Construction method and application of semiliquidambar cathayensis fingerprint spectrum

Abstract

The invention discloses a construction method and application of a semiliquidambar cathayensis fingerprint spectrum, and belongs to the technical field of traditional Chinese medicine analysis. The construction method of the semiliquidambar cathayensis fingerprint spectrum comprises the following steps: (1) extracting total triterpenoid saponins in roots or leaves of semiliquidambar cathayensis; and (2) detecting the total triterpenoid saponins of the semiliquidambar cathayensis prepared in the step (1) by adopting high performance liquid chromatography to obtain a chromatogram of the semiliquidambar cathayensis, and the chromatogram of the semiliquidambar cathayensis is the fingerprint spectrum of the semiliquidambar cathayensis. The invention provides the construction method of the semiliquidambar cathayensis fingerprint spectrum, the fingerprint spectrum can be used for evaluating the batch quality stability of the semiliquidambar cathayensis, and reliable technical support is provided for standardized application of the semiliquidambar cathayensis.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, and in particular relates to a method for constructing a fingerprint spectrum of Hemiptera: Paniculata and its application. Background Art

[0002] Semiliquidambar cathayensis (HT Chang) is a traditional Chinese medicinal herb with properties such as dispelling wind and dampness, relaxing tendons and activating blood circulation. It is widely used to treat ailments such as rheumatism, arthralgia, and traumatic injuries. However, the plant origin of Semiliquidambar cathayensis is complex, potentially involving species from the Hamamelidaceae (Semiliquidambar cathayensis) or Araliaceae (Dendropanax dentiger), resulting in significant differences in its chemical composition. Currently, quality control of Semiliquidambar cathayensis relies primarily on morphological identification and determination of a few active ingredients (such as flavonoids and terpenes). These methods fail to fully reflect its intrinsic quality and are easily affected by factors such as origin and harvest season, hindering its standardized application.

[0003] Semiliquidambar cathayensis Chang is an evergreen tree in the genus Semiliquidambar of the Fungiaceae family. Compounds isolated and detected from various parts of the plant (roots, leaves, stems, and nectar) primarily include flavonoids, alkaloids, terpenes, phenols and their derivatives, steroids, fatty acids, polysaccharides, organic acids, vitamins, and ellagic acid derivatives. Inflammation is the body's natural response and protective mechanism against various harmful stimuli, such as lipopolysaccharide (LPS). Left untreated, it can lead to the development of cancer. Currently, a wide range of anti-inflammatory drugs, such as anti-inflammatory drugs, ibuprofen, and aspirin, are available. However, these drugs often cause adverse reactions, particularly kidney damage. Therefore, the search for new anti-inflammatory drugs from botanical sources is of great significance. Traditional Chinese medicine, renowned for its rich source of bioactive natural products, is widely used to treat various inflammatory conditions with multiple components, targets, functions, and pathways. As a traditional folk medicine, Herba Hedychium is used to treat diseases related to inflammation, such as rheumatoid arthritis, lumbar muscle degeneration, postpartum achalasia, hemiplegia and hepatitis. The chemical components of its alcohol extract are mainly triterpenoid compounds and ellagic acid derivatives.

[0004] Currently, the quality analysis methods for Hemiptera: (1) Traditional microscopic or physicochemical identification has poor specificity and cannot distinguish closely related species; (2) Single component content determination (e.g., quercetin, syringin) cannot reflect the synergistic effects of multiple components in traditional Chinese medicine; (3) Conventional chromatographic methods (e.g., HPLC-UV) have limited separation effects and are difficult to characterize complex component systems. In addition, existing research lacks a systematic construction of a characteristic fingerprint of Hemiptera, resulting in a lack of scientific basis for its authenticity identification and batch consistency evaluation.

[0005] Fingerprint technology can comprehensively characterize the overall characteristics of the chemical components of traditional Chinese medicine through modern analytical methods (such as HPLC, GC-MS or spectral coupling technology), and has been listed as an important method for quality control of traditional Chinese medicine in the "Chinese Pharmacopoeia". Summary of the Invention

[0006] The present invention provides a method for constructing a fingerprint of Hemiptera chinensis, comprising the following steps:

[0007] (1) Take 5g of Hemiptera helichrysum dry powder, extract with 80% ethanol under reflux for 1.5-2.5h, with a solid-liquid ratio of g / ml of 1:28-32, extract once or twice, filter, combine the filtrates, and concentrate under reduced pressure to a solid-liquid ratio of g / ml of 1:14-16, add 20% H2SO4 by mass to make a solution containing 2-4% sulfuric acid, reflux and acidify for 1.5-2.5h, cool to room temperature, adjust pH to 5, and use 90-110 ml ethyl acetate for 4-6 times, the extracts are combined and distilled to dryness to obtain triterpenoid sapogenin extract, the triterpenoid sapogenin extract is dissolved in 80% ethanol, 30-50 ml of the pretreated AB-8 macroporous adsorption resin is added to the above-dissolved triterpenoid sapogenin extract solution, the mixture is sealed and subjected to constant temperature oscillation adsorption for at least 20 hours, filtered, the liquid on the resin surface is dried, and 70-80 ml of 95% ethanol is added, the mixture is shaken and eluted for at least 20 hours, and filtered to obtain the purified total triterpenoid saponins of Helianthus annuus.

[0008] (2) Detecting the total triterpenoid saponins of the Heterocarpus serratus obtained in step (1) by high performance liquid chromatography to obtain a chromatogram of Heterocarpus serratus, wherein the chromatogram of Heterocarpus serratus is the fingerprint of Heterocarpus serratus; wherein the chromatographic conditions are as follows:

[0009] Chromatographic column: Agilent Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm);

[0010] Column temperature: 30°C;

[0011] Mobile phase: Phase A: 0.1% formic acid / water solution; Phase B: acetonitrile;

[0012] Flow rate: 1.0 mL / min;

[0013] Injection volume: 10 μL;

[0014] Pool temperature: 40°C;

[0015] Detection wavelength: 203nm;

[0016] The gradient elution program is shown in the following table: .

[0017] In one embodiment of the present invention, the dry powder of Panax notoginseng in step (1) is the dry powder of the roots or leaves of Panax notoginseng.

[0018] In one embodiment of the present invention, in step (1), 80% ethanol is refluxed for extraction for 2 h.

[0019] In one embodiment of the present invention, the solid-liquid ratio of the Helianthus annuus dry powder to 80% ethanol in step (1) is 1:30 g / ml.

[0020] In one embodiment of the present invention, 20% by mass of H2SO4 is added in step (1) to prepare a 3% sulfuric acid solution.

[0021] In one embodiment of the present invention, in step (1), extraction is performed five times with 100 ml of ethyl acetate.

[0022] The present invention also provides a method for constructing a standard fingerprint of Panax notoginseng, the method comprising:

[0023] Take R portions of Hemiptera root or leaf, and obtain fingerprints of the R portions of Hemiptera root or leaf according to any of the methods described above; analyze the fingerprints of the R portions of Hemiptera root or leaf using traditional Chinese medicine chromatographic fingerprint similarity evaluation software to obtain a standard fingerprint of the Hemiptera root or leaf; wherein R≥5.

[0024] The present invention also provides a method for constructing the fingerprint of the above-mentioned Hemiptera: Anemone serrata and its use in the quality control of Hemiptera serrata; the standard fingerprint of the Hemiptera serrata leaf / root includes 6 characteristic chromatographic peaks, and the retention times are: 25.838±0.05min, 31.105±0.05min, 33.769±0.05min, 40.352±0.05min, 41.389±0.05min, and 44.112±0.05min, respectively.

[0025] The present invention also provides use of the method for constructing a standard fingerprint of Hemiptera: Scutellaria baicalensis in quality control of Hemiptera: the standard fingerprint of Hemiptera: Scutellaria baicalensis leaves / roots comprises 6 characteristic chromatographic peaks, with retention times being, in sequence, 25.838±0.05 min, 31.105±0.05 min, 33.769±0.05 min, 40.352±0.05 min, 41.389±0.05 min, and 44.112±0.05 min.

[0026] The present invention also provides a quality control method for Banfenghe, the method comprising:

[0027] (1) Taking the leaf or root of the to-be-tested Hedera japonica, and obtaining the fingerprint of the leaf or root of the to-be-tested Hedera japonica according to any of the methods described above;

[0028] (2) The fingerprint of the test Hemiptera leaf or root obtained in step (1) is evaluated for similarity with the standard fingerprint of the Hemiptera leaf or root constructed according to the above method for constructing the standard fingerprint of Hemiptera. If the similarity is ≥0.90, the quality standard of the Hemiptera to be tested is determined.

[0029] We selected terpenoids from six species of Hemiptera as indicators for constructing fingerprints of Hemiptera from different provenances. These six compounds are:

[0030] (1) Centella asiatica

[0031] Asiatic acid is the main active ingredient (triterpenoid) of Centella asiatica, and its effects cover multiple areas:

[0032] ① Skin health: inhibits ultraviolet (UVA)-induced skin damage (such as photoaging), promotes wound healing (better than ingredients such as Centella asiatica), and improves acne and sensitive skin (antibacterial, regulates sebaceous gland secretion).

[0033] ② Anti-inflammatory and antioxidant: inhibit the release of inflammatory factors (such as TNF-α, IL-8), scavenge free radicals, and reduce oxidative stress.

[0034] ③ Liver protection and anti-fibrosis: inhibit the activation of hepatic stellate cells, reduce collagen synthesis, and resist liver fibrosis; protect liver cell mitochondria and scavenge free radicals.

[0035] ④Others: antidepressant (shortening the immobility time of mice forced to swim), hypoglycemic (increasing glucose excretion in diabetic mice), antibacterial (inhibiting Staphylococcus aureus, Candida albicans, etc.), immune regulation (inhibiting the formation of autoantibodies).

[0036] (2) Atractylodes lactone

[0037] Atractylodes lactone is the core active ingredient of Atractylodes macrocephala (lactones), mainly including Atractylodes lactone I and III, which have the following effects:

[0038] ① Anti-inflammatory and anti-tumor: inhibit inflammatory response and have inhibitory effects on various tumor cells.

[0039] ② Gastrointestinal regulation: promote nutrient absorption and improve gastrointestinal function (such as Atractylodes lactone I can regulate gastrointestinal function).

[0040] ③ Antioxidant: Atractylodes lactone III has antioxidant activity and helps prevent chronic diseases.

[0041] (3) Maslinic acid

[0042] Maslinic acid is an important organic acid component of hawthorn, and its main functions are:

[0043] ① Anti-tumor: It has a good inhibitory effect on the proliferation of cancer cells (such as synergistically inhibiting cancer cells with betulinic acid).

[0044] ② Auxiliary effects: It may be involved in the effects of hawthorn on "strengthening the stomach and digestion, promoting qi and dispersing blood stasis, lowering blood pressure and lipids" (maslinic acid is one of the active ingredients of hawthorn), but its direct mechanism of action is not clear in the search results.

[0045] (4) Betulinic acid (betulinic acid)

[0046] Betulinic acid is a pentacyclic triterpenoid component of plants such as birch, which has a wide range of effects:

[0047] ① Anti-tumor: It has a strong toxic effect on various tumors such as melanoma, neuroectodermal tumors and malignant brain tumors (it has no killing effect on normal cells).

[0048] ② Anti-infection: anti-inflammatory, antibacterial, anti-malarial, and anti-HIV-1 virus activity is stronger than some clinical drugs.

[0049] ③Skin care: moisturizing (locking in moisture, repairing the stratum corneum), anti-oxidation (neutralizing free radicals), improving pigmentation (inhibiting melanin production).

[0050] (5) Oleanolic acid

[0051] Oleanolic acid is a pentacyclic triterpenoid component of plants such as Gentianaceae, with "liver protection" as its core function:

[0052] ① Liver protection: Reduce serum alanine aminotransferase (ALT), promote liver cell regeneration, repair necrotic tissue, and be used for auxiliary treatment of acute and chronic hepatitis.

[0053] ② Anti-inflammatory and anti-tumor: inhibit the release of inflammatory factors and have inhibitory effects on various tumor cells (inducing apoptosis and inhibiting proliferation).

[0054] ③Metabolic regulation: lowering blood lipids (lowering cholesterol and triglycerides), anti-diabetic (increasing insulin sensitivity).

[0055] (6) Oleanolic acid

[0056] The search results did not specify the specific pharmacological effects of oleanolic acid, but only mentioned that it is one of the organic acid components in red dates (it is an active ingredient in red dates along with oleanolic acid, hawthorn acid, etc.).

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The present invention provides a method for constructing a fingerprint of Panax notoginseng. The fingerprint can be used for batch quality stability evaluation of Panax notoginseng, providing reliable technical support for the standardized application of Panax notoginseng. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the fingerprint of the root of Hedera chinensis in Example 2.

[0060] Figure 2 This is the fingerprint of the half-maple lotus leaf in Example 2. DETAILED DESCRIPTION

[0061] Example 1

[0062] (1) Optimization of the extraction method of total triterpenoid saponins from Hemiptera

[0063] ① Pretreatment

[0064] Weigh the roots and leaves of Hedera chinensis separately, dry them separately, grind them into powder, and pass the coarse powder through a 0.4 mm sieve for later use.

[0065] ②Extraction method

[0066] Take 5g of dried powder from Hemiptera chinensis, extract it with ethanol under reflux, filter it, combine the filtrates, and concentrate them under reduced pressure. Add an appropriate amount of 20% H₂SO₄ (to make a 3% sulfuric acid solution), reflux and acid-hydrolyze it. Cool to room temperature, adjust the pH to 5 with 20% NaOH solution, and extract it with ethyl acetate. Combine the extracts and evaporate them to dryness to obtain a triterpenoid sapogenin extract. Dissolve it in 80% ethanol to prepare test solutions of varying concentrations. Calculate the extraction yield of triterpenoid saponins in Hemiptera chinensis using a colorimetric method.

[0067] A single-factor experiment was conducted according to the design in Table 1, examining the effects of ethanol concentration, initial solid-liquid ratio, reflux extraction time (h), number of extractions, concentrated solid-liquid ratio, reflux acid hydrolysis extraction time (h), and number of ethyl acetate extractions on the extraction yield of triterpenoid saponins from Hemiptera. When one factor was examined, the other factors were set to the second level as described in the table. Three replicates were used.

[0068] Table 1 Multifactor experimental plan

[0069]

[0070] (2) Enrichment and purification of total triterpenoid saponins from Hemiptera

[0071] Soak each X-type macroporous adsorption resin in twice the volume of 95% ethanol for 24 hours, then filter. Wet-load the resin onto a column (Φ2.0 cm × 30 cm). Wash with ethanol until the effluent, mixed with water (1:3, v / v), is free of white turbidity and absorbs zero at a wavelength of 200-600 nm. Rinse with distilled water until the alcohol odor is eliminated. Store the resin in a wet state until ready for use.

[0072] Static adsorption-desorption method for resin screening: 5 g of each pretreated resin was placed in an Erlenmeyer flask. 40 ml of a solution of the semi-arid test sample (containing a triterpene aglycone concentration of XXX mg / ml (this concentration was determined by colorimetry)) was precisely added. The flask was sealed and placed in a constant temperature shaker for 24 hours of adsorption. The mixture was filtered and the triterpene aglycone content in the filtrate was determined. The specific adsorption capacity and adsorption rate were calculated. After absorbing the liquid on the surface of the resin, the resin was transferred to an Erlenmeyer flask and 75 ml of 95% ethanol was added to each flask. The mixture was shaken and eluted for 24 hours. The mixture was filtered and the triterpene aglycone content in the filtrate was determined. The specific elution capacity and elution rate were calculated. The calculation formulas for each value are as follows:

[0073] Specific adsorption capacity = (C0V0-C1V1) / W

[0074] Adsorption rate (%) = (C0V0-C1V1) / C0V0×100%

[0075] Specific elution volume = C2 V2 / W

[0076] Elution rate (%) = C2 V2 / (C0 V0-C1 V1) × 100%;

[0077] Transfer rate = adsorption rate × elution rate

[0078] Where: C0 is the mass concentration of the solution before adsorption (mg / ml), C1 is the mass concentration of the solution after adsorption (mg / ml), V is the corresponding solution volume (ml), V1 is the volume of the solution after adsorption (ml), V2 is the volume of the solution after elution (ml), V2 is the mass concentration of the solution after elution (mg / ml), and W is the mass of the resin (g).

[0079] (3) Determination of the content of total triterpenoid saponins in Hemiptera

[0080] Take a 0.2 mg / mL solution of the test sample and oleanolic acid reference solution, scan the chromatograms over the 200-600 nm wavelength range. Record the chromatograms. Separately, take 0.2 mL of each 0.2 mg / mL solution of the test sample and reference solution in a test tube. After evaporating the solvent, add 0.2 mL of a 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid. Heat in a 70°C water bath for 20 minutes. After cooling, add 5 mL of glacial acetic acid, shake well, and scan the chromatograms over the 200-600 nm wavelength range. The color-developed extract and the reference solution share a common absorption peak at 549 nm, so 549 nm was used as the detection wavelength.

[0081] To prepare a standard curve, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of oleanolic acid reference solution were taken, and the absorbance (A) was measured after color development. A linear regression was performed with oleanolic acid content as the abscissa and A value (Y) as the ordinate. The regression equation was Y = 8.6744X + 0.0066, with R² = 0.9993. Good linearity was observed within the oleanolic acid content range of 10 to 100 μg.

[0082] (4) Results and analysis

[0083] ①Effects of ethanol concentration, initial solid-liquid ratio and reflux extraction time on the extraction yield of total triterpenoid saponins from Helianthus annuus

[0084] When the ethanol concentration is 80%, the extraction rate of triterpenoid saponins reaches the maximum. After that, as the ethanol concentration increases, the triterpenoid extraction rate decreases. This may be because the increase in ethanol concentration increases the dissolution of impurities and hinders the dissolution of triterpenoid saponins. Therefore, the experiment selected 80% ethanol concentration as the optimized value, the initial solid-liquid ratio was 1:30, and the reflux extraction time was 2h.

[0085] ②The effect of extraction times on the extraction yield of total triterpenoid saponins from Hemiptera

[0086] When the number of extractions varied from 1 to 3, the extraction rate of triterpenoid saponins increased significantly. When the number of extractions was greater than 3, the change in the extraction rate of triterpenoid saponins tended to be flat as the number of extractions increased, and there was little significance in continuing to increase the number of extractions. Therefore, the experiment selected 1 extraction as the optimal value.

[0087] ③Effects of the ratio of concentrate to liquid and reflux acid hydrolysis time on the extraction yield of total triterpenoid saponins from Hemiptera japonica

[0088] When the liquid-to-material ratio is small, there is less solvent, so the extraction yield is small. As the solvent increases, the solvent contacts the material very fully, but the dissolution of triterpenoid saponins in the material will reach the upper limit. Therefore, the experiment selected a liquid-to-material ratio of 1:15 as the best optimization value.

[0089] When the reflux acid hydrolysis time was changed between 1 and 3 h, there was no significant difference in the extraction yield of triterpenoid saponins; when the extraction time was 2 h, the extraction yield of triterpenoid saponins increased significantly.

[0090] ④The effect of ethyl acetate extraction times on the extraction yield of total triterpenoid saponins from Hemiptera japonica

[0091] When the number of ethyl acetate extractions varied from 4 to 5, the yield of triterpenoid saponins gradually increased; however, when the number of ethyl acetate extractions reached 6, the yield of triterpenoid saponins did not increase significantly. Therefore, the experiment selected 5 ethyl acetate extractions as the optimal value.

[0092] ⑤Effect of macroporous adsorption resin on the enrichment of total triterpenoid saponins in Helianthus annuus

[0093] The adsorption and elution performance of three macroporous adsorption resins, AB-8, D101 and ADS-17, on the total triterpenoid saponins from Hemiptera were studied respectively. The best separation resin was screened and the effluent was collected to determine the triterpenoid content. The best macroporous adsorption resin was AB-8.

[0094] Table 2 Static adsorption rate and elution rate of triterpenoid sapogenins by three types of macroporous adsorption resins

[0095]

[0096] Table 3 Optimal extraction methods for triterpenoid saponins from different tissues of Hemiptera japonica

[0097]

[0098] The optimal process flow is as follows: take 5 g of dry powder of Hemiptera japonica, reflux extract with 80% ethanol for 2 h, the solid-liquid ratio is 1:30 (g / ml), extract 1 or 2 times, filter, combine the filtrates, and concentrate under reduced pressure to a solid-liquid ratio of about 1:15 (g / ml), add an appropriate amount of 20% H2SO4 (to make a 3% sulfuric acid solution), reflux acid hydrolyze for 2 h, cool to room temperature, adjust the pH to 5 with 20% NaOH solution, extract with ethyl acetate (100 ml×5 times), combine the extracts, and distill to dryness to obtain triterpenoid sapogenin extract. Dissolve the triterpenoid sapogenin extract in 80% ethanol, take the pretreated AB-8 macroporous adsorption resin, place it in a conical flask, add 40ml of the Hemiptera quinata test sample solution, seal it, place it on a constant temperature shaker for 24 hours of adsorption, filter it, dry the liquid on the resin surface, transfer it to a conical flask, add 75ml of 95% ethanol, shake and elute it for 24 hours, and filter it to obtain the purified Hemiptera quinata total triterpenoid saponins. This method has good reproducibility, low instrument requirements, and is stable and reliable, providing a reference for the industrial extraction, further development, and utilization of Hemiptera quinata triterpenoid saponins.

[0099] Example 2

[0100] 1. Standards and reagents

[0101] Standards: Asiatic acid (CAS: 464-92-6), Atractylodes lactone (CAS: 73069-13-3), Maslinic acid (CAS: 4373-41-5), Betulinic acid (CAS: 472-15-1), Oleanolic acid (CAS: 508-02-1), Oleanolic acid (CAS: 17990-42-0).

[0102] Reagents: HPLC-acetonitrile (Shanghai Xingke), experimental water (Watsons).

[0103] 2. Draw a standard curve

[0104] Prepare the above-mentioned standard substances into a 1 mg / mL standard stock solution with methanol and store it in a refrigerator at -20°C. Then dilute the standard stock solution with methanol to prepare a series of standard solutions with concentrations of 10, 20, 50, 100, and 200 mg / L, which are prepared and used immediately.

[0105] 3. Chromatographic conditions

[0106] Chromatographic column: Agilent Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm);

[0107] Column temperature: 30°C;

[0108] Mobile phase: Phase A: 0.1% formic acid / water solution; Phase B: acetonitrile;

[0109] Flow rate: 1.0 mL / min;

[0110] Injection volume: 10 μL;

[0111] Pool temperature: 40°C;

[0112] Detection wavelength: 203nm;

[0113] The gradient elution program is shown in Table 4:

[0114] Table 4

[0115]

[0116] Table 5 Different sources of Banfenghe samples

[0117]

[0118] The samples in Table 5 were treated by the method of Example 1 to prepare the test solutions, and the fingerprints of batches S1-S10 were obtained. The results are shown in Figure 1-Figure 2 ,in Figure 1 This is the fingerprint of the root of Hedera chinensis. Figure 2 The fingerprint of Heliconia sibiricum leaves is shown in Table 6.

[0119] Table 6 Retention time of characteristic chromatographic peaks in fingerprint

[0120]

[0121] Example 3 Precision Test

[0122] Take the half-leaf maple leaf of batch S1 and the half-leaf maple root of batch S2 in Table 5, prepare the test solution according to the method of Example 1, and detect the test solution according to the chromatographic conditions of Example 2. The sample was injected 6 times continuously to obtain chromatograms including 6 common peaks, wherein the 6 common peaks were numbered in sequence, and the peak areas and retention times of the 6 common peaks were obtained, and the relative standard deviation (RSD) was calculated. The results are shown in Tables 7 and 8. The results show that the precision of the method of the present invention is good.

[0123] Table 7 Precision results of half-maple lotus leaf

[0124]

[0125] Table 8 Precision results of semi-maple root

[0126]

[0127] Example 4 Repeatability Test

[0128] Take the half-leaf maple leaf of batch S1 and the half-leaf maple root of batch S2 in Table 5, prepare the test solution according to the method of Example 1, prepare 6 parallel portions, and detect the test solution according to the chromatographic conditions of Example 2, and obtain chromatograms including 6 common peaks, wherein the 6 common peaks are numbered in sequence, and the peak areas and retention times of the 6 common peaks are obtained, and the relative standard deviation (RSD) is calculated. The results are shown in Tables 9 and 10. The results show that the method of the present invention has good repeatability.

[0129] Table 9 Repeatability results of half-maple lotus leaf

[0130]

[0131] Table 10 Repeatability results of Hemiptera root

[0132]

[0133] Example 5 Stability Test

[0134] The leaves of the Hemiptera japonica from batch S1 and the roots of the Hemiptera japonica from batch S2 in Table 5 were sampled and tested according to the chromatographic conditions of Example 2 at 1, 4, 8, 16, 24, and 48 hours, respectively. Chromatograms including 6 common peaks were obtained, and the 6 common peaks were numbered in sequence. The peak areas and retention times of the 6 common peaks were obtained, and the relative standard deviations (RSDs) were calculated. The results are shown in Tables 11 and 12. The results show that the test solutions were stable after being placed at room temperature for 48 hours.

[0135] Table 11 Stability results of half-maple lotus leaf

[0136]

[0137] Table 12 Stability results of semi-maple root

[0138]

[0139] Example 6 Construction of standard fingerprints of leaves and roots of Hemiptera chinensis

[0140] The fingerprints of 5 batches of Hemiptera leaves and 5 batches of Hemiptera roots obtained in Example 2 were respectively taken and analyzed using Chinese medicine chromatographic fingerprint similarity evaluation software. The fingerprints of Hemiptera leaves of batch S1 and Hemiptera roots of batch S2 were used as reference maps. The average method was used with a time window width of 0.1. After multi-point correction, the chromatographic peaks were matched to obtain the standard fingerprints of Hemiptera leaves and Hemiptera roots. After analysis, the fingerprints of 5 batches of Hemiptera leaves and 5 batches of Hemiptera roots all had 6 common peaks, and the retention times of the 6 characteristic chromatographic peaks are shown in Table 13. The fingerprints of 5 batches of Hemiptera leaves and 5 batches of Hemiptera roots had a similarity of more than 0.920 relative to the standard fingerprint. The similarity results are shown in Table 14, which shows that the fingerprints of Hemiptera roots or leaves from each production area have high consistency.

[0141] Table 13 Fingerprint test results of Heliconia obesum leaves and roots from five provenances

[0142]

[0143]

[0144] Table 14 Similarity evaluation results of leaf and root fingerprints of Hemiptera chinensis from five provenances

[0145]

[0146] Example 7 Quality determination of the semi-maple to be tested

[0147] Take the leaf or root of the Herba Cyperi to be tested, and obtain a chromatogram of the leaf or root of the Herba Cyperi to be tested according to the method of Example 2. According to the retention time of Example 2, determine the six characteristic chromatographic peaks of the Herba Cyperi to be tested and obtain their peak areas. Use traditional Chinese medicine chromatographic fingerprint similarity evaluation software to perform similarity evaluation on the chromatogram of the Herba Cyperi to be tested and the standard fingerprint of the Herba Cyperi obtained in Example 2. If the similarity is ≥0.90, it is determined that the quality of the Herba Cyperi is qualified.

[0148] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing a fingerprint of Panfenghe, characterized in that: The following steps are involved: (1) Take 5g of Hemiptera helichrysum dry powder, extract with 80% ethanol under reflux for 1.5-2.5h, with a solid-liquid ratio of g / ml of 1:28-32, extract once or twice, filter, combine the filtrates, and concentrate under reduced pressure to a solid-liquid ratio of g / ml of 1:14-16, add 20% H2SO4 by mass to make a solution containing 2-4% sulfuric acid, reflux and acidify for 1.5-2.5h, cool to room temperature, adjust pH to 5, and use 90-110 ml ethyl acetate for 4-6 times, the extracts are combined and distilled to dryness to obtain triterpenoid sapogenin extract, the triterpenoid sapogenin extract is dissolved in 80% ethanol, 30-50 ml of the pretreated AB-8 macroporous adsorption resin is added to the above-dissolved triterpenoid sapogenin extract solution, the mixture is sealed and subjected to constant temperature oscillation adsorption for at least 20 hours, filtered, the liquid on the resin surface is dried, and 70-80 ml of 95% ethanol is added, the mixture is shaken and eluted for at least 20 hours, and filtered to obtain the purified total triterpenoid saponins of Helianthus annuus. (2) Detecting the total triterpenoid saponins of the Heterocarpus serratus obtained in step (1) by high performance liquid chromatography to obtain a chromatogram of Heterocarpus serratus, wherein the chromatogram of Heterocarpus serratus is the fingerprint of Heterocarpus serratus; wherein the chromatographic conditions are as follows: Chromatographic column: Agilent Eclipse Plus C18 (4.6 mm × 250 mm, 5 μm); Column temperature: 30°C; Mobile phase: Phase A: 0.1% formic acid / water solution; Phase B: acetonitrile; Flow rate: 1.0 mL / min; Injection volume: 10 μL; Pool temperature: 40°C; Detection wavelength: 203nm; The gradient elution program is shown in the following table: 。 2. The construction method according to claim 1, characterized in that The Banfenghe dry powder in step (1) is the dry powder of the roots or leaves of Banfenghe dry powder.

3. The construction method according to claim 2, characterized in that In step (1), 80% ethanol was refluxed for 2 h.

4. The construction method according to claim 3, characterized in that In the step (1), the solid-liquid ratio of the Helianthus annuus dry powder to 80% ethanol is 1:30 in g / ml.

5. The construction method according to claim 4, characterized in that: In the step (1), 20% by mass of H2SO4 is added to prepare a 3% sulfuric acid solution.

6. The construction method according to claim 5, characterized in that: In step (1), extraction was performed five times with 100 ml of ethyl acetate.

7. A method for constructing a standard fingerprint of Panax notoginseng, characterized in that: The method comprises: Take R portions of Hemiptera root or leaf, and obtain fingerprints of the R portions of Hemiptera root or leaf according to the method according to any one of claims 1 to 6; analyze the fingerprints of the R portions of Hemiptera root or leaf using traditional Chinese medicine chromatographic fingerprint similarity evaluation software to obtain a standard fingerprint of the Hemiptera root or leaf; wherein R≥5.

8. Use of the method for constructing the fingerprint of Hemiptera as described in any one of claims 1 to 6 in the quality control of Hemiptera; the standard fingerprint of Hemiptera leaves / roots comprises 6 characteristic chromatographic peaks with retention times of 25.838±0.05min, 31.105±0.05min, 33.769±0.05min, 40.352±0.05min, 41.389±0.05min, and 44.112±0.05min, respectively.

9. Use of the method for constructing a standard fingerprint of Hemiptera as claimed in claim 7 in quality control of Hemiptera; the standard fingerprint of Hemiptera leaves / roots comprises 6 characteristic chromatographic peaks with retention times of 25.838±0.05 min, 31.105±0.05 min, 33.769±0.05 min, 40.352±0.05 min, 41.389±0.05 min, and 44.112±0.05 min, respectively.

10. A quality control method for Banfenghe, characterized in that: The method comprises: (1) taking a leaf or root of the to-be-tested Hedera japonica, and obtaining a fingerprint of the leaf or root of the to-be-tested Hedera japonica according to the method according to any one of claims 1 to 6; (2) The fingerprint of the test Hemiptera leaf or root obtained in step (1) and the standard fingerprint of Hemiptera leaf or root constructed according to the method for constructing a standard fingerprint of Hemiptera according to claim 7 are evaluated for similarity. If the similarity is ≥0.90, the quality standard of the test Hemiptera is determined.

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