stem2 is the lower section of the tender stem, Root is the fibrous root.

[0081] Figure 8 Correlations between total amino acids, theanine, glutamic acid and unknown amino acids in different tea plants pairwise; (A) Correlations between total amino acids, theanine, glutamic acid and unknown amino acids in spring pairwise; (B) Correlations between total amino acids, theanine, glutamic acid and unknown amino acids in summer pairwise; (C) Correlations between total amino acids, theanine, glutamic acid and unknown amino acids in autumn pairwise; Taa is total amino acids, The is theanine, Glu is glutamic acid, Unk is the unknown amino acid in Example 3; CTN1 is Camellia ptilophylla No. 1, CTN2 is Camellia ptilophylla No. 2, CTN3 is Camellia ptilophylla No. 3, ZJ is Zijuan, YH9 is Yinghong No. 9, YH12 is Hongyan No. 12.

[0082] Figure 9 Contents of unknown amino acids in different tea plants; C. ptilophylla H.T.Chang is Camellia ptilophylla, C. sinensis (L.) O.Kuntze is tea, C. sinensis var. assamica (Masters) Kitamura is Assam tea, C. sinensis var. pubilimba Chang is white hair tea, C. assamica var. Kucha is bitter tea, C. sinensis var. assamicacv. Hainan-dayezhong is Hainan large-leaf tea.

[0083] Figure 10 One-dimensional hydrogen spectrum of the derivative of the unknown amino acid in Example 3.

[0084] Figure 11 The biological Cosy spectrum of the unknown amino acid in Example 3.

[0085] Figure 12 The HSQC spectrum of the derivative of the unknown amino acid in Example 3.

[0086] Figure 13 The DEPT spectrum of the derivative of the unknown amino acid in Example 3.

[0087] Figure 14 The carbon spectrum of the derivative of the unknown amino acid in Example 3.

[0088] Figure 15 The HMBC spectrum (1) of the derivative of the unknown amino acid in Example 3.

[0089] Figure 16 The HMBC spectrum (2) of the derivative of the unknown amino acid in Example 3.

[0090] Figure 17 The HMBC spectrum (2) of the derivative of the unknown amino acid in Example 3.

[0091] Figure 18 Mass spectrometry identification of the derivative (DNFB-4-amino-5-hydroxypentanoic acid) of the unknown amino acid in Example 3 and chemical structure verification of 4-amino-5-hydroxypentanoic acid; (A) High-resolution mass spectrometry identification of DNFB-4-amino-5-hydroxypentanoic acid; (B) HPLC analysis of Sample 1 (pure product of DNFB-4-amino-5-hydroxypentanoic acid obtained by separation and purification); (C) HPLC chart of Sample 2 (product obtained by derivatizing 4-amino-5-hydroxypentanoic acid standard with DNFB); (D) HPLC chart of the mixture of Sample 1 and Sample 2 in a ratio of 1:1; E HPLC chart of the mixture of Sample 2 and water in a ratio of 1:1 Detailed implementation mode

[0092] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0093] Tea germplasm resource materials

[0094] The tea germplasm resources adopted are stored in the "Guangdong Tea Germplasm Resource Bank" of the Tea Research Institute of Guangdong Academy of Agricultural Sciences in Yingde City, Guangdong Province.

[0095] 400 tea germplasm resources are involved, all of which are tea and its varieties, including: 300 Camellia ptilophylla H.T.Chang resources, 40 Camellia sinensis (L.) O.Kuntze resources, 29 Camellia sinensis var. assamica (Masters) Kitamura resources, 19 Camellia sinensis var. pubilimba Chang resources, 2 Camellia assamica var. kucha resources, and 10 Camellia.sinensis var. assamica cv. Hainan-dayezhong resources;

[0096] Among them, the Camellia assamica var. kucha resources and the Camellia sinensis var. assamica cv. Hainan-dayezhong resources are both varieties of Camellia sinensis var. assamica. Among the Camellia ptilophylla resources, Camellia ptilophylla No. 1, Camellia ptilophylla No. 2, and Camellia ptilophylla No. 3 are new strains, and the remaining 297 are individual plants with good growth;

[0097] Among the 100 tea tree resources other than Camellia ptilophylla, they are all newly developed tea tree varieties or selected new strains;

[0098] Each of the new varieties or new strains is planted in 3 rows, randomly distributed in the resource nursery, and each row represents a biological replicate;

[0099] The materials of the individual plants of Camellia ptilophylla are first collected uniformly and then evenly divided into three parts, and each part is a biological replicate. All resources are managed according to the same cultivation method.

[0100] Example 1 Analysis of the differences in the flavors and amino acid components of different tea categories between the Camellia ptilophylla population and the Camellia sinensis var. assamica resource Yinghong No. 9

[0101] I. Experimental methods

[0102] 1. Tea trial production

[0103] Using the one-bud two-leaf of Yinghong No. 9 in summer and the Camellia ptilophylla population (300 individual plants) as raw materials, the trial production of black tea, white tea, and green tea was carried out respectively.

[0104] Black tea trial production: The raw materials are withered at 35°C until the water content is 60%, then rolled until the tea leaves are tightly curled and the tea juice can be squeezed out between the fingers. After rolling, the tea leaves are fermented in the fermentation room until the aroma of the tea leaves changes from a green smell to a floral and fruity aroma. The fermented tea leaves are baked at 80°C for 2.5 h.

[0105] Green tea trial production: The raw materials are withered at 35°C until the water content reaches 70%, and then undergo fixation in a stir-frying pan at 220°C. When the water content of the fixed leaves is about 58% and the floral and fruity aroma emerges, the tea leaves are taken out and spread out to cool. Subsequently, the tea leaves are kneaded until they are wet and sticky and tightly coiled into strips. The kneaded tea leaves are baked at 80°C for 2.5 h.

[0106] White tea trial production: The raw materials are withered at 35°C until the water content reaches 30%, dried at 30°C until almost dry, and then dried at 80°C for 2.5 h.

[0107] Preparation of tea soup: Grind the tea leaves into powder, weigh 1 g of the powder into an Erlenmeyer flask, and add 90 ml of boiling water. Place the Erlenmeyer flask in a water bath at 100°C and heat for 45 minutes. After filtering the tea soup to remove the tea dregs, make the volume of the tea soup up to 100 ml.

[0108] 2. Sensory evaluation of tea

[0109] The sensory evaluation of tea is carried out in accordance with "GB / T 23776-2009 Method for Sensory Evaluation of Tea".

[0110] 3. Determination of total free amino acids in tea soup (detected by ninhydrin colorimetry)

[0111] The determination of total amino acids refers to the national standard of the People's Republic of China "GB / T 8314-2013 Determination of Total Free Amino Acids in Tea".

[0112] 4. Determination of dry matter content

[0113] The determination of dry matter content refers to the national standard of the People's Republic of China "GB / T 8303-2013 Preparation of Ground Tea Samples and Determination of Their Dry Matter Content".

[0114] 5. Determination method of amino acid components in tea soup (OPA derivatization method)

[0115] (1) Respectively draw borate buffer solution, ultrapure water, OPA (o-phthalaldehyde, derivatization reagent), FMOC (9-fluorenylmethoxycarbonyl, derivatization reagent), diluent (2 ml aqueous solution added with 10 μl phosphoric acid), ultrapure water, 50% acetonitrile (acetonitrile: water = 1:1) solution into 1.5 mL sample bottles, and place them at positions 61, 62, 63, 64, 65, 66 and 67 on the sample rack of Shimadzu LC-30AD ultra-high performance liquid chromatograph.

[0116] (2) Place 500 μL of the prepared tea soup or amino acid mixed standard (containing 2.5 μmol / ml of aspartic acid, 2.5 μmol / ml of glutamic acid, 2.5 μmol / ml of arginine, 2.5 μmol / ml of serine, 2.5 μmol / ml of phenylalanine, 2.5 μmol / ml of threonine, 2.5 μmol / ml of lysine, 2.5 μmol / ml of alanine, 2.5 μmol / ml of isoleucine, 2.5 μmol / ml of tyrosine, 2.5 μmol / ml of valine, 2.5 μmol / ml of methionine, 2.5 μmol / ml of histidine, 2.5 μmol / ml of glycine, 2.5 μmol / ml of leucine, and 1.25 μmol / ml of cysteine) into a 1.5 mL sample vial, accurately add 50 μL of the internal standard (2.5 μmol / ml of norvaline), shake well, and place it in the sample rack.

[0117] (3) Sample derivation follows the procedure of Shimadzu Corporation; chromatographic column: Durashell-AA special column, 3 μm, 4.6 x 150 mm; column temperature: 50 °C; detection wavelength: 338 nm for primary amino acids and 262 nm for secondary amino acids.

[0118] (4) The sample elution procedure is shown in Table 1.

[0119] Table 1 Amino acid component analysis procedure

[0120]

[0121] (5) Calculate the content of 16 amino acids in the sample based on the concentration and peak area of the amino acid internal standard (norvaline), as well as the peak area and concentration of the 16 amino acid (aspartic acid, glutamic acid, arginine, serine, phenylalanine, threonine, lysine, alanine, isoleucine, tyrosine, valine, methionine, histidine, cysteine, glycine, and leucine) standards.

[0122] II. Experimental results

[0123] Analyze the amino acid components of green tea, white tea, and black tea made from the Camellia ptilophylla population and Yinghong No. 9 using the OPA derivation method. The results Figure 1 are shown as follows. Theanine is the main free amino acid in green tea, white tea, and black tea of Yinghong No. 9, with contents of 11.90 mg / g, 8.90 mg / g, and 6.54 mg / g respectively. The theanine content in Camellia ptilophylla is extremely significantly lower than that of Yinghong No. 9, with theanine contents in its green tea, white tea, and black tea being 0.03 mg / g, 0.05 mg / g, and 0.04 mg / g respectively. In addition to theanine, the other main free amino acids in green tea, white tea, and black tea made from Yinghong No. 9 also include aspartic acid and glutamic acid, and their contents have no obvious difference from those in Camellia ptilophylla.

[0124] The results of the ninhydrin colorimetric method showed that the total amino acid contents of green tea, white tea and black tea made from the Camellia ptilophylla population were 2.42%±0.22%, 2.18%±0.15% and 2.09%±0.09% of the dry matter content respectively, while the total amino acid contents of green tea, white tea and black tea made from Yinghong No. 9 were 2.32%±0.11%, 2.52%±0.29% and 2.32%±0.02% respectively. From the p-value of the significant difference analysis, it can be seen that there is no obvious difference in the total amino acid contents between the green tea, black tea and white tea made from the Camellia ptilophylla population and Yinghong No. 9( Figure 2 ).

[0125] Content of 35 amino acids in the steamed green samples of the new shoots of the Camellia ptilophylla population and 10 other tea group plants in Example 2

[0126] I. Experimental method

[0127] To analyze the amino acid components in Camellia ptilophylla more comprehensively, the new shoots of the Camellia ptilophylla population (a mixture of 300 individual Camellia ptilophylla plants) and 10 other non-Camellia ptilophylla tea group plants were analyzed for 35 amino acid components.

[0128] The 10 non-Camellia ptilophylla tea group plants are: Lingyun No. 2, Daba Baimao No. 1, Kenya No. 7, Yinghong No. 9, Kucha No. 6, Kucha No. 11, Hainan Daye No. 1, Hainan Daye No. 2, Huangjinye and Hongyan No. 12; among them, Lingyun No. 2 and Daba Baimao No. 1 belong to the white tea resources, Kenya Daye No. 7 and Yinghong No. 9 belong to the Assam tea resources, Kucha No. 6, Kucha No. 11, Hainan Daye No. 1 and Hainan Daye No. 2 belong to the Assam species variety, and Huangjinye and Hongyan No. 12 belong to the Chinese species resources.

[0129] The one-bud-two-leaf of 10 tea group plants were collected to make steamed green samples. At the same time, the one-bud-two-leaf of different individual Camellia ptilophylla plants were mixed and analyzed for 35 amino acid components using an automatic amino acid analyzer (Sykam S433D, Sykam Company, Germany).

[0130] The chromatographic conditions used a 4.6mm×60mm column filled with 2622SC cation resin, and a 0.2mol / L sodium citrate buffer solvent system. The external standard method was used to quantify 35 free amino acid components.

[0131] II. Experimental results

[0132] The results are as Figure 3As shown in the figure, 14 kinds of amino acids were detected in the new shoots of the Camellia ptilophylla population. The dry matter contents from high to low were glutamic acid (2.92 mg / g), aspartic acid (0.55 mg / g), β - aminoisobutyric acid (0.46 mg / g), theanine (0.17 mg / g), arginine (0.05 mg / g), phosphoserine (0.03 mg / g), serine (0.03 mg / g), alanine (0.03 mg / g), tryptophan (0.02 mg / g), threonine (0.02 mg / g), valine (0.02 mg / g), leucine (0.02 mg / g), β - alanine (0.01 mg / g), and γ - aminobutyric acid (0.01 mg / g).

[0133] Among the 35 amino acid substances, hydroxyproline, 3 - methylhistidine, 1 - methylhistidine, carnosine, and α - aminoadipic acid were not detected in the Camellia ptilophylla population and 10 tea section varieties (lines).

[0134] Citrulline, asparagine, α - aminobutyric acid, phosphoethanolamine, lysine, taurine, proline, glycine, cystine, methionine, isoleucine, tyrosine, phenylalanine, histidine, and ornithine were detected in some resources of the 10 tea section plants, but not in Camellia ptilophylla.

[0135] β - alanine was only detected in the Camellia ptilophylla population and not in the other 10 tea section plants. The content of β - aminoisobutyric acid in Camellia ptilophylla (0.46 mg / g) was significantly higher than that in the other 10 tea section plants (0 - 0.02 mg / g). The contents of aspartic acid, threonine, serine, and theanine in Camellia ptilophylla were significantly lower than those in the other tea section plants. Especially for the content of theanine, the theanine content in Camellia ptilophylla was only 0.18 mg / g, and the theanine contents of the 10 other tea section resources were 63 - 168 times that of Camellia ptilophylla ( Figure 3 ).

[0136] Example 3 Atlas Characteristics of Amino Acid Components in Different Tissue Parts and Distribution of Unknown Amino Acids in Tissues

[0137] I. Experimental Method

[0138] Using the OPA derivation method, 3 new strains of Camellia ptilophylla (Camellia ptilophylla No. 1, Camellia ptilophylla No. 2, and Camellia ptilophylla No. 3) and 3 cultivated tea tree varieties (Zijuan, Yinghong No. 9, and Hongyan 12) were selected in three time periods of spring, summer, and autumn to carry out the determination of amino acid components in different tissue parts (fibrous roots, lower part of young stems, upper part of young stems, old leaves on semi - lignified stems, the second leaf of new shoots, the first leaf of new shoots, and buds); at the same time, in summer, the fruits and flower buds of 6 varieties (lines) were selected to determine their amino acid components using the OPA derivation method. The sampling standards are as Figure 4As shown in Figure A. The specific method of the OPA derivatization method for determining amino acid composition is the same as that in Example 1.

[0139] II. Experimental Results

[0140] There are differences in the spectra of 3 new strains of Camellia ptilophylla and 3 cultivated tea tree varieties. From the detection spectra, it can be seen that the fibrous root spectrum of Camellia ptilophylla No. 1 is similar to that of the cultivated variety Yinghong No. 9, while there are significant differences in the amino acid spectra of Camellia ptilophylla and Yinghong No. 9 in buds, the first leaf, the second leaf, old leaves, the upper section of young stems, fruits and flower buds ( Figure 4 Figure B). From the amino acid detection spectra of 9 tissue parts of Yinghong No. 9, it can be seen that in the cultivated tea tree varieties, theanine is the main free amino acid in the tea tree body, and its peak time is about 10.4 minutes. There is no obvious peak at the 10.4 peak in the amino acid detection peak map of the above-ground tissues of Camellia ptilophylla, while there is a relatively obvious peak at 7.6 minutes. The peak time of this peak is different from that of the standard products of 16 common amino acids in tea leaves. This peak is the product of the reaction with the derivatization reagent OPA (o-phthalaldehyde). It can be seen that this substance may be an uncommon amino acid or a new amino acid (unknown amino acid), and it is specifically present in Camellia ptilophylla.

[0141] By measuring the amino acid composition of different tissue parts in spring ( Figure 5 ), summer ( Figure 6 ), and autumn ( Figure 7 ) and analyzing the main free amino acids, it was found that theanine ( Figure 5 Figure B is for spring, Figure 6 Figure B is for summer, Figure 7 Figure B is for autumn), aspartic acid ( Figure 5 Figure C is for spring, Figure 6 Figure C is for summer, Figure 7 Figure C is for autumn) and glutamic acid ( Figure 5 Figure D is for spring, Figure 6 Figure D is for summer, Figure 7 Figure D is for autumn) are the main free amino acids in the 3 cultivated varieties, while the most abundant in Camellia ptilophylla is the unknown amino acid ( Figure 5 Figure S is for spring, Figure 6 Figure S is for summer, Figure 7 Figure S is for autumn), glutamic acid ( Figure 5 Figure D is for spring, Figure 6 Figure D is for summer, Figure 7 Figure D is for autumn) and aspartic acid ( Figure 5 Figure C is for spring, Figure 6 Figure C is for summer, Figure 7 Figure C is for autumn).

[0142] This unknown amino acid was found to be specifically present in Camellia ptilophylla and was not detected in different tissues of three developed tea tree varieties. Trace amounts of the unknown amino acid of Example 3 were detected in the roots of Camellia ptilophylla, and relatively more of this unknown amino acid was contained in the new shoots. Among them, the content of this unknown amino acid was the highest in the second leaves of Camellia ptilophylla No. 1 and Camellia ptilophylla No. 3 in autumn, and the content of this unknown amino acid was the highest in the old leaves of Camellia ptilophylla No. 2( Figure 5 S represents spring, Figure 6 S represents summer, Figure 7 S represents autumn).

[0143] Correlation analysis of the unknown amino acid in Example 4 with total amino acids, theanine, and glutamic acid

[0144] I. Experimental method

[0145] The detection method and detection data are the same as those in Example 3.

[0146] II. Experimental results

[0147] Since theanine and glutamic acid are related in the metabolic pathway and there may be a relationship between the unknown amino acid and theanine, the correlation among the three was compared emphatically.

[0148] The results showed that there were differences in the changes of the total amino acid content and amino acid components in different tissues of different tea tree varieties in different seasons( Figure 5 represents spring, Figure 6 represents summer, Figure 7 represents autumn). Based on the amino acid components in different varieties and tissues in spring, summer, and autumn, correlation analysis was carried out pairwise between the total amino acids and the main free amino acids (theanine, glutamic acid, and the unknown amino acid in Example 3). The results showed that there was a certain correlation among the total amino acids, theanine, the unknown amino acid in Example 3, and glutamic acid in the three seasons. The correlation between theanine and the total amino acids, and theanine and the unknown amino acid in Example 3 was relatively high in all three quarters( Figure 8 ), and the correlation coefficients between theanine and the total amino acids in the three quarters were 0.68, 0.47, and 0.84 respectively, and the correlation coefficients between theanine and the unknown amino acid in Example 3 in the three quarters were -0.53, -0.4, and -0.5 respectively. The correlation between theanine and glutamic acid in spring and summer was relatively low, with correlations of -0.17 and -0.1( Figure 8 A, Figure 8 B), and the correlation in autumn was relatively high, with a correlation of -0.4( Figure 8C). The correlation between the unknown amino acid in Example 3 and glutamic acid is relatively low in spring and summer, with the correlation coefficients being 0.1 and 0.2 respectively, and relatively high in autumn, with the correlation coefficient being 0.77. The correlation between the unknown amino acid in Example 3 and the total amount of amino acids in spring, summer and autumn is -0.29, 0.08 and -0.30 respectively.

[0149] Therefore, the unknown amino acid in Example 3 is an amino acid with a high negative correlation with theanine.

[0150] Isolation and Identification of the Unknown Amino Acid in Example 3 in Camellia ptilophylla

[0151] I. Isolation of the Unknown Amino Acid in Example 3 in Camellia ptilophylla

[0152] The contents of the unknown amino acid in Example 3 and theanine in 300 individual plants of Camellia ptilophylla and 100 other tea group plants were determined by the OPA derivatization method in Example 1. For the separation and extraction of amino acids, the steamed green sample extract of Camellia ptilophylla needs to be derivatized with DNFB (2,4-dinitrofluorobenzene). Two individual plants of Camellia ptilophylla with high unknown amino acid content and two with low unknown amino acid content were selected for amino acid detection by the DNFB (2,4-dinitrofluorobenzene) derivatization method. By comparing the peak shapes, it was determined that the elution time of the unknown amino acid in Example 3 in the DNFB (2,4-dinitrofluorobenzene) derivatization method was 42 minutes.

[0153] 1. DNFB Large-scale Derivatization of Amino Acids

[0154] (1) Experimental Method

[0155] The steamed green sample of the Camellia ptilophylla population was extracted at a ratio of 1 kg of steamed green sample to 4 L of water at 100 °C for 2 hours. After sample extraction, it was preliminarily filtered through two layers of gauze to remove tea residues to obtain the primary extract; the primary extract was centrifuged at 3500 rpm to collect the supernatant as the tea soup.

[0156] The DNFB derivatization method (large-scale derivatization method) for amino acid separation and extraction was as follows: Dilute 8 L of the prepared tea soup by 5 times, measure 300 ml of the tea soup into a 1 L beaker, and simultaneously add 300 ml of a 10 mL / L DNFB acetonitrile solution and 30 ml of a 0.5 M pH 9.0 NaHCO3 buffer solution. After mixing, it was derivatized in a 60 °C water bath in the dark for 1 h to obtain the derivatized product. The tea soup was derivatized through multiple reactions, and all derivatized products were concentrated to 20 L (the product after large-scale DNFB derivatization of the tea soup) for subsequent separation and purification.

[0157] The product obtained by extensive derivatization of the tea soup with DNFB was analyzed by HPLC. The elution peak at 42 minutes was observed to determine whether the unknown amino acid in Example 3 existed in the product of extensive derivatization of the tea soup with DNFB (2,4-dinitrofluorobenzene). The specific HPLC analysis method was as follows:

[0158] Liquid chromatograph (Agilent, Agilent 1200), chromatographic column (Eclipse Plus C18, 250 mm × 4.6 mm, 5 μm).

[0159] The mobile phase A for liquid chromatographic analysis was 0.05 M NaAc buffer solution (pH 6.5, containing 10 mL / L N,N-dimethylformamide), and the mobile phase B was an aqueous acetonitrile solution (V / V = 1:1).

[0160] The flow rate of the mobile phase was 1.0 mL / min, and the gradient elution program was as shown in Table 2.

[0161] The column temperature of the chromatographic column was 28 °C; the sample injection volume was 5 μL; the detection wavelength of the instrument was 360 nm.

[0162] Table 2 Elution program for HPLC analysis of amino acids extensively derivatized with DNFB

[0163]

[0164] (2) Experimental results

[0165] By detecting the derivatized product using HPLC, it was found that there was an obvious peak at 42 minutes, indicating successful derivatization.

[0166] 2. First purification

[0167] Take 20 L of the product obtained by extensive derivatization of the tea soup with DNFB, adjust the pH to about 3.0 with phosphoric acid, remove the organic solvent by rotary evaporation, and then load it onto a 100 × 500 mm HP-20 macroporous resin column. Wash it successively with 10% (V / V) acetonitrile-aqueous solution, 30% (V / V) acetonitrile-aqueous solution, 50% (V / V) acetonitrile-aqueous solution, 70% (V / V) acetonitrile-aqueous solution, and 90% (V / V) acetonitrile-aqueous solution for 30 minutes each. Detect the effluent by HPLC and collect the effluent of the target peak at 42 minutes.

[0168] The collected effluent containing the target peak was rotary evaporated to dryness, dissolved in 2 L of 10% (V / V) acetonitrile-aqueous solution, and filtered to remove the filter residue to obtain a crude separation and extraction solution. The crude separation and extraction solution was further purified by preparative liquid chromatography (Hanbang high-pressure preparative chromatography DAC100).

[0169] 3. Second purification

[0170] The preparative chromatographic conditions for secondary purification are as follows: chromatographic column (DAC chromatographic column, 100*250nm, 10um), packing material (YMCC18), wavelength of 360nm, and flow rate of 80ml / min.

[0171] The mobile phase solution system for secondary purification is as follows: mobile phase A is acetonitrile, mobile phase B is an aqueous solution of 0.1% acetic acid. By volume, the gradient elution program is: 0min - 10% A, 40min - 10% A, 100min - 15% A, 140min - 20% A, 170min - 25% A, 195min - 25% A, 200min - 90% A, 220min - 90% A.

[0172] Collecting solutions were detected by HPLC, and the collecting solutions with the purity of the unknown amino acid in Example 3 greater than 90% were combined.

[0173] 4. Tertiary purification

[0174] The collecting solutions obtained after secondary purification were combined, diluted by a factor of two with pure water, and then pumped for sample loading for tertiary purification:

[0175] The chromatographic conditions and mobile phase solution system for tertiary purification are the same as those for secondary purification.

[0176] By volume, the gradient elution program is: 0min - 15% A, 5min - 15% A, 15min - 30% A, 25min - 30% A, 40min - 50% A.

[0177] Collecting solutions were detected by HPLC, and the collecting solutions with the purity of the unknown amino acid greater than 95% were combined.

[0178] The collecting solutions were rotary evaporated to remove acetonitrile and then freeze-dried to obtain the target substance.

[0179] 5. NMR detection of the target substance (unknown amino acid derivative in Example 3)

[0180] (1) Experimental method

[0181] The target substance (unknown amino acid derivative in Example 3) was analyzed by hydrogen spectrum, COSY spectrum, HSQC spectrum, DEPT spectrum, carbon spectrum, and HMBC spectrum using 600MHz nuclear magnetic resonance. According to 1 the 1H-NMR spectrum, combined with the results of the COSY spectrum, HSQC spectrum, and DEPT spectrum, the 1 1H spectrum of the compound was assigned.

[0182] The target substance (the unknown amino acid derivative in Example 3) was dissolved in an acetonitrile solution and subjected to GC-MS / MS detection on an ultra-high resolution liquid chromatography-mass spectrometer, and the positive ion mode of its first-order mass spectrum was analyzed.

[0183] (2) Experimental results

[0184] The unknown amino acids in 300 individual plants of Camellia ptilophylla were detected by the OPA method. The results showed that the unknown amino acids in Example 3 were detected in all 300 Camellia ptilophylla resources, and were not detected in the other 100 resources (partial results are shown in Figure 9 ). Among them, the peak areas of the unknown amino acids in Example 3 of three individual plants M260, M386 and M420 in the Camellia ptilophylla population were relatively large, and their peak areas were 314.1, 281.3, and 298.2 respectively. The peak areas of the unknown amino acids in Example 3 of three individual plants M314, M338 and M598 were relatively small, and their peak areas were 24.80, 28.70 and 18.60 respectively. The detection results showed that: the unknown amino acids in Example 3 existed in all 300 Camellia ptilophylla, and did not exist in the 100 other tea group plants. The contents of the unknown amino acids in Example 3 in different individual plants of Camellia ptilophylla were different. The elution positions of the unknown amino acids in Example 3 in the DNFB derivatization method could be determined by three individual plants of Camellia ptilophylla with high and three with low contents of the unknown amino acids in Example 3.

[0185] The hydrogen spectrum of the unknown amino acid derivative in Example 3 is as shown in Figure 10 , the COSY spectrum is as shown in Figure 11 , the HSQC spectrum is as shown in Figure 12 , the DEPT spectrum is as shown in Figure 13 , the carbon spectrum is as shown in Figure 14 , and the HMBC spectrum is as shown in Figure 15 , Figure 16 and Figure 17 .

[0186] The hydrogen spectrum shows 10 sets of hydrogens, and the integral ratios of hydrogens from low field to high field are 1:1:1:1:1:1:1:2:2:2 respectively, corresponding to 13 protons in the compound molecule. The hydrogen at δ12.150 is a broad singlet with 1 proton. The COSY spectrum shows no hydrogen-proton correlation, and the HSQC and DEPT spectra show no carbon correlation for this hydrogen, which is assigned to COOH-11; the hydrogen at δ8.860 is a doublet with 1 proton. The COSY spectrum shows no hydrogen-proton correlation, and the HSQC and DEPT spectra show that this hydrogen is correlated with a methine carbon (δ124.19), which is assigned to H-3; the hydrogen at δ8.695 is a doublet with 1 proton. The COSY spectrum shows a correlation with the hydrogen at δ3.972, and the HSQC and DEPT spectra show no carbon correlation for this hydrogen, which is assigned to H-7; the hydrogen at δ8.238 is a dd peak with 1 proton. The COSY spectrum shows a correlation with the hydrogen at δ7.343, and the HSQC and DEPT spectra show that this hydrogen is correlated with a methine carbon (δ130.38), which is assigned to H-5; the hydrogen at δ7.343 is a doublet with 1 proton. The COSY spectrum shows a correlation with the hydrogen at δ8.238, and the HSQC and DEPT spectra show that this hydrogen is correlated with a methine carbon (δ116.19), which is assigned to H-6; the hydrogen at δ5.152 is a broad singlet with 1 proton. The COSY spectrum shows a correlation with the hydrogen at δ3.592, and the HSQC and DEPT spectra show no carbon correlation for this hydrogen, which is assigned to OH-12; the hydrogen at δ3.972 is a multiplet with 1 proton. The COSY spectrum shows correlations with the hydrogens at δ8.695, δ3.593, and δ1.872, and the HSQC and DEPT spectra show that this hydrogen is correlated with a methine carbon (δ54.43), which is assigned to H-8; the hydrogen at δ3.592 is a multiplet with 2 protons. The COSY spectrum shows correlations with the hydrogens at δ3.972 and δ5.152, and the HSQC and DEPT spectra show that this hydrogen is correlated with a methylene carbon (δ62.38), which is assigned to H-12; the hydrogen at δ2.352 is a triplet with 2 protons. The COSY spectrum shows a correlation with the hydrogen at δ1.872, and the HSQC and DEPT spectra show that this hydrogen is correlated with a methylene carbon (δ30.50), which is assigned to H-10; the hydrogen at δ1.872 is a multiplet with 2 protons. The COSY spectrum shows correlations with the hydrogens at δ2.352 and δ3.972, and the HSQC and DEPT spectra show that this hydrogen is correlated with a methylene carbon (δ26.40), which is assigned to H-9.

[0187] According to 13 the C-NMR spectrum, combined with the HSQC spectrum, DEPT spectrum, and HMBC spectrum, the 13 C spectrum of the compound can be assigned. The DEPT spectrum and two-dimensional spectra assist in the analysis of the carbon spectrum as follows: There are 11 effective peaks in total, including 3 secondary carbon peaks, 4 tertiary carbon peaks, and 4 quaternary carbon peaks.

[0188] δ26.40, δ30.50, and δ62.38 are secondary carbon signals; the HSQC spectrum shows that δ26.40 is correlated with the hydrogen proton at δ1.872, and the HMBC spectrum ( Figure 15 ) shows correlation with the hydrogen protons at δ8.695, δ3.972, δ3.592, and δ2.352. Therefore, it is assigned to C-9. The HSQC spectrum shows that δ30.50 is correlated with the hydrogen proton at δ2.352, and the HMBC spectrum ( Figure 15 ) shows correlation with the hydrogen protons at δ3.972 and δ1.872. Therefore, it is assigned to C-10. The HSQC spectrum shows that δ62.38 is correlated with the hydrogen proton at δ3.592, and the HMBC spectrum ( Figure 15 ) shows correlation with the hydrogen protons at δ8.695, δ3.972, and δ1.872. Therefore, it is assigned to C-12.

[0189] δ54.43, δ116.19, δ124.19, and δ130.38 are four tertiary carbon signals. The HSQC spectrum shows that δ54.43 is correlated with the hydrogen proton at δ3.972, and the HMBC spectrum ( Figure 16 ) shows correlation with the hydrogen protons at δ8.695, δ3.592, δ2.352, and δ1.872. Therefore, it is assigned to C-8. The HSQC spectrum shows that δ116.19 is correlated with the hydrogen proton at δ7.343, and the HMBC spectrum ( Figure 16 ) shows long-range correlation with δ8.695. Therefore, it is assigned to C-6 on the benzene ring. The HSQC spectrum shows that δ124.19 is correlated with the hydrogen proton at δ8.860, and the HMBC spectrum ( Figure 16 ) shows long-range correlation with δ8.238 and δ7.343. Therefore, it is assigned to C-3 on the benzene ring.

[0190] The HSQC spectrum shows that δ130.38 is correlated with the hydrogen proton at δ8.238, and the HMBC spectrum ( Figure 17 ) shows long-range correlation with δ8.860 and δ7.343. Therefore, it is assigned to C-5 on the benzene ring.

[0191] δ130.25, δ135.23, δ148.76, and δ174.68 are four quaternary carbon signals. The HMBC spectrum ( Figure 17 ) shows that δ130.25 has long-range correlation with δ8.860, δ8.695, and δ7.343. Therefore, it is assigned to C-2 on the benzene ring. The HMBC spectrum ( Figure 17 ) shows that δ135.23 has long-range correlation with δ8.860, δ8.238, and δ7.343. Therefore, it is assigned to C-4 on the benzene ring. The HMBC spectrum ( Figure 17 ) shows that δ148.76 has long-range correlation with δ8.860, δ8.238, δ7.343, and δ3.972. Therefore, it is assigned to C-1 on the benzene ring. The HMBC spectrum ( Figure 17) It shows that δ174.68 is remotely correlated with δ2.352 and δ1.872, so it is attributed to C-11.

[0192] Therefore, the structural formula of the unknown amino acid is as shown in formula (I)

[0193]

[0194] 6. Mass spectrometry analysis of the target substance (unknown amino acid derivative of Example 3)

[0195] (1) Experimental method

[0196] The obtained target substance (unknown amino acid derivative of Example 3) was analyzed on an ultra-high resolution liquid-mass spectrometer. The mass spectrometry conditions are as follows: chromatographic column (ACQUITY UPLC BEH C18, 2.1x100 mm, 1.7 μm).

[0197] The mobile phase solution system is as follows: mobile phase A is 0.1% formic acid, and mobile phase B is acetonitrile.

[0198] The elution program is shown in Table 3: 0 min - 90% A, 10 min - 5% A, 15 min - 5% A, 15.1 min - 90% A, 20 min - 90% A, and the flow rate is 0.3 mL / min.

[0199] The ionized secondary fragments were analyzed by Mass Frontier software.

[0200] Table 3 High-resolution mass spectrometry analysis elution system of the unknown amino acid derivative of Example 3:

[0201]

[0202] (2) Experimental results

[0203] The results of the primary mass spectrometry detection showed that the molecular weight of the unknown amino acid derivative of Example 3 was 300.1. Through the chemical reaction formula of the amino acid and the DNFB derivative reagent, the chemical formula of the DNFB derivative reagent, the chemical structural formula obtained by nuclear magnetic resonance analysis, and the molecular weight identified by the primary mass spectrometry, it was finally confirmed that the unknown amino acid of Example 3 was 4-amino-5-hydroxypentanoic acid ( Figure 18 A).

[0204] 7. Verification of the structural formula of 4-amino-5-hydroxypentanoic acid

[0205] (1) Experimental method

[0206] Use the pure product of the derivative of the unknown amino acid in Example 3 obtained from the previous step of separation and purification (i.e., the DNFB derivative of 4-amino-5-hydroxypentanoic acid, denoted as DNFB-4-amino-5-hydroxypentanoic acid) as Sample 1; chemically synthesize a standard of 4-amino-5-hydroxypentanoic acid, and perform DNFB derivatization according to the small-scale derivatization method. The derivatized product is used as Sample 2; Samples 1 and 2 are separately subjected to HPLC analysis.

[0207] In addition, mix the solutions of Samples 1 and 2 in equal volumes as Sample 3, and then perform HPLC detection.

[0208] Mix Sample 2 and water in equal volumes as Sample 4, and then perform HPLC analysis.

[0209] The HPLC analysis method is the same as the DNFB derivatized product analysis conditions in Example 5.

[0210] Among them, the specific method of the small-scale derivatization method is as follows: Take a 25 mL volumetric flask, add 2.0 mL of tea soup, and at the same time take 2.0 mL of a 10 mL / L acetonitrile solution of DNFB, and then add 2 mL of a 0.5 M NaHCO3 buffer solution with pH 9.0. Derivatize in the dark in a 60 °C water bath for 1 h. After cooling, dilute to volume with a 0.01 M KH2PO4 buffer solution with pH 7.0, shake well, filter through a 0.45 μm filter head, and perform HPLC analysis on the machine.

[0211] The specific method of HPLC analysis is as follows:

[0212] Liquid chromatograph (Agilent, Agilent 1200), chromatographic column (Eclipse Plus C18, 250 mm × 4.6 mm, 5 μm).

[0213] The mobile phase A for liquid chromatographic analysis is a 0.05 M NaAc buffer solution (pH 6.5, containing 10 mL / L N,N-dimethylformamide), and the mobile phase B is an acetonitrile aqueous solution (V / V = 1:1).

[0214] The flow rate of the mobile phase is 1.0 mL / min, and the gradient elution program is shown in Table 2.

[0215] The column temperature of the chromatographic column is 28 °C; the sample injection volume is 5 μL; the instrument detection wavelength is 360 nm.

[0216] (2) Experimental results

[0217] It was found that the peak emergence times of Sample 1 and Sample 2 were the same ( Figure 18 B and Figure 18 C), and they both emerged at about 42 minutes.

[0218] In addition, the solutions of Sample 1 and Sample 2 were mixed in equal volumes and then subjected to HPLC detection. The mixed sample showed a single peak at 42 minutes, and its peak area was close to the average of Sample 1 and Sample 2 ( Figure 18 D).

[0219] Sample 2 and water were mixed in equal volumes and then subjected to HPLC analysis. The peak area at 42 minutes was about half of that in Sample 2 ( Figure 18 E).

[0220] Through Figure 18 the experiments from B to Figure 18 E, the accuracy of nuclear magnetic resonance and mass spectrometry analysis for the structural identification of 4-amino-5-hydroxypentanoic acid was proven.

[0221] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. For those of ordinary skill in the art, based on the above description and ideas, other different forms of changes or modifications can be made. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of a compound in the identification of the variety of Camellia ptilophylla, characterized in that, The structural formula of the compound is shown in formula (I).

2. A method for identifying Camellia ptilophylla, characterized in that, Detect the compound with the structural formula shown in formula (I) in the sample to be tested. The sample to be tested is Camellia ptilophylla.

3. The method according to claim 2, wherein Perform HPLC analysis on the DNFB derivatization product of the aqueous extract of the sample to be tested.

4. The method according to claim 3, wherein The chromatographic column is Ecllipse Plus C18, and the mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is an NaAc buffer solution containing N,N-dimethylformamide, and mobile phase B is an aqueous acetonitrile solution; gradient elution is carried out. Use of the detection reagent for the compound with the structural formula shown in formula (I) in the preparation of a kit for identifying Camellia ptilophylla.

6. The application according to claim 5, characterized in that, The detection reagent is a derivatization reagent and / or an HPLC analysis reagent.

7. The application according to claim 6, wherein The derivatization reagent is an acetonitrile solution of DNFB and a NaHCO3 buffer solution.

8. The application according to claim 7, wherein The HPLC analysis reagent contains a mobile phase, and the mobile phase includes mobile phase A and mobile phase B. Among them, mobile phase A is an NaAc buffer solution containing N,N-dimethylformamide, and mobile phase B is an aqueous acetonitrile solution.

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