A quality detection method for goat milk leaf medicinal material

Abstract

The present invention discloses a quality detection method for goat milk leaf medicinal materials, including identification and / or content determination; the identification of goat milk leaf medicinal materials is a thin layer chromatography identification method; the content determination is to determine the content of silver forging glycosides in goat milk leaf medicinal materials by high performance liquid chromatography. The thin layer identification method of goat milk leaf medicinal materials adopted in the present application has the characteristics of high sensitivity, strong specificity and good separation effect, is simple and feasible, has strong reproducibility, and can accurately identify the authenticity of medicinal materials; the method for determining the content of silver forging glycosides in goat milk leaf medicinal materials adopted in the present application can accurately and quickly determine the content of goat milk leaf medicinal materials, and provide effective qualitative and quantitative analysis means for goat milk leaf medicinal materials, so as to facilitate the quality control of goat milk leaf medicinal materials. The quality detection method of goat milk leaf medicinal materials provided by the present invention can effectively control the overall quality of goat milk leaf medicinal materials and ensure the safety and effectiveness of goat milk leaf medicinal materials in clinical use.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis, in particular to a quality detection method for a goat milk leaf medicinal material. Background Art

[0002] Sheep milk leaf (Elaeagnus oleraceus leaf): named according to the "Quality Standards for Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province".

[0003] [Origin] This product is the dried leaves of Elaeagnus pungens Thunb. or Elaeagnus henryi Warb. of the Elaeagnaceae family. It is harvested in summer and sun-dried.

[0004] Regarding the origin of goat milk leaf, the existing standards include the Chinese Pharmacopoeia (1977), Shanghai Chinese Medicinal Materials Standard, Guangdong Chinese Medicinal Materials Standard, Hubei Chinese Medicinal Materials Standard, Hunan Chinese Medicinal Materials Standard, Guizhou Chinese Medicinal Materials and Ethnic Medicinal Materials Quality Standard, Yunnan Chinese Medicinal Materials Standard (Yi Medicine) and Fujian Chinese Medicinal Materials Standard, which contain three medicinal materials varieties of "goat milk leaf / Elaeagnus leaf", "white green leaf" and "Elaeagnus angustifolia leaf" for medicinal use, involving six origin plants (Elaeagnus pungens, Yichang Elaeagnus henryi, E. glabra, Fujian Elaeagnus oldhami, green leaf Elaeagnus viridis, Elaeagnus angustifolia), with E. pungens being the most widely used. According to field investigations and literature records, there are also many varieties of milk seeds such as E. umbellata Thunb. used as "goat milk leaf" in Guizhou (Miao) and Hubei (Tujia).

[0005] The component analysis (component composition and content) of a total of 7 species of Elaeagnus showed that the distribution area of ​​Fujian Elaeagnus was small and its composition was quite different from that of Elaeagnus leaves; the component compositions of Elaeagnus and Yichang Elaeagnus were relatively similar; the component compositions of the other 5 species of Elaeagnus were quite different from those of Elaeagnus and Yichang Elaeagnus. After consulting the literature and conducting field investigations, it was found that Elaeagnus sibiricum and Elaeagnus yichangensis were widely distributed, rich in resources, and included in the standard. Elaeagnus fujianensis was only distributed in Fujian and Guangdong, and was mainly used by the people in Fujian. Green leaf Elaeagnus sibiricum (distributed in Shaanxi and Hubei) was a common medicinal material used by the Yi people. Elaeagnus angustifolia (distributed in the west and North China, etc.) was a common medicinal material used by the Uygur people. Other species, except for Elaeagnus ramblingensis, had no standard inclusion. Although Elaeagnus ramblingensis was also widely distributed and included in the standard, its composition was quite different from that of Elaeagnus sibiricum and Elaeagnus yichangensis. Elaeagnus badongensis (widely distributed), milk fruit (widely distributed), and long-cleft Elaeagnus sibiricum (only distributed in Guizhou) had no standard inclusion. With reference to the existing standard inclusion, after comprehensive consideration, it was determined that the origin of "E. pungens Thunb. / Elaeagnus sibiricum leaf" was Elaeagnus pungens Thunb. and Elaeagnus henryi Warb.

[0006] According to the "Guizhou Province Traditional Chinese Medicine and Ethnic Medicine Quality Standards", the harvesting period is determined as "summer harvesting".

[0007] [Properties] The properties are specified in the Chinese Pharmacopoeia (1977) and the local standards of Hunan, Shanghai and Guizhou. According to the observation of samples, there are certain differences between the medicinal materials (leaves) of Elaeagnus oleraceus and Elaeagnus yichangensis, so the original standard was revised to specify the properties of the two species respectively.

[0008] The leaves of Goat Milk mainly contain flavonoids, organic acids and saponins: the flavonoid components are mainly derivatives of three flavonoid aglycones: kaempferol, quercetin and isorhamnetin. They mainly include: kaempferol, quercetin, isorhamnetin, argentin, rutin, white ramie glycosides, etc.

[0009] The Quality Standards for Traditional Chinese and Ethnic Chinese Medicines in Guizhou Province, the Quality Standards for Traditional Chinese Medicines in Guangdong Province, and the Quality Standards for Traditional Chinese Medicines in Hunan Province stipulate that reference medicinal materials should be used as reference substances for TLC identification, while the Quality Standards for Traditional Chinese Medicines in Hubei Province stipulates that oleanolic acid should be used as reference substance for TLC identification. The original Hubei Provincial Standard stipulates that oleanolic acid should be used as reference substance for TLC identification, but since this component is a common component of many medicinal materials, its specificity is poor.

[0010] The leaves of many plants of the genus Elaeagnus are used medicinally by the Han, Miao, Tujia, Yi, and Uygur ethnic groups, and there are also finished medicines using these medicinal materials in prescriptions that have been put on the market. However, regarding the quality standards of its medicinal materials, the above-mentioned relevant standards only have provisions for [identification] (properties, powder, thin layer of control medicinal materials) and [inspection], and the standards are still imperfect, which also restricts the quality control of its finished preparations. Therefore, it is urgent to develop a quality detection method for goat milk leaf medicinal materials to effectively control the overall quality of goat milk leaf medicinal materials and ensure the safety and effectiveness of goat milk leaf medicinal materials in clinical use. Summary of the invention

[0011] The purpose of the present invention is to provide a quality detection method for goat milk leaf medicinal material. The present invention has the characteristics of being able to effectively control the overall quality of goat milk leaf medicinal material and ensure the safety and effectiveness of goat milk leaf medicinal material in clinical use.

[0012] The technical solution of the present invention is a quality detection method for goat milk leaf medicinal material, including identification and / or content determination items; wherein the identification of goat milk leaf medicinal material is carried out by a thin layer chromatography identification method; and the content determination is carried out by using a high performance liquid chromatography method to determine the content of arginine in the goat milk leaf medicinal material.

[0013] In the aforementioned quality detection method of goat milk leaf medicinal material, the thin layer chromatography identification method comprises the following steps:

[0014] (1) Weigh 2g of the powder of this product, add 10 times the amount of water and boil for 30 minutes, filter, evaporate the filtrate on a water bath, add 0.5ml of anhydrous ethanol to dissolve the residue, and use it as the test solution; take 2g of the control medicinal material of goat milk leaf, and prepare the control medicinal material solution in the same way;

[0015] (2) Thin layer chromatography test

[0016] Pipette 5 μl of each of the above two solutions and place them on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as the adhesive, use a mixture of ethyl acetate: water: formic acid = 57:10:8.2 as the developing agent, develop, take out, dry, and examine under a 365nm ultraviolet lamp to obtain the test sample chromatogram.

[0017] In the aforementioned quality detection method for the medicinal material of goat milk leaf, in the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions in the chromatogram of the control medicinal material.

[0018] In the aforementioned quality detection method of goat milk leaf medicinal material, the method for determining the content of arginine in the goat milk leaf medicinal material is determined by high performance liquid chromatography, comprising the following steps:

[0019] 1) Chromatographic conditions of liquid chromatograph

[0020] The chromatographic column is inertsustain C18, with octadecylsilane bonded silica gel as the filler; acetonitrile and 0.4% formic acid water are used as the mobile phase, the elution time is 0.01 to 25 minutes, 25% to 29% acetonitrile is used for isocratic elution, the column temperature is 30°C, the flow rate is 1.0 ml / min, and the detection wavelength is 314 nm to determine argentin;

[0021] 2) Preparation of the reference solution for content determination: Accurately weigh an appropriate amount of the silver forging glycoside reference substance and dissolve it in methanol to make a solution containing

[0022] 0.22392 mg of assay reference solution;

[0023] 3) Preparation of test solution for content determination: accurately weigh 1.0 g of the powder of the product, place it in a stoppered conical flask, add 30 ml of 75% methanol, weigh it, reflux it in a water bath for 1.5 hours, cool it quickly, weigh it, make up the weight loss with 75% methanol, shake it well, filter it and take the filtrate to obtain the test solution for content determination;

[0024] 4) Determination method: Accurately pipette 10 μl of the above-mentioned reference substance and test substance solutions, inject into liquid chromatography, and determine the content of arginine in goat milk leaf medicinal material to obtain.

[0025] In the aforementioned quality inspection method for goat milk leaf medicinal material, the goat milk leaf medicinal material, calculated on the basis of dry product, shall contain not less than 0.16% arginine C30H26O13.

[0026] In the above-mentioned quality detection method of goat milk leaf medicinal material, in the chromatographic conditions of step 1) the liquid chromatograph, the theoretical plate number calculated based on arginine should be no less than 7500, and calculated based on the peaks of kaempferol, quercetin and isorhamnetin should be no less than 5000.

[0027] Compared with the prior art, the quality detection method of the present invention includes a thin layer identification method of goat milk leaf medicinal materials and a method for determining the content of silver forging glycosides in goat milk leaf medicinal materials. Specifically, the thin layer identification method of goat milk leaf medicinal materials adopted in this application has the characteristics of high sensitivity, strong specificity and good separation effect, is simple and feasible, has strong reproducibility, and can accurately identify the authenticity of medicinal materials; the method for determining the content of silver forging glycosides in goat milk leaf medicinal materials adopted in this application can accurately and quickly determine the content of goat milk leaf medicinal materials, and provide effective qualitative and quantitative analysis means for goat milk leaf medicinal materials, which is conducive to controlling the quality of goat milk leaf medicinal materials. The quality detection method of goat milk leaf medicinal materials provided by the present invention can effectively control the overall quality of goat milk leaf medicinal materials and ensure the safety and effectiveness of clinical use of goat milk leaf medicinal materials.

[0028] The method of the invention is of great significance for further quality control of goat milk leaf medicinal materials, and can provide a scientific basis for the formulation of quality tables for goat milk leaf medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a thin layer chromatogram of the test sample preparation method. Figure 1 1. Argentoside reference substance; 2. Ultrasonic extraction of Elaeagnus leaves; 3. Ethyl acetate water reflux extraction of Elaeagnus leaves; 4. Butanol water reflux extraction of Elaeagnus leaves; 5. Ultrasonic extraction of Elaeagnus yichangensis leaves; 6. Ethyl acetate water reflux extraction of Elaeagnus yichangensis leaves; 7. Butanol water reflux extraction of Elaeagnus yichangensis leaves; 8. Ultrasonic extraction of Elaeagnus leaves; 9. Ethyl acetate water reflux extraction of Elaeagnus yichangensis leaves);

[0030] Figure 2 This is a thin layer chromatogram of sample spot quantity investigation. (In the figure, 1, argyrocoside reference substance; 2, 5μl of Elaeagnus elaeagnus leaf spot sample; 3, 10μl of Elaeagnus elaeagnus leaf spot sample; 4, 15μl of Elaeagnus elaeagnus leaf spot sample; 5, 5μl of Elaeagnus yichang leaf spot sample; 6, 10μl of Elaeagnus yichang leaf spot sample; 7, 15μl of Elaeagnus yichang leaf spot sample; 8, 5μl of Elaeagnus elaeagnus leaf spot sample; 9, 10μl of Elaeagnus elaeagnus leaf spot sample; 10, 15μl of Elaeagnus elaeagnus leaf spot sample; 11, 10μl of Elaeagnus elaeagnus leaf spot sample; 12, 10μl of Elaeagnus elaeagnus leaf spot sample).

[0031] Figure 3 It is a thin layer chromatogram of the ethyl acetate-2-butanone-formic acid (8:3:0.25) development system (in the figure, 1, argentin; 2, Elaeagnus elaeagnus leaves; 3, Elaeagnus yichangensis leaves; 4, Elaeagnus elaeagnus leaves; 5, Elaeagnus elaeagnus leaves; 6, Elaeagnus elaeagnus leaves);

[0032] Figure 4 It is a thin layer chromatogram of the chloroform-acetone-formic acid (5:5:0.125) development system (in the figure, 1, argentin; 2, Elaeagnus elaeagnus leaves; 3, Elaeagnus yichangensis leaves; 4, Elaeagnus elaeagnus leaves; 5, Elaeagnus elaeagnus leaves; 6, Elaeagnus elaeagnus leaves);

[0033] Figure 5 It is a thin layer chromatogram of the chloroform-methanol-formic acid (36:4:0.75) development system (in the figure, 1, arginine; 2, Elaeagnus elaeagnus leaves; 3, Elaeagnus yichangensis leaves; 4, Elaeagnus elaeagnus leaves);

[0034] Figure 6 It is the thin layer chromatogram at 30% relative humidity;

[0035] Figure 7 It is the thin layer chromatogram at 72% relative humidity;

[0036] Figure 8 This is the thin layer chromatogram at room temperature 6°C;

[0037] Fig. 9 This is the TLC at 25°C under air conditioning;

[0038] Fig.10 It is the thin layer chromatogram of the goat milk leaf samples of batches S1-S10 (in the figure, 1 represents silver forging glycoside, and 2-11 represents the goat milk leaf samples of batches S1-S10);

[0039] Fig.11 It is the thin layer chromatogram of the goat milk leaf samples of batches S11-S18 (in the figure, 1 represents silver forging glycoside, and 2-9 represent the goat milk leaf samples of batches S11-S18);

[0040] Fig.12 It is the UV scanning chromatogram of the silver forging glycoside reference substance;

[0041] Fig.13 It is a chromatogram of samples under different gradient conditions (in the figure, A gradient 1 (acetonitrile-0.4% formic acid water isocratic elution); B gradient 2 (acetonitrile-0.4% formic acid water isocratic elution); C gradient 3 (methanol-0.4% formic acid water); D gradient 4 (acetonitrile-0.4% formic acid water gradient elution); E gradient 5 (acetonitrile-0.4% formic acid water gradient elution); 1, silver forging glycoside);

[0042] Fig.14 It is the chromatogram of the reference substance and the test substance of gradient 5 (in the figure, A is the test substance; B is the reference substance; 1 is silver forging glycoside);

[0043] Fig.15 is the linear regression curve of arginine;

[0044] Fig.16 This is the UV spectrum of kaempferol;

[0045] Fig.17 is the UV spectrum of quercetin;

[0046] Fig.18 is the UV spectrum of isorhamnetin;

[0047] Fig.19 is the gradient chromatogram of each mobile phase;

[0048] Fig. 20 It is the HPLC chromatogram of the sample and the mixed reference substance (in the figure, A is the test sample; B is the mixed reference substance; 1 is quercetin; 2 is kaempferol; 3 isorhamnetin);

[0049] Fig.21 is the standard curve graph of quercetin;

[0050] Fig. 22 is the linear curve graph of kaempferol;

[0051] Fig.23is the linear graph of isorhamnetin;

[0052] Fig.24 This is the cluster analysis diagram of 29 samples;

[0053] Fig.25 is a bar graph of arginine content;

[0054] Fig.26 This is a statistical chart of the contents of three aglycones in 29 batches of medicinal materials. DETAILED DESCRIPTION

[0055] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0056] Embodiment. A quality detection method for goat milk leaf medicinal material,

[0057] instrument:

[0058] Shimadzu LC-20AT liquid chromatograph, including autosampler and DAD detector. Labsolution chromatography system; chromatographic column: inertsustain C18 (4.6mm×250mm, 5μm); AL204 electronic analytical balance (Mettler Toledo Instrument (Shanghai) Co., Ltd.); VORTEX3 vortex mixer (IKA, Germany); SHHW21 constant temperature water bath (Beijing Kewei Yongxing Instrument Co., Ltd.); BS224S electronic balance: 1 / 10, Sartorius; KQ-5200DB ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Company); DHG-9140A electric constant temperature blast drying oven: Gongyi Yuhua Instrument Co., Ltd.; 8-10 box-type resistance furnace: manufactured by Shenyang Energy Saving Electric Furnace Factory, China; three-purpose ultraviolet analyzer: Shanghai Gucun Electro-Optical Instrument Factory.

[0059] Reagents and test drugs

[0060] 1. Reference

[0061] The reference substances of arginine (Z30J7S16999), kaempferol (RO3F6C1), quercetin (C2016Y1722), and isorhamnetin (PO8J7F15965) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity of ≥98%.

[0062] 2. Reagents

[0063] Acetonitrile was of chromatographic grade (Merck, Germany), formic acid was of chromatographic grade (Tianjin Damao Chemical Reagent), concentrated hydrochloric acid (Xilong Chemical Co., Ltd.), chloroform, acetone, formic acid, ethyl acetate, 2-butanone, and methanol were of analytical grade (Sinopharm Chemical Reagent Co., Ltd.); silica gel GF254 (for thin layer chromatography). Ultrapure water (Millipore-Simplicity Ultrapure Water Treatment System, Merck Millipore, Germany).

[0064] Medicinal Materials

[0065] There are many species of medicinal plants in the genus Elaeagnus. There are 6 species of "Yang Nai Ye Ye / Elaeagnus Leaves / Elaeagnus Elaeagnus Leaves" whose leaves are used as medicine in the existing standards. The 29 batches of samples (including leaves, roots, stems, and fruits) used in this study were all collected by ourselves or commissioned for collection and purchase. They were identified by Lecturer Mu Zejing of the Center for Ethnic Medicine Research of Jiangxi University of Traditional Chinese Medicine. There are 7 species in total, including 4 species included in the standards and 3 species recorded as medicinal in literature. The specific source information of the samples is shown in Table 1.

[0066] Table 1 Sample information table

[0067]

[0068] 9

Medicinal material name

[0069] Sheep milk leaf (Elaeagnus oleraceus leaf): named according to the "Quality Standards for Traditional Chinese and Ethnic Medicinal Materials in Guizhou Province".

[0070] In this study, the TLC identification of the reference medicinal materials specified in the original Guizhou Provincial Standard was repeated in accordance with the experiment, and the results showed that the method was feasible. In addition, the TLC identification with oleanolic acid as the reference specified in the original Hubei Provincial Standard had poor specificity, considering that this component is a common component of many medicinal materials. Considering that flavonoids are the main component type of Rheum officinale, a TLC identification method with linaloside as the reference was established in the study, and the results showed that the method was feasible. Comprehensive considerations showed that the content of linaloside in Rheum officinale was relatively high, and it was finally determined that this component would be used as an indicator component for content measurement. Therefore, this revised standard still uses the TLC identification of reference medicinal materials and uses linaloside as an indicator component for content measurement.

[0071] Thin layer identification: Take 2g of the powder of this product, add 10 times the amount of water and boil for 30 minutes, filter, evaporate the filtrate on a water bath, add 0.5ml of anhydrous ethanol to dissolve the residue as the test solution. Take another 2g of the reference medicinal material of goat milk leaf, and prepare the reference medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the Fourth Part of the 2015 Edition of the Chinese Pharmacopoeia), take 5μl of each of the above two solutions, respectively, on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as the adhesive, use ethyl acetate-water-formic acid (57:10:8.2) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test product, at the corresponding position of the chromatogram of the reference medicinal material, a fluorescent spot of the same color appears.

[0072] 1) Preparation method investigation

[0073] Weigh 1.0 g of goat milk leaf sample powder, and examine the effects of 30 ml water + 30 ml ethyl acetate, 30 ml water + 30 ml n-butanol reflux extraction for 1 h, and 60 ml 75% methanol ultrasonic extraction on the spotting effect. The results are shown in Figure 1 .

[0074] Thin layer plate: silica gel G plate; spotting volume: 5 μl; developing agent: chloroform-acetone-formic acid (5:5:0.25); developing method and distance: upward development 7 cm; color development: spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored; inspection: inspection under 365nm ultraviolet light.

[0075] Depend on Figure 1 It can be seen that the spot color obtained by direct ultrasonic extraction is lighter, while the spots obtained by the reflux of n-butanol water and ethyl acetate aqueous solution are clear and the difference is not great. Considering that n-butanol is highly toxic and has a higher boiling point, ethyl acetate water is selected as the test sample to prepare the extraction solvent. Therefore, the test sample preparation method is determined as: take goat milk leaf powder 1.0g, accurately weighed, placed in a plugged conical flask, accurately add 30ml water and 30ml ethyl acetate solution, shake well, water bath reflux extraction 60min, filter rapidly, the subsequent filtrate is placed in a separating funnel, stratification, the supernatant is evaporated, and methanol is dissolved and fixed to volume in a 5ml volumetric flask, to obtain.

[0076] 2) Sample quantity inspection

[0077] The amount of sample was investigated based on the extraction of ethyl acetate and water reflux. Figure 2 The results showed that the spotting volume could be 5-15μl, and 10μl was chosen.

[0078] Thin layer plate: silica gel G plate; developing agent: chloroform-acetone-formic acid (5:5:0.125); developing method and developing distance: upward development 8 cm; color development: spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored; inspection: inspection under 366nm ultraviolet light.

[0079] 3) Development agent investigation

[0080] Using silver forging glycoside as the reference substance, three developing systems were investigated: chloroform-acetone-formic acid (5:5:0.125), chloroform-methanol-formic acid (36:4:0.75), and ethyl acetate-2-butanone-formic acid (8:3:0.25). Figure 3-5 As a result, the separation effects of the three systems were all good, but the spots under the condition of chloroform-acetone-formic acid (5:5:0.125) were concentrated and obvious.

[0081] Thin layer plate: silica gel G plate; development method and development distance: upward development 7cm; color development: spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored; inspection: inspection under 365nm ultraviolet light.

[0082] 4) Durability test under environmental conditions

[0083] Under the condition that the development system was selected as chloroform-acetone-formic acid (5:5:0.125), the durability test of environmental conditions was carried out. The relative humidity of 30% and 72%, as well as the temperature of room temperature 6℃ and air conditioning 25℃ were tested respectively, and good separation effects were achieved. Figures 6 to 9 .

[0084] Thin layer plate: silica gel G plate; developing agent: chloroform-acetone-formic acid (5:5:0.125); developing method and developing distance: 7 cm upward; color development: spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored; inspection: inspection under 365nm ultraviolet light.

[0085] 5) Thin layer chromatograms of 18 batches of medicinal materials

[0086] Select the powder of goat milk leaf samples from batches S1-S18 from the medicinal materials, prepare the test solution according to the conditions, use chloroform-acetone-formic acid (5:5:0.125) as the development system, and simultaneously draw 10 μl of the test and reference solutions onto the silica gel G plate. The test and reference samples show the same fluorescent spots at the same distance on the thin layer plate. Fig.10 and 11 .

[0087]

Content Determination

[0088] Antitussive and antiasthmatic is one of the main effects of E. pungens leaves. According to literature reports, plants of the genus Elaeagnus contain volatile oils, flavonoids, saponins, terpenes and steroids, fatty acids, alkaloids, phenols, tannins, lignans, organic acids, glycosides and other ingredients; pharmacological studies on E. pungens have shown that the n-butanol extract of its leaves has good antitussive and expectorant effects, and significantly inhibits the contraction of guinea pig tracheal smooth muscle, which is consistent with the medicinal effects of E. pungens leaves and is one of its effective parts, but there is little research on the components of E. pungens. In view of this, UPLC / Q-TOF-MS / MS was used in this revision to conduct a preliminary component analysis of the n-butanol extract of E. pungens. The results showed that this part mainly contained flavonoid glycosides, phenolic acids and saponins, and 49 components were deduced. Combined with reference substance comparison and structural analysis, 6 flavonoid components were confirmed, namely quercetin, kaempferol, isorhamnetin, quercetin-3-O-sophoroside, isorhamnetin-3-O-sophoroside, and silver forging glycoside (kaempferol glycoside).

[0089] According to the above research results, flavonoid components were tentatively selected as the index components for content determination. At the same time, considering the need for comparative identification with other species of Elaeagnus and the need to improve the specificity of the index components, the contents of four components, including linalool, kaempferol, quercetin, and isorhamnetin, were investigated. Since there are dozens of flavonoid glycosides in "sheep milk leaves", and the content of each monomer component is low, and most of them are flavonoid oxygen glycosides, which may be affected by many factors such as the plant growth environment, the content in the medicinal material is not stable, so the samples were hydrolyzed with hydrochloric acid to determine the aglycones such as kaempferol, quercetin, and isorhamnetin.

[0090] Test solution: weigh about 1.0 g of goat milk leaf powder, accurately weigh it, place it in a 50 ml conical flask, add 30 ml of 75% methanol, weigh it, reflux it in a water bath for 1.5 hours, cool it quickly, make up the weight loss with 75% methanol, shake it well, filter the subsequent filtrate through a 0.45 μm microporous filter membrane, and obtain it.

[0091] Reference solution: Accurately weigh an appropriate amount of silver glycoside reference substance and dissolve it in methanol to prepare a reference solution containing 0.22392 mg per ml.

[0092] Accurately weigh appropriate amounts of quercetin, kaempferol, and isorhamnetin reference substances, add methanol to dissolve them to prepare a mixed reference substance solution containing 0.0888 mg kaempferol, 0.1208 mg quercetin, and 0.0414 mg isorhamnetin per ml.

[0093] The chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as filler; acetonitrile and 0.4% formic acid water are used as mobile phases, column temperature is 30°C, flow rate is 1ml / min, and detection wavelength is 314nm. The theoretical plate number should be no less than 7500 when calculated based on arginine, and no less than 5000 when calculated based on kaempferol, quercetin, and isorhamnetin peaks. Acetonitrile (A) and 0.4% formic acid water (B) are used as mobile phases (0.01-25min, 25%-29% A isocratic elution), column temperature is 30°C, flow rate is 1.0ml / min, and detection wavelength is 314nm to determine arginine.

[0094] 1. Determination of the content of arginine

[0095] 1.1 Selection of detection wavelength and investigation of mobile phase

[0096] According to the UV scanning results of silver forging glycoside (see Fig.12 ), silver forging glycoside has maximum absorption at 314nm, so it is selected as the detection wavelength. Considering that flavonoid glycosides have more hydroxyl groups, adding a certain amount of acid can effectively improve the peak shape. With an elution flow rate of 1.0ml·min-1, the elution ratios of mobile phases such as acetonitrile-0.4% formic acid water isocratic elution, acetonitrile-0.4% formic acid water gradient elution, and methanol-0.4% formic acid water gradient elution were investigated. The chromatographic gradient is shown in Table 7, and the corresponding liquid chromatogram is shown in Fig.13 .

[0097] Table 7 Mobile phase gradient settings

[0098]

[0099] From the results in the above figure, it can be seen that gradients 1 and 2 are both acetonitrile-0.4% formic acid water isocratic elution. Under the two conditions, the separation of the silver forging glycoside chromatographic peak is about 1.0, which cannot achieve a good separation effect. Continuing to extend the time may not have much effect on improving the separation. Gradient 3 uses methanol-0.4% formic acid water as the gradient elution mobile phase. The separation of the silver forging glycoside chromatographic peak is above 1.5, but the chromatographic peak is wide and the time is relatively long; Gradients 4 and 5 are both acetonitrile-0.4% formic acid water gradient elution. Among them, the separation of the silver forging glycoside chromatographic peak of gradient 4 is less than 1.5, and the separation of the silver forging glycoside chromatographic peak of gradient 5 is above 1.5, and the peak time is moderate, and the peak shape is sharp and symmetrical. Therefore, gradient 5 is selected as the mobile phase gradient for the determination of the silver forging glycoside content in goat milk leaves.

[0100] 1.2. Investigation of different flow rates and column temperatures

[0101] Taking gradient 5 as the mobile phase elution gradient, the effects of three different flow rates of 0.8ml / min, 1.0ml / min, and 1.2ml / min and three different temperatures of 20℃, 30℃, and 40℃ on the separation of chromatographic peaks of silver forgingin were investigated. The results showed that different flow rates and different temperatures had little effect on the separation of chromatographic peaks of silver forgingin. Considering factors such as efficiency and energy, 1.0ml / min and 30℃ were selected as the mobile phase flow rate and column oven temperature for the determination of silver forgingin content in goat milk leaves.

[0102] 1.3. Determination of chromatographic conditions

[0103] According to the above test results, the chromatographic conditions for the determination of the content of arginine in goat milk leaves were determined as follows: the chromatographic column is inertsustain C18 (4.6mm×250mm, 5μm); the mobile phase is acetonitrile (A)-0.4% formic acid water (B); gradient elution (0.01~25min, 25%~29% A); flow rate: 1.0ml·min-1; column temperature: 30℃; detection wavelength 314nm; injection volume 10μl; detection time 25min. Under the above gradient, the chromatograms of the reference and test samples are shown in Fig.14 .

[0104] 1.4 Investigation of extraction methods

[0105] 1.4.1 Investigation of extraction methods

[0106] Weigh 2 portions of S14 and S17 powder of goat milk leaf, about 1.0g each, accurately weigh, add 40ml methanol, weigh, reflux in a 75℃ water bath for 1.5h, cool, make up the weight loss with methanol, shake well, filter, take the filtrate and filter it with a 0.45μm microporous membrane to obtain the test solution. Take 2 portions of S14 and S17 sample goat milk leaf powder, about 1.0g each, accurately weigh, add 40ml methanol, weigh, ultrasonic for 1h, cool, make up the weight loss with methanol, shake well, filter, take the filtrate and filter it with a 0.45μm microporous membrane to obtain the test solution. Sample injection was determined according to the chromatographic conditions of 1.3, and the results are shown in Table 8.

[0107] Table 8 Investigation of extraction methods

[0108]

[0109] From the results in the above table, it can be seen that the extraction efficiency of silver forging glycosides in the reflux extraction of the two samples is more than 30% higher than that of ultrasound. Therefore, reflux extraction is selected as the extraction method for preparing the test solution.

[0110] 1.4.2 Investigation of extraction solvent

[0111] Weigh 6 portions of the same batch of goat milk leaf powder, 1.0 g each, accurately weigh, put into a stoppered conical flask, add 50% methanol, 75% methanol, methanol, 50% ethanol, 75% ethanol, 40 ml of ethanol solution, weigh, extract under water bath reflux for 1.5 hours, cool, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate and filter with a 0.45 μm microporous membrane to obtain. Sample injection and determination according to the chromatographic conditions of 1.3, the determination results are shown in Table 9.

[0112] Table 9 Investigation of extraction solvents

[0113]

[0114] From the results in Table 3, it can be seen that the extraction efficiency of methanol is slightly higher than that of ethanol, and the extraction efficiency of 75% methanol is the best. Therefore, 75% methanol is selected as the extraction solvent for preparing the test solution.

[0115] 1.4.3 Examination of solvent dosage

[0116] Weigh 3 portions of the same batch of samples of goat milk leaf powder, 1.0g each, accurately weigh, add 30ml, 40ml, 50ml 75% methanol respectively, weigh, extract under water bath reflux for 1.5h, cool, add 75% methanol to make up the weight loss, shake well, filter, take the filtrate and filter it with a 0.45μm microporous membrane to obtain. Sample injection and determination according to the chromatographic conditions of 1.3, the results are shown in Table 10.

[0117] Table 10 Investigation of the amount of extraction solvent

[0118]

[0119] Based on the results in the above table, it can be seen that when the solvent dosage is 50ml, the extraction efficiency of silver forging glycoside is the best, followed by 30ml, but the difference between the two is not much. Considering the reasons such as saving solvent, 30ml is selected as the extraction solvent dosage for the preparation of the test solution.

[0120] 1.4.4 Extraction time investigation

[0121] Weigh 3 samples of goat milk leaf from the same batch, 1.0 g each, accurately weigh, add 30 ml of 75% methanol, weigh, extract under water bath reflux for 1 h, 1.5 h, 2 h, add 75% methanol to make up for the weight loss, shake well, filter, and filter the filtrate with a 0.45 μm microporous membrane to obtain. Inject according to the chromatographic conditions of 1.3, and the results are shown in Table 11.

[0122] Table 11 Investigation of extraction time

[0123]

[0124] The results in the above table show that as the extraction time increases, the extraction efficiency of silver forging glycosides increases slowly. Considering the extraction time and extraction efficiency comprehensively, 1.5 h was selected as the extraction time for the preparation of the test solution.

[0125] Based on the above investigation results, the method for preparing the test solution was finally determined as follows: weigh about 1.0 g of goat milk leaf powder, accurately weigh it, place it in a 50 ml conical flask, add 30 ml of 75% methanol, weigh it, reflux it in a water bath for 1.5 hours, cool it quickly, make up for the weight loss with 75% methanol, shake it well, filter the subsequent filtrate, and filter it with a 0.45 μm microporous filter membrane to obtain the solution.

[0126] 1.5 Methodological validation

[0127] 1.5.1 Linear relationship investigation

[0128] Accurately weigh 11.196 mg of dried silver forging glycoside reference substance, place it in a 50 ml volumetric flask, add methanol to dissolve, make up to volume, shake well, and make a reference substance stock solution containing 0.22392 mg per ml. The stock solution was diluted 2, 4, 8, 16, 32, and 64 times in sequence. Take 10 μl of the stock solution and each dilution according to item 1.3, inject three times, measure and calculate the average peak area, perform linear regression on the peak area with the reference substance concentration, and draw a standard curve and regression equation. The results of the linear investigation of silver forging glycoside are shown in Table 12, and the regression curve is shown in Fig.15 .

[0129] Table 12 Results of investigation on the linear relationship of silver forging glycosides

[0130]

[0131] From the above chart, we can see that the concentration of silver forging glycoside is 3.499μg·ml -1 ~223.92μg·ml -1 In the range, there is a good linear relationship with the peak area (R 2 =0.9999), the regression curve is y=27410087x-28046.

[0132] 1.5.2 Precision test

[0133] Accurately pipette the 4-fold diluted stock solution, inject it into the liquid chromatograph, and measure it according to the conditions in 1.3 for a total of six times, and record the peak area. The results are shown in Table 13.

[0134] Table 13 Precision investigation

[0135]

[0136] From the results in the above table, it can be seen that the RSD of the peak area of ​​argentopsis glycoside measured 6 times is less than 1%, indicating that the precision of the instrument is good.

[0137] 1.5.3 Repeatability test

[0138] According to the method for preparing the test solution determined above, 6 samples of goat milk leaf medicinal material test solution were prepared in parallel for determination, and their RSD values ​​were calculated. The results are shown in Table 14.

[0139] Table 14 Repeatability test results

[0140]

[0141] The results showed that 6 samples of test solution of Goat Milk Leaf were prepared in parallel for determination, and the RSD value of arginine was 1.032%, indicating that the method had good repeatability.

[0142] 1.5.4 Repeatability Experiment

[0143] Take 6 samples of the same batch of goat milk leaf samples, each with 1.0g, accurately weigh, prepare the test solution according to the method in 1.4, inject and measure according to the conditions in 1.3, and record the peak area. The results are shown in Table 15.

[0144] Table 15 Repeatability test

[0145]

[0146] From the results in the above table, it can be seen that the RSD of the content determination values ​​in the 6 goat milk leaf samples is less than 1%, indicating that the test method has good repeatability.

[0147] 1.5.5 Solution stability test

[0148] Take the test sample of goat milk leaf and prepare the test sample solution according to the method in 1.4. After 0, 1, 2, 4, 8, 12, and 24 hours, respectively, inject and measure according to the conditions in 1.3, and record the peak area. The results are shown in Table 16.

[0149] Table 16 Stability Study

[0150]

[0151] From the results in the above table, it can be seen that the RSD of the peak area of ​​the test sample measured within 24 hours is 0.47%, which is less than 1%, indicating that the test solution has good stability within 24 hours.

[0152] 1.5.6 Sample recovery rate

[0153] Take 9 portions of the same batch of goat milk leaf samples with known content, each portion is about 1.0g, accurately weighed, divided into three groups, and add about 80%, 100%, and 120% of the content of arginine in the medicinal materials to the reference substance, respectively, prepare the test solution according to 2.2.5, and inject and measure according to the conditions in 2.1.3, and calculate the recovery rate of the sample. The results are shown in Table 17.

[0154] Table 17 Silver forging glycosides sample recovery experimental results

[0155]

[0156]

[0157] From the results in the above table, it can be seen that the average recoveries of silver forging glycoside at the three test levels were 98.24%, 98.80% and 102.69%, respectively, and the RSDs were all less than 2%, indicating that the sample recovery rate of the test method was good.

[0158] 1.5.7 Sample content determination

[0159] Take 0.5 g of each of 10 batches of goat milk leaf medicinal materials, accurately weigh them, prepare them according to the preparation method of the test solution, take 10 μl of each sample, record the peak area, and calculate the mass fraction of each component using the regression equation. The results are shown in Table 12.

[0160] Take 1.0 g of each sample powder from 29 batches, prepare the test solution according to 1.4, and inject and measure according to the conditions in 1.3. The results are shown in Table 18.

[0161] Table 18 Determination results of silver forging glycosides in 29 batches of samples

[0162]

[0163] The results showed that among the seven species of Elaeagnus, the content of E. pungens and E. henryi in leaves was higher, while the content of other species and parts was significantly lower than that of leaves. The content of E. pungens leaves was extremely low and not within the linear range.

[0164] 2 Determination of the content of quercetin, kaempferol and isorhamnetin

[0165] 2.1 Investigation of mobile phase

[0166] The UV scanning results of kaempferol, quercetin and isorhamnetin (see Figure 16 to Figure 18 ) It can be seen that the three reference substances all have maximum absorption at around 370nm, so the detection wavelength is determined to be 370nm. Using acetonitrile-0.4% formic acid aqueous solution as the mobile phase, the effects of isocratic elution and gradient elution with different gradient mobile phases on chromatographic separation were investigated. The gradient settings are shown in Table 19, and the gradient chromatograms are shown in Fig.19 .

[0167] Table 19 Mobile phase gradient settings

[0168]

[0169] From the above spectrum, it can be seen that gradient 1 and gradient 2 are gradient elutions, and kaempferol and isorhamnetin cannot reach the ideal separation state. Gradients 3-5 all reach above 1.5. In view of the fact that the separation of each target peak of gradient 5 is above 2, and the peak elution time is moderate and the peak shape is symmetrical, gradient 5 is selected as the gradient for the determination of goat milk leaf content.

[0170] 2.2 Determination of chromatographic conditions

[0171] Gradient 5 was used as the mobile phase elution gradient. After investigating the separation effects of different flow rates and column temperatures on each component, the chromatographic conditions were finally determined as follows: chromatographic column inertsustain C18 (4.6mm×250mm, 5μm). Isocratic elution: acetonitrile (29% A)-0.4% formic acid water (71% B); flow rate: 1.0ml·min-1; column temperature: 30℃; detection wavelength: 370nm. Injection volume: 10μl. Sampling time: 35min. Under the above chromatographic conditions, the chromatograms of the sample and mixed reference are shown in Fig. 20 .

[0172] 2.3 Investigation of test sample preparation methods

[0173] 2.3.1 Investigation of extraction methods

[0174] Weigh about 1.0g of sample No. 14 powder, accurately weigh, add 40ml75% methanol, weigh, ultrasonic for 60min, cool, make up the weight loss with the corresponding solvent, shake well, filter, take 20ml of the filtrate, add 3ml of concentrated hydrochloric acid, weigh, reflux in a water bath at 80℃ for 1h, cool rapidly, make up the weight loss, shake well, filter the subsequent filtrate and filter it with a 0.45μm microporous membrane to obtain. Take about 1.0g of sample No. 14 sheep milk leaf powder, accurately weigh, add 40ml75% methanol, weigh, reflux in a water bath for 1.5h, cool, make up the weight loss with the corresponding solvent, shake well, filter, take 20ml of the filtrate, add 3ml of concentrated hydrochloric acid, weigh, reflux in a water bath at 80℃ for 1h, cool rapidly, make up the weight loss, shake well, filter the subsequent filtrate and filter it with a 0.45μm microporous membrane to obtain. Take the test sample prepared above and analyze it according to the method in 2.2. The results are shown in Table 20.

[0175] Table 20 Investigation of extraction methods

[0176]

[0177] The results in the table above show that the extraction efficiency of reflux extraction for the three compounds is more than 30% higher than that of ultrasonic extraction, which may be due to the high solubility of flavonoid glycosides in hot solvents. Therefore, reflux extraction was selected as the extraction method for preparing the test solution.

[0178] 2.3.2 Investigation of extraction solvent

[0179] Weigh 6 portions of sample powder from the same batch (passed through a 60-mesh sieve), 1.0 g each, accurately weigh, put into a stoppered conical flask, add 50% methanol, 75% methanol, methanol, 50% ethanol, 75% ethanol, and 40 ml of ethanol solution respectively, weigh, extract under reflux in a water bath for 1.5 h, cool, and make up for the weight loss with the corresponding solvent. For the rest, follow the operations under 3.3.1 and the sample is measured according to the chromatographic conditions under 2.2. The measurement results are shown in Table 21.

[0180] Table 21 Extraction solvent investigation

[0181]

[0182] From the results in the table above, we can see that with the increase of methanol concentration, the extraction efficiency of the three compounds gradually increases. With the increase of ethanol concentration, the extraction efficiency of the three compounds first increases and then decreases, among which 75% ethanol has the best extraction efficiency for the three compounds. Therefore, 75% ethanol is selected as the extraction solvent.

[0183] 2.3.3 Examination of solvent dosage

[0184] Weigh 3 portions of sample powder from the same batch (pass through a 60-mesh sieve), 1.0 g each, accurately weigh, add 25 ml, 40 ml, and 55 ml of 75% ethanol respectively, weigh, extract under reflux in a water bath for 1.5 h, cool, make up the weight loss with the corresponding solvent, shake well, filter, and perform the rest according to the operations under 2.3.1 and the sample injection and determination according to the chromatographic conditions under 2.2. The determination results are shown in Table 22.

[0185] Table 22 Investigation of solvent dosage

[0186]

[0187] From the results in the table above, it can be seen that as the amount of extraction solvent increases, the extraction efficiency of the three compounds first increases and then decreases. Based on this, 40 ml is selected as the solvent extraction amount.

[0188] 2.3.4 Investigation of the amount of hydrochloric acid used

[0189] Weigh 3 portions of the same batch of goat milk leaf powder (passed through a 60-mesh sieve), about 1.0 g, accurately weigh, place in a 100-ml conical flask, add 40 ml of 75% ethanol, weigh, reflux in a water bath for 1 h, cool rapidly, make up for the weight loss, shake well, filter, take 20 ml of the filtrate and place in a stoppered conical flask, add 3, 4, and 5 ml of concentrated hydrochloric acid, and prepare the rest according to the operation under 2.3.1. Sample injection and determination are carried out according to the chromatographic conditions in 2.2. The determination results are shown in Table 23.

[0190] Table 23 Investigation of hydrochloric acid dosage

[0191]

[0192] From the results in the above table, it can be seen that with the increase of the amount of hydrochloric acid, the hydrolysis efficiency of the three compounds gradually increased. However, considering that the over-acidic test samples may affect the column efficiency of the chromatographic column, 5 ml of hydrochloric acid was selected as the preparation amount of the test samples.

[0193] 2.3.5 Investigation of extraction time

[0194] Under the above conditions, the effect of water bath reflux for 60 min, 90 min and 120 min on the extraction of the three components was investigated. The results are shown in Table 24.

[0195] Table 24 Reflow time investigation

[0196]

[0197] The results showed that among the different extraction times, 120 min had the highest extraction efficiency, but the effects of different extraction times on the extraction rate were not significant, and the change trends were inconsistent, which may be related to the hydrolysis process of different components. After comprehensive consideration, 60 min was determined as the water bath reflux extraction time.

[0198] 2.3.6 Determination of test sample preparation method

[0199] Based on the results of the above investigation items, the preparation method of the test sample is determined as follows: take about 1.0g of the test sample powder, accurately weigh it, place it in a stoppered conical flask, add 40ml of 75% ethanol, weigh it, reflux it in a water bath for 1h, cool it rapidly, make up for the weight loss, shake it well, filter it, take 20ml of the filtrate and place it in a stoppered conical flask, add 5ml of hydrochloric acid, weigh it, reflux it in a water bath at 80℃ for 1h, cool it rapidly, make up for the weight loss, shake it well, filter it, and filter the subsequent filtrate through a 0.45μm microporous filter membrane to obtain the test sample solution.

[0200] 2.4 Investigation of linear range

[0201] Accurately weigh 4.44 mg of kaempferol reference substance, 6.04 mg of quercetin reference substance, and 2.07 mg of isorhamnetin reference substance, dilute to 50 ml with methanol, dissolve with ultrasonic aid, and filter through a 0.45 μm microporous membrane to prepare a mixed reference substance stock solution containing 0.0888 mg of kaempferol, 0.1208 mg of quercetin, and 0.0414 mg of isorhamnetin per ml. The stock solution was diluted 2, 4, 8, 16, 32, 64, and 128 times in sequence. Take 10 μl of the stock solution and each dilution and inject 3 times each according to the conditions in item 2.2. Determine and calculate the average peak area, perform linear regression on the peak area using the reference substance concentration, and draw a standard curve and regression equation. The linearity test results of each compound are shown in Tables 25 to 27, and the regression curve is shown in Figures 21 to 23 .

[0202] Table 25 Quercetin linear relationship investigation results

[0203]

[0204] Table 26 Kaempferol linear relationship investigation table

[0205]

[0206] Table 27 Isorhamnetin linear relationship investigation table

[0207]

[0208] The results showed that quercetin was in the range of 0.94 to 120.8 μg ml -1 , kaempferol 0.69~88.8μg·ml -1 , isorhamnetin 0.32~41.4μg·ml -1 In the range, it has a good linear relationship with the peak area, R 2 They are 0.9995, 0.9999 and 0.9999 respectively.

[0209] 2.5 Precision experiment

[0210] Accurately pipette the reference solution and inject it into the liquid chromatograph. Perform the injection analysis according to the conditions in 2.2 for a total of 6 times and record the peak area. The results are shown in Table 28.

[0211] Table 28 Precision Investigation

[0212]

[0213] Based on the results in the above table, the RSD of the peak area of ​​each compound measured 6 times was less than 1%, indicating that the precision of the instrument was good.

[0214] 2.6 Stability test

[0215] Take the test sample and prepare the test solution according to the method in 2.3.6. After 0, 1, 2, 4, 8, 12, and 24 hours, inject the sample and measure according to the conditions in 2.2 respectively, and record the peak area. The results are shown in Table 29.

[0216] Table 29 Stability Study

[0217]

[0218]

[0219] The above results show that the peak area RSD of the three test components measured multiple times within 24 hours is less than 2%, indicating that the test solution has good stability within 24 hours.

[0220] 2.7 Repeatability Study

[0221] Take 6 portions of the same batch of goat milk leaf samples (passed through a 60-mesh sieve), each 1.0 g, accurately weigh, prepare the test solution according to the method in 2.3.6, inject and measure according to the conditions in 2.2, and record the peak area. The results are shown in Table 30.

[0222] Table 30 Repeatability test

[0223] sequence 1 2 3 4 5 6 RSD% <![CDATA[Quercetin (mg·g -1 )]]> 0.4118 0.4192 0.4297 0.4213 0.4140 0.4081 1.85 <![CDATA[Kaempferol (mg·g -1 )]]> 0.6832 0.6941 0.7139 0.6999 0.6755 0.6636 2.61 <![CDATA[Isorhamnetin (mg·g -1 )]]> 0.6850 0.6908 0.7109 0.6925 0.6813 0.6768 1.73

[0224] From the results in the above table, it can be seen that the RSDs of the contents of the three components to be tested in six repeated tests were all less than 3%, indicating that the repeatability of the method was good.

[0225] 2.8 Sample recovery experiment

[0226] Take 6 portions of the same batch of goat milk leaf samples with known content, each portion is about 0.5g, accurately weighed, and add about 100% of the reference substance of the three aglycones in the medicinal materials respectively, prepare the test solution according to 2.3.6, and inject and measure according to the conditions in 2.2, and calculate the recovery rate of the sample. The results are shown in Table 31.

[0227] Table 31 Sample recovery

[0228]

[0229] The results showed that the recoveries of the three compounds were good.

[0230] 2.9 Content determination

[0231] Take 1.0g of each of the 29 batches of sample powder, weigh accurately, prepare the test solution according to 2.3.6, and inject and measure according to the conditions in 2.2. The results are shown in Table 32.

[0232] Table 32 Content determination of 29 batches of samples

[0233]

[0234] As shown in the table above, the contents of the three flavonoid aglycones are higher in leaves, lower in stems, and not contained (or measured) in roots. The contents, composition ratios, and total amounts of the three aglycones in the leaves of different plants vary greatly.

[0235] 3 Comparison of components from different Elaeagnus plants

[0236] 3.1 Cluster analysis

[0237] As shown in Table 18 and Table 29, there are significant differences in the contents of arginine and three flavonoid aglycones among the various original species and different medicinal parts. In order to explore whether this difference can be used to distinguish the various original species, SPSS18.0 software was used to perform a systematic cluster analysis on all samples using the ward method and the Euclidean distance clustering method, with the contents of arginine and three aglycones as indicator variables. The results are shown in Fig.24 .

[0238] The cluster analysis diagram showed that the determined components could well distinguish different original species. Fifteen of the 16 batches of Elaeagnus oleraceus leaves were clustered into one large category, and only sample S14 and Badong Elaeagnus oleraceus leaves were clustered into one category. Sample S14 was a large amount of 20kg commercial medicinal materials, which was different from the other 15 batches of 1kg small samples. This may be due to the large amount of medicinal materials collected, the wide collection area, and the admixture of leaves of other plants of the same genus, resulting in differences in chemical components, especially the content of isorhamnetin aglycone, which was significantly higher than that of other batches of samples. Yichang Elaeagnus oleraceus and Elaeagnus oleraceus were clustered into one large category, indicating that the contents of the two types of components in Yichang Elaeagnus oleraceus and Elaeagnus oleraceus leaves were more similar. Three batches of Elaeagnus oleraceus were clustered into one small category, and two batches of milk seeds were clustered into one small category. The remaining long-cleft Elaeagnus oleraceus leaves and Fujian Elaeagnus oleraceus leaves were each clustered into one small category, indicating that the chemical components of different original species were significantly different. The above results show that the content and composition of linaloside and three aglycone components can well distinguish the seven species of Elaeagnus oleraceus. The stems, roots, fruits and leaves of medicinal materials are not grouped together, indicating that their components are quite different from those of the leaves, indicating that there is a certain scientific basis for the different clinical indications of the roots, leaves and fruits when used separately as medicine.

[0239] 3.2 Comparison of argyroside and three aglycones among different species

[0240] Comparison of the contents of arginine, quercetin, kaempferol and isorhamnetin in seven species of Elaeagnus and their different parts is shown in Fig.25 , 26 .

[0241] The content of arginine in the leaves of Elaeagnus yichangensis was the highest, reaching 3.1704 mg·g-1, followed by Elaeagnus yichangensis leaves, and the contents of the other species were all lower, indicating that there were large differences between species. Whether they can be mixed with each other still needs to be studied in depth in combination with market and clinical use surveys, variety and origin sorting, activity and quality evaluation, etc. The content in stems and fruits is less than one-tenth of that in leaves, and the roots do not contain arginine, suggesting that different parts should be used as different medicinal materials.

[0242] The corresponding ratios of quercetin, kaempferol and isorhamnetin among different species may have certain regularities due to different metabolic pathways in plants. The content of kaempferol in Elaeagnus cotyledons is kaempferol>quercetin>isorhamnetin, and when the quercetin content is 1, the ratio of quercetin to kaempferol and isorhamnetin in 16 batches of Elaeagnus cotyledons, except for S14, is stable at 1:1.31~2.38:0.16~0.64, and the ratio of Yichang Elaeagnus cotyledons is close to this. The isorhamnetin content in Elaeagnus vine leaves is significantly higher than kaempferol and quercetin. The ratio of kaempferol to quercetin in milk cotyledons is greater than that in Elaeagnus cotyledons, and the isorhamnetin content is extremely low, with a ratio of 1:2.94~3.89:0.05. The quercetin content in the leaves of Fujian Elaeagnus was the highest, and quercetin>kaempferol>isorhamnetin. The ratio of isorhamnetin to kaempferol in the leaves of Badong Elaeagnus was close to 1:1. The total amount of the three aglycones in the leaves of Fujian Elaeagnus was the highest, followed by the leaves of vine Elaeagnus, Elaeagnus, Yichang Elaeagnus and long-lobed Elaeagnus. The cotyledons of milk and Badong Elaeagnus were the lowest. The content in stems, leaves, and fruits was lower than that in leaves, and the root did not contain it.

[0243] 3.3 Selection of original species

[0244] The contents of the two components in the leaves of Elaeagnus sibiricum are generally high, and the leaves are widely distributed and have a long history of medicinal use. Therefore, they are used as the main basal species for comparison with other species. The Guizhou Provincial Standard (2003 Edition) once included the basal plants of the leaves of Goat's Milk Leaf (Elaeagnus sibiricum) as E. pungens Thunb., E. henryi Warb., and E. glabra Thunb.. The Fujian Provincial Standard (2006 Edition) also uses the Fujian Elaeagnus sibiricum, which is widely distributed locally, as a medicinal Elaeagnus sibiricum. From the cluster analysis diagram and the contents of the two components, it can be seen that the components of Yichang Elaeagnus sibiricum and Elaeagnus sibiricum are relatively similar, so it is retained as the basal species. The components of E. henryi Warb. and E. glabra Thunb. are quite different from those of Elaeagnus sibiricum. The milk fruit, Badong Elaeagnus sibiricum, and long-cleft Elaeagnus sibiricum, which are also used as medicinal Elaeagnus sibiricum in Guizhou and other places, are also quite different from Elaeagnus sibiricum. In summary, the basal plants of goat milk leaf (Elaeagnus leaf) are preferably E. pungens Thunb. and E. henryi Warb., while whether it is reasonable to use E. glabra Thunb. at the same time remains to be studied, and they are not used as basal plants for the time being.

[0245] 4. Selection and Limits of Components for Content Determination

[0246] Flavonoids are the main components of Elaeagnus plants and one of their effective parts. The above studies have shown that Elaeagnus leaves contain dozens of flavonoid components, all of which are flavonoid glycosides formed by kaempferol, quercetin and isorhamnetin as aglycones with various sugars or coumarins, ferulic acid or sinapinic acid. The content analysis of linaloside and the three aglycones of kaempferol, quercetin and isorhamnetin showed that, in general, the content of linaloside in Elaeagnus and Yichang Elaeagnus was high and relatively consistent, while the three aglycones and their composition ratios were quite different in various species. Literature reports that linaloside is also one of the components with biological activity. Taking comprehensive considerations, linaloside was selected as the index component for content determination of Elaeagnus leaves.

[0247] In Table 17, among the 17 samples of leaves of E. pungens and E. henryi, sample No. 14 is an abnormal value (0.05%). The content of arginine in the other 16 samples is between 0.1471% and 0.31704%, and the average content is 0.1961%. The average is 0.16%, which is 20% lower than the average. 14 of the 17 batches of samples are qualified, and the qualified rate is 82%. Based on this, it is determined that the content of arginine in the leaves of E. pungens shall not be less than 0.16%.

Claims

1. A quality detection method for goat milk leaf medicinal material, characterized in that: It includes identification and content determination items; the identification of the medicinal material of goat milk leaf is carried out by thin layer chromatography identification method; the content determination is to determine the content of silver forging glycosides in the medicinal material of goat milk leaf by high performance liquid chromatography; The method for determining the content of arginine in the medicinal material of goat milk leaf is determined by high performance liquid chromatography, comprising the following steps: 1) Chromatographic conditions of liquid chromatograph The chromatographic column is inertsustain C18, with octadecylsilane bonded silica gel as the filler; acetonitrile and 0.4% formic acid water are used as the mobile phase, the elution time is 0.01 to 25 minutes, 25% to 29% acetonitrile is used for isocratic elution, the column temperature is 30°C, the flow rate is 1.0 ml / min, and the detection wavelength is 314 nm to determine argentin; 2) Preparation of the content determination reference solution: accurately weigh an appropriate amount of the arginine reference substance, add methanol to dissolve it and prepare a content determination reference solution containing 0.22392 mg per ml; 3) Preparation of test solution for content determination: accurately weigh 1.0 g of the powder of the product, place it in a stoppered conical flask, add 30 ml of 75% methanol, weigh it, reflux it in a water bath for 1.5 hours, cool it quickly, weigh it, make up the weight loss with 75% methanol, shake it well, filter it and take the filtrate to obtain the test solution for content determination; 4) Determination method: Accurately pipette 10 μl of the above-mentioned reference substance and test substance solutions, inject into liquid chromatography, and determine the content of arginine in goat milk leaf medicinal material to obtain.

2. The quality detection method of a goat milk leaf medicinal material according to claim 1, characterized in that: The thin layer chromatography identification method includes the following steps: (1) Weigh 2g of the powder of this product, add 10 times the amount of water and boil for 30 minutes, filter, evaporate the filtrate on a water bath, add 0.5ml of anhydrous ethanol to dissolve the residue, and use it as the test solution; take 2g of the control medicinal material of goat milk leaf, and prepare the control medicinal material solution in the same way; (2) Thin layer chromatography test Pipette 5 μl of each of the above two solutions and place them on the same silica gel G thin layer plate with sodium carboxymethyl cellulose as the adhesive, use a mixture of ethyl acetate: water: formic acid = 57:10:8.2 as the developing agent, develop, take out, dry, and examine under a 365nm ultraviolet lamp to obtain the test sample chromatogram.

3. The quality detection method of a goat milk leaf medicinal material according to claim 2, characterized in that: In the chromatogram of the test sample, fluorescent spots of the same color should appear at the corresponding positions in the chromatogram of the control medicinal material.

4. The quality detection method of the goat milk leaf medicinal material according to claim 1, characterized in that: The content of arginine C in goat milk leaf medicinal material is calculated based on the dry product. 30 H 26 O 13 Not less than 0.16%.

5. The quality detection method of the goat milk leaf medicinal material according to claim 1, characterized in that: In step 1), in the chromatographic conditions of the liquid chromatograph, the theoretical plate number calculated based on arginine should be no less than 7500, and the theoretical plate number calculated based on the peaks of kaempferol, quercetin and isorhamnetin should be no less than 5000.

Images