Dicliptera chinensis detection method

Abstract

The invention provides a dicliptera chinensis detection method, and belongs to the technical field of analysis and detection. The detection method comprises a thin-layer chromatography identification method, and the thin-layer chromatography identification method comprises the following steps: preparing a thin-layer test sample solution; preparing a thin-layer reference medicinal material solution; preparing a thin-layer reference substance solution; and performing thin-layer chromatographic analysis. According to the method, the dicliptera chinensis sample and the fake Japanese peristrophe herb can be accurately distinguished, the separation effect is good, the specificity is good, and the durability is high.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly relates to a detection method for Dicliptera chinensis (L.) Nees. Background Art

[0002] Dicliptera chinensis (L.) Nees is the dried whole herb of the plant Dicliptera chinensis (L.) Nees in the family Acanthaceae. The roots are fibrous and light yellow. The stems are much branched and zigzag, 30-80 cm long and 0.2-0.3 cm in diameter; the surface is grayish green, covered with sparse pubescence, with 4-6 obtuse edges, and the nodes are slightly swollen. The leaves are opposite, the leaf blades are mostly shrunken and broken, and when flattened, they are ovate or broadly ovate, 2.5-6 cm long and 1.5-3.5 cm wide; dark green or grayish green, the apex is acuminate, the base is broadly cuneate or slightly decurrent, and the margin is entire; both surfaces are nearly glabrous or the midrib on the lower surface is covered with sparse pubescence; the petiole is 0.2-2.5 cm long. Some are with flowers, the flowers are axillary, and are composed of several capitula into a corymbose or paniculate inflorescence: the bracts are opposite, leaf-like, broadly ovate or nearly round, of unequal size, 0.6-1 cm long, and there are several two-lipped flowers inside. It has a slight odor and tastes light and slightly sweet.

[0003] Dicliptera chinensis (L.) Nees has the effects of clearing heat, cooling blood, promoting diuresis, and detoxifying. It is mainly used to treat cold and fever, febrile macules, hematemesis, epistaxis, hematochezia, hematuria, metrorrhagia and metrostaxis, cough due to lung heat, sore throat, red eyes due to liver heat, infantile convulsion, dysuria, leukorrhea, herpes zoster, carbuncles, furuncles, snake and dog bites. It has extremely high medicinal value.

[0004] Peristrophe japonica (Thunb.) Bremek. is a herbaceous plant in the genus Peristrophe of the family Acanthaceae. Dicliptera chinensis (L.) Nees and Peristrophe japonica (Thunb.) Bremek. are often misused because of their similar appearances, and it is difficult to distinguish them with the naked eye. Therefore, it is necessary to establish a method for identifying Dicliptera chinensis (L.) Nees. Summary of the Invention

[0005] The purpose of the present invention is to provide a detection method for Dicliptera chinensis (L.) Nees.

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

[0007] The present invention provides a detection method for Dicliptera chinensis (L.) Nees, and the detection method includes a thin-layer chromatography identification method, and the thin-layer chromatography identification method includes the following steps:

[0008] Preparation of the thin-layer test solution: The sample to be tested is extracted with an organic solvent to obtain an organic solvent extract, which is evaporated to dryness to obtain a residue, and the residue is dissolved in an organic solvent to obtain the thin-layer test solution;

[0009] Preparation of thin-layer control medicinal material solution: Extract the Dicliptera chinensis (Osbeck) Nees control medicinal material with an organic solvent to obtain an organic solvent extract. Evaporate the extract to dryness to obtain a residue. Dissolve the residue in an organic solvent to obtain the thin-layer control medicinal material solution;

[0010] Preparation of thin-layer reference substance solution: Dissolve the Dicliptera chinensis (Osbeck) Nees reference substance in methanol to obtain the thin-layer reference substance solution;

[0011] Thin-layer chromatography analysis: Spot the thin-layer test sample solution, the thin-layer control medicinal material solution, and the thin-layer reference substance solution on the same thin-layer plate respectively. Develop with a developing agent, take out, air dry, and examine; the developing agent is ethyl acetate - butanone - formic acid - water, and the volume ratio of ethyl acetate, butanone, formic acid, and water is 4 - 6:0.8 - 1.2:0.8 - 1.2:0.8 - 1.2.

[0012] Preferably, the Dicliptera chinensis (Osbeck) Nees reference substance isschaftoside.

[0013] Preferably, the organic solvent is methanol with a concentration of 40 - 60%, and the mass - volume ratio of the test sample or the Dicliptera chinensis (Osbeck) Nees control medicinal material to the organic solvent during extraction is 0.8 - 1.2 g:15 - 25 mL;

[0014] The extraction method is heating under reflux, and the heating reflux time is 20 - 40 min;

[0015] When the residue is dissolved in an organic solvent, the mass - volume ratio of the organic solvent to the test sample or the Dicliptera chinensis (Osbeck) Nees control medicinal material is 0.8 - 1.2 mL:0.8 - 1.2 g.

[0016] Preferably, dissolve the Dicliptera chinensis (Osbeck) Nees reference substance in methanol to form a solution with a concentration of 0.4 - 0.6 mg / mL;

[0017] The thin-layer plate is a silica gel G thin-layer plate, and the dosage of each solution during thin-layer chromatography analysis is 2 - 15 μL;

[0018] The examination method is: Spray the thin-layer plate with aluminum trichloride solution, dry it with hot air, and examine it under an ultraviolet lamp.

[0019] Preferably, the detection method further includes a fingerprint detection method, and the fingerprint detection method includes the following steps:

[0020] Preparation of liquid-phase test sample solution: Extract the test sample with an organic solvent, filter, and take the filtrate to obtain the liquid-phase test sample solution;

[0021] Preparation of liquid-phase control medicinal material reference solution: Extract the Dicliptera chinensis (Osbeck) Nees control medicinal material with an organic solvent, filter, and take the filtrate to obtain the liquid-phase control medicinal material reference solution;

[0022] Preparation of liquid-phase reference substance solution: Dissolve the Dicliptera chinensis (Osbeck) Nees reference substance in methanol to obtain the liquid-phase reference substance solution;

[0023] High-performance liquid chromatography detection: Inject the liquid-phase test sample solution, the liquid-phase reference crude drug reference substance solution, and the liquid-phase reference substance solution into a liquid chromatograph for determination.

[0024] Preferably, the Dicliptera chinensis (Osbeck) Nees reference substance isschaftoside.

[0025] Preferably, the organic solvent is ethanol with a concentration of 40-60%, and the mass-volume ratio of the test sample or the Dicliptera chinensis (Osbeck) Nees reference crude drug to the organic solvent during extraction is 0.4-0.6 g: 15-25 mL;

[0026] The extraction method is heating under reflux, and the heating reflux time is 20-40 min;

[0027] The Dicliptera chinensis (Osbeck) Nees reference substance is dissolved in methanol to form a solution with a concentration of 15-25 μg / mL.

[0028] Preferably, the chromatographic column used in the high-performance liquid chromatography detection is CAPCELLPAK MGⅡ, 5.0 μm, 4.6×250 mm; the packing material is octadecylsilane chemically bonded silica; mobile phase A is acetonitrile, and mobile phase B is 0.08-0.12% phosphoric acid solution; gradient elution is adopted; the detection wavelength is 335 nm, the flow rate is 0.7-0.9 mL / min, and the column temperature is 23-27 °C.

[0029] Preferably, the conditions for gradient elution are as follows:

[0030] 0-20 min, the volume percentage of mobile phase A changes from 13% to 15%;

[0031] 20-35 min, the volume percentage of mobile phase A is maintained at 15%;

[0032] 35-40 min, the volume percentage of mobile phase A changes from 15% to 16%.

[0033] The present invention also provides an application of the above method in the detection of Dicliptera chinensis (Osbeck) Nees.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention provides a detection method for Dicliptera chinensis (Osbeck) Nees, and the method includes a thin-layer chromatography identification method and a fingerprint detection method. The method of the present invention can accurately distinguish Dicliptera chinensis (Osbeck) Nees samples from the fake Dicliptera polycephala (Champ. ex Benth.) C. B. Clarke, has a good separation effect, good specificity, and strong durability. Description of the Drawings

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0037] Figure 1 It is the thin-layer chromatography identification result in Example 1;

[0038] Figure 2 It is the result of thin-layer chromatography identification using thin-layer silica gel G plates produced by different manufacturers in Example 2;

[0039] Figure 3 It is the result of thin-layer chromatography identification under different developing temperatures and different relative humidity conditions in Example 2;

[0040] Figure 4 It is the result of thin-layer chromatography identification with different sample application amounts in Example 2;

[0041] Figure 5 It is the result of thin-layer chromatography identification of test solution samples prepared with different extraction solvents in Example 3;

[0042] Figure 6 It is the result of thin-layer chromatography identification of test solution samples prepared with different extraction methods in Example 3;

[0043] Figure 7 It is the thin-layer chromatography identification result obtained with different color development methods in Example 3;

[0044] Figure 8 It is the thin-layer chromatography identification result obtained with different sample application methods in Example 3;

[0045] Figure 9 It is the result of thin-layer chromatography identification of 18 batches of Dicliptera chinensis samples in Example 4;

[0046] Figure 10 It is the result of thin-layer chromatography identification of Dicliptera chinensis samples and Peristrophe japonica samples in Example 4;

[0047] Figure 11 It is the high performance liquid chromatography detection result in Example 5;

[0048] Figure 12 It is the result of high performance liquid chromatography detection using methanol - 0.1% phosphoric acid system and methanol - water system in Example 6;

[0049] Figure 13Results of high performance liquid chromatography (HPLC) detection using acetonitrile - water system and acetonitrile - 0.1% phosphoric acid system in Example 6;

[0050] Figure 14 Results of HPLC detection of test samples prepared with water and 50% ethanol as extraction solvents in Example 7;

[0051] Figure 15 Results of HPLC detection of test samples prepared with 50% methanol and absolute ethanol as extraction solvents in Example 7;

[0052] Figure 16 Results of HPLC detection obtained under the optimal test sample solution preparation conditions in Example 7;

[0053] Figure 17 Standard curves of dicaffeoylquinic acid - 2 and dicaffeoylquinic acid - 1 in Example 8;

[0054] Figure 18 Standard curves of acteoside and isoacteoside in Example 8;

[0055] Figure 19 Results of HPLC detection of 50% ethanol in Example 8;

[0056] Figure 20 Characteristic fingerprints of 18 batches of Dicliptera chinensis samples in Example 9;

[0057] Figure 21 Characteristic fingerprints of Dicliptera chinensis samples and samples of the fake Clinacanthus nutans in Example 9. Detailed implementation manners

[0058] The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0059] The sources of the Dicliptera chinensis samples used in the following examples are shown in Table 1. Among them, the Dicliptera chinensis sample 2 (GGC002) was collected in the wild by the staff of the Traditional Chinese Medicine Laboratory of the Guangxi Zhuang Autonomous Region Institute for Drug Control. After identification, it was determined to be Dicliptera chinensis, a plant of the genus Dicliptera in the Acanthaceae family. In the experiment, this sample was used as the control medicinal material of Dicliptera chinensis for comparison with other samples. The sources of the samples of the fake Clinacanthus nutans are shown in Table 2. All samples were pulverized and passed through a No. 3 sieve for standby.

[0060] Table 1 Information of Dicliptera chinensis samples

[0061] Number Name Origin Harvest Location GGC001 Dicliptera chinensis (L.) Juss. Guangzhou, Guangdong Guangzhou, Guangdong GGC002 Dicliptera chinensis (L.) Juss. Meizhou, Guangdong Meizhou, Guangdong GGC003 Dicliptera chinensis (L.) Juss. Meizhou, Guangdong Meizhou, Guangdong GGC004 Dicliptera chinensis (L.) Juss. Liuzhou, Guangxi Tianshuizhai, Sanjiang, Liuzhou, Guangxi GGC005 Dicliptera chinensis (L.) Juss. Guilin, Guangxi Longji Town, Longsheng, Guangxi GGC006 Dicliptera chinensis (L.) Juss. Zhangzhou, Fujian Bozhou, Anhui GGC007 Dicliptera chinensis (L.) Juss. Fuzhou, Fujian Lotus Pond, Sichuan GGC008 Dicliptera chinensis (L.) Juss. Lipu, Guangxi Lipu, Guangxi GGC009 Dicliptera chinensis (L.) Juss. Lipu, Guangxi Lipu, Guangxi GGC010 Dicliptera chinensis (L.) Juss. Guangdong Lipu, Guangxi GGC011 Dicliptera chinensis (L.) Juss. Shenzhen, Guangdong Yulin, Guangxi GGC012 Dicliptera chinensis (L.) Juss. Nanjing, Fujian Yulin, Guangxi GGC013 Dicliptera chinensis (L.) Juss. Nanjing, Fujian Yulin, Guangxi GGC014 Dicliptera chinensis (L.) Juss. Huaiji, Guangdong Yulin, Guangxi GGC015 Dicliptera chinensis (L.) Juss. Yulin, Guangxi Yulin, Guangxi GGC016 Dicliptera chinensis (L.) Juss. Maling, Lipu, Guangxi Maling, Lipu, Guangxi GGC017 Dicliptera chinensis (L.) Juss. Qingshan, Lipu, Guangxi Qingshan, Lipu, Guangxi GGC018 Dicliptera chinensis (L.) Juss. Pulu, Lipu, Guangxi Pulu, Lipu, Guangxi

[0062] Table 2 Information of samples of the fake Clinacanthus nutans

[0063]

[0064]

[0065] The equipment and materials used in thin-layer chromatography identification are: Kunshan KQ800D type numerical control ultrasonic cleaner (320W, 40KHZ), Mettler AB204-S electronic balance, CAMAG thin-layer ultraviolet digital imaging system, andschaftoside reference substance (National Institutes for Food and Drug Control, batch number 111912-202204).

[0066] The equipment and materials used in high-performance liquid chromatography detection are: Agilent 1260 high-performance liquid chromatograph; Agilent1260 Infinity II high-performance liquid chromatograph; waters 2690 high-performance liquid chromatograph; DAD ultraviolet detector; Milli-Q pure water processor; ultrasonic cleaner (Jiangsu Kunshan Ultrasonic Instrument Co., Ltd.); Kromasil 100-5-C18 (250×4.6mm) chromatographic column; CAPCELLPAK C18 MGⅢ(250×4.6mm) chromatographic column; CAPCELLMGⅡ(250×4.6mm) chromatographic column; CP224S electronic analytical balance (Sartorius AG, Germany), XS205 electronic analytical balance (Mettler Toledo AG, Switzerland).schaftoside reference substance (National Institutes for Food and Drug Control, batch number 111912-202204, content 94.9%), wiesenin-1 (Wuhan Tianzhi Biotechnology Co., Ltd., CFN92030, content ≥98%), wiesenin-2 (Wuhan Tianzhi Biotechnology Co., Ltd., CFN92031, content ≥98%), isoschaftoside (Dest Bio, DST231216-019, content ≥98%). Acetonitrile is of chromatographic purity, water is of high purity, and other reagents are of analytical purity.

[0067] Example 1

[0068] A method for thin-layer chromatography identification of Dicliptera chinensis (L.) Juss., the steps are as follows:

[0069] Thin-layer test sample solution: Weigh 1g of the powder of the Dicliptera chinensis (L.) Juss. sample to be tested, add 20 mL of 50% methanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the thin-layer test sample solution.

[0070] Thin-layer control medicinal material solution: Weigh 1g of the powder of the control medicinal material (GGC002), add 20 mL of 50% methanol, heat under reflux for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 mL of methanol to obtain the thin-layer control medicinal material solution.

[0071] Thin-layer reference solution: Take the reference substance of shafto-side and make a solution with methanol at a concentration of 0.5 mg per 1 mL to obtain the thin-layer reference solution.

[0072] Test according to the thin-layer chromatography method (General Principles 0502, Volume IV, Chinese Pharmacopoeia 2020 Edition). Pipette 5 μL of each of the above three solutions and spot them on the same silica gel G thin-layer plate (Qingdao Ocean Chemical Co., Ltd., 20240821) respectively. Use ethyl acetate - butanone - formic acid - water (5:1:1:1) as the developing solvent. Develop at a temperature of 25 °C, a relative humidity of 40%, and a developing distance of 8 cm. Take out, dry in air, immediately spray with aluminum trichloride solution, dry with hot air, and examine under an ultraviolet lamp (365 nm). In the chromatogram of the test sample, at the positions corresponding to the chromatograms of the reference medicinal material and the reference substance, fluorescent spots of the same color should appear.

[0073] The results of thin-layer chromatography identification are as Figure 1 shown. Among them, band 1 is the negative sample (the reagent for extracting Dicliptera chinensis, i.e., 50% methanol), band 2 is the reference medicinal material of Dicliptera chinensis GGC002, band 3 is the reference substance of shafto-side, band 4 is the Dicliptera chinensis sample 10 (GGC010), band 5 is the Dicliptera chinensis sample 11 (GGC011), and band 6 is the Dicliptera chinensis sample 17 (GGC017). It can be seen that in the chromatogram of the test sample, at the positions corresponding to the chromatograms of the reference medicinal material of Dicliptera chinensis and the reference substance, fluorescent spots of the same color appear. The thin-layer chromatography separation effect is good, the spots are clear, the retention factor is moderate, and there is no interference from the negative sample, and the specificity is good.

[0074] Example 2 Durability Experiment Investigation

[0075] The purpose of this example is to investigate the effects of thin-layer silica gel G plates produced by different manufacturers, different developing temperatures, different relative humidities, and different sample application amounts on the thin-layer chromatography identification effect. Except for the above differences, the other methods and parameters are the same as those in Example 1.

[0076] The results of thin-layer chromatography identification using thin-layer silica gel G plates produced by different manufacturers are as Figure 2 shown. From left to right are the results of thin-layer chromatography identification using thin-layer silica gel G plates produced by Qingdao Ocean Chemical Co., Ltd. (20240821), Yantai Jiangyou Silica Gel Development Co., Ltd. (020210308), and MERCK (HX33139821); among them, band 1 is the reference medicinal material of Dicliptera chinensis GGC002, band 2 is the Dicliptera chinensis sample 10 (GGC010), band 3 is the Dicliptera chinensis sample 11 (GGC011), band 4 is the Dicliptera chinensis sample 17 (GGC017), and band 5 is the reference substance of shafto-side.

[0077] The results of thin-layer chromatography identification under different developing temperatures and different relative humidities are as Figure 3As shown in the figure, the left side shows the thin-layer chromatography identification results under the conditions of a temperature of 4°C and a relative humidity of 25%, and the right side shows the thin-layer chromatography identification results under the conditions of a temperature of 25°C and a relative humidity of 40%. Among them, band 1 is the control medicinal material of Dicliptera chinensis (GGC002), band 2 is the sample 10 of Dicliptera chinensis (GGC010), band 3 is the sample 11 of Dicliptera chinensis (GGC011), band 4 is the sample 17 of Dicliptera chinensis (GGC017), and band 5 is the reference substance of harpagoside.

[0078] The results of chromatographic identification using different sample application amounts are as Figure 4 shown. Among them, band 1 is the control medicinal material of Dicliptera chinensis (GGC002), band 2 is the reference substance of harpagoside, and bands 3-6 are the results of sample application of 2 μL, 5 μL, 10 μL, and 15 μL of the sample 17 of Dicliptera chinensis (GGC017), respectively.

[0079] It can be seen that Dicliptera chinensis can be accurately identified by thin-layer chromatography using thin-layer silica gel G plates produced by different manufacturers, different developing temperatures, different relative humidities, and different sample application amounts. After development, the spots are clear, the resolution is good, and there is no tailing. This shows that the method of the present invention has good durability.

[0080] Investigation of thin-layer chromatography conditions in Example 3

[0081] The purpose of this example is to investigate the effects of extraction solvents, extraction methods, color development methods, and sample application methods on the thin-layer chromatography identification effect. Except for the above differences, the remaining methods and parameters are the same as those in Example 1.

[0082] 1. Investigation of extraction solvents

[0083] Using methanol, 50% methanol, and water as solvents respectively, prepare test solution samples with 3 different extraction solvents according to the preparation method of the test solution. Detect according to the thin-layer chromatography conditions described in Example 1. The results are as Figure 5 shown. Among them, bands 1-3 are the detection results of the test solution (GGC017) extracted using 50% methanol, methanol, and water as solvents respectively. It can be seen that when using 50% methanol as the extraction solvent, the main spot is clearer and there are fewer impurities. Therefore, 50% methanol is used as the extraction solvent.

[0084] 2. Investigation of extraction methods

[0085] Using 50% methanol as the solvent, prepare test solution samples using 2 different extraction methods: ultrasonic and reflux. Detect according to the thin-layer chromatography conditions described in Example 1. The results are as Figure 6 shown. Among them, band 1 is the result of the reflux method, and band 2 is the result of the ultrasonic method. The results show that when using reflux as the extraction method, the main spot is clearer. Therefore, reflux is used as the extraction method.

[0086] 3. Investigation of the color development method

[0087] After the thin layer plate development was completed, take it out, dry it in the air, compare the effects on the test results of immediately spraying with aluminum trichloride solution, drying with hot air, and examining under an ultraviolet lamp (365 nm), and spraying with aluminum trichloride solution after standing for 1 hour, drying with hot air, and examining under an ultraviolet lamp (365 nm). The results are as Figure 7 shown. Among them, the left figure shows the results of immediate color development, and the right figure shows the results of color development after standing for 1 hour. Band 1 is the control medicinal material of Dicliptera chinensis (GGC002), Band 2 is the sample 10 of Dicliptera chinensis (GGC010), Band 3 is the sample 11 of Dicliptera chinensis (GGC011), Band 4 is the sample 17 of Dicliptera chinensis (GGC017), and Band 5 is the reference substance of harpagide. It can be seen that after standing for 1 hour, the color of the main spot changes from green to blue, and it is not as clear as immediate color development. Therefore, immediately spraying with aluminum trichloride solution after drying in the air, drying with hot air, and examining under an ultraviolet lamp (365 nm) is used as the display condition.

[0088] 4. Investigation of the spotting method

[0089] Take the test solution respectively and spot it in dot and strip shapes for comparison. The results are as Figure 8 shown. Among them, Bands 1-3 are the results of dot spotting of the samples GGC010, GGC011, and GGC017 of Dicliptera chinensis respectively, Band 4 is the reference substance of harpagide, and Bands 5-7 are the results of strip spotting of the samples GGC010, GGC011, and GGC017 of Dicliptera chinensis respectively. It can be seen that the spots of strip spotting are clear and the resolution is good. Therefore, strip spotting is used as the spotting method.

[0090] Example 4

[0091] According to the method described in Example 1, thin layer chromatography identification was carried out on 18 batches of Dicliptera chinensis samples in Table 1. The results are as Figure 9 shown. Among them, Band 1 is the control medicinal material of Dicliptera chinensis (GGC002), Band 2 is harpagide, Band 3 is GGC001, Band 4 is GGC002, Band 5 is GGC003, Band 6 is GGC004, Band 7 is GGC005, Band 8 is GGC006, Band 9 is GGC007, Band 10 is GGC008, Band 11 is GGC009, Band 12 is GGC0010, Band 13 is GGC011, Band 14 is GGC012, Band 15 is GGC013, Band 16 is GGC014, Band 17 is GGC015, Band 18 is GGC016, Band 19 is GGC017, and Band 20 is GGC018.

[0092] Meanwhile, according to the method described in Example 1, thin-layer chromatography identification was performed on the Dicliptera chinensis samples and Peristrophe japonica samples, and the results are as Figure 10 shown. Among them, band 1 is JTSZC001, band 2 is JTSZC002, band 3 is JTSZC003, band 4 is JTSZC004, band 5 is JTSZC005, band 6 is JTSZC006, band 7 is harpagide, band 8 is GGC003, band 9 is GGC004, band 10 is GGC005, band 11 is GGC006, band 12 is GGC007, and band 13 is the control medicinal material of Dicliptera chinensis GGC002.

[0093] It can be seen that the thin-layer chromatograms of the Dicliptera chinensis samples and Peristrophe japonica samples are quite different, and the method of the present invention can distinguish the two well.

[0094] Example 5

[0095] A method for detecting the fingerprint of Dicliptera chinensis is as follows:

[0096] Preparation of the liquid-phase test sample solution: Take 0.5 g of the sample to be tested, add 20 mL of 50% ethanol solution, heat under reflux for 30 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain the liquid-phase test sample solution.

[0097] Preparation of the liquid-phase reference medicinal material reference solution: Place 0.5 g of the Dicliptera chinensis control medicinal material in a conical flask, add 20 mL of 50% ethanol solution, heat under reflux for 30 minutes, filter, and take the subsequent filtrate to obtain the liquid-phase reference medicinal material reference solution.

[0098] Preparation of the liquid-phase reference substance reference solution: Take an appropriate amount of harpagide reference substance, add methanol to make a solution containing 20 μg per 1 mL to obtain the liquid-phase reference substance reference solution.

[0099] Perform high-performance liquid chromatography detection according to the high-performance liquid chromatography method (General Principles 0512, Volume IV, Chinese Pharmacopoeia 2020 Edition): Precisely pipette 10 μL each of the reference solution and the test sample solution, and inject them into the liquid chromatograph for determination. Among them, the chromatographic column used in the high-performance liquid chromatography detection is CAPCELLPAK MGⅡ, 5.0 μm, 4.6×250 mm; the filler is octadecylsilane-bonded silica gel; mobile phase A is acetonitrile, and mobile phase B is 0.1% phosphoric acid solution; gradient elution is adopted, and the gradient elution conditions are: 0 - 20 min, the volume percentage of mobile phase A changes from 13% to 15%, 20 - 35 min, the volume percentage of mobile phase A is maintained at 15%, 35 - 40 min, the volume percentage of mobile phase A changes from 15% to 16%; the detection wavelength is 335 nm, the flow rate is 0.8 mL / min, and the column temperature is 25°C.

[0100] Six characteristic peaks should appear in the chromatogram of the test sample, and their retention times should correspond to those of the six characteristic peaks in the chromatogram of the reference medicinal material. Among them, the retention time of peak 5 should correspond to that of the reference peak ofschaftoside. The peak corresponding to the reference peak ofschaftoside is the S peak. Calculate the relative retention times of peaks 1, 2, 3, 4, and 6 with respect to the S peak, and the relative retention times should be within the range of ±10% of the specified values, which are 0.39 (peak 1), 0.66 (peak 2), 0.75 (peak 3), 0.89 (peak 4), and 1.29 (peak 6). The theoretical plate number calculated based onschaftoside should be not less than 5000. The obtained chromatogram is as shown in Figure 11 shown, where the abscissa ranges from 0 to 40, representing time (min); the ordinate ranges from 0 to 165, representing signal (mV); R(6).

[0101] Investigation of the chromatographic conditions in Example 6

[0102] The purpose of this example is to investigate the influence of the chromatographic conditions (detection wavelength, mobile phase composition, mobile phase elution program) in Example 5 on the results of high-performance liquid chromatography detection. Except for the above differences, the remaining steps and parameters are the same as those in Example 5.

[0103] 1. Investigation of the detection wavelength

[0104] Using a diode array detector for detection and extracting the spectral diagrams of each component, it was found that wogonin-1, wogonin-2,schaftoside, and isoschaftoside all had maximum absorption at 270 nm and 335 nm. Comparing the chromatograms under the two wavelength conditions, it was found that when measured at 335 nm, the separation of each component was good, there was no tailing phenomenon, and the interference was minimal. Therefore, the sample was determined under the condition of 335 nm.

[0105] 2. Investigation of the mobile phase composition

[0106] The effects of methanol-water system, acetonitrile-water system, methanol-0.1% phosphoric acid system, and acetonitrile-0.1% phosphoric acid solution system on the results of high-performance liquid chromatography detection were investigated respectively. The results are as shown in Figure 12 and Figure 13 shown. It can be seen that using the acetonitrile-0.1% phosphoric acid solution system as the mobile phase system for content determination can result in better peak shapes and separation degrees of each component, and the elution time is relatively appropriate.

[0107] 3. Investigation of the mobile phase elution program

[0108] In the experiment, isocratic elution and gradient elution programs were explored. To shorten the analysis time and achieve effective separation of each component, combined with the analysis of the experimental results, it was shown that gradient elution could meet the experimental requirements. The gradient elution conditions were as follows: acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B; the gradient elution conditions are shown in Table 3.

[0109] Table 3 Mobile Phase Gradient Elution Conditions

[0110] Time min Acetonitrile 0.1% Phosphoric Acid 0～20 13→15 87→85 20～35 15 85 35～40 15→16 85→84

[0111] Investigation on the Preparation of Test Solution in Example 7

[0112] The purpose of this example is to investigate the effects of the extraction solvent, extraction method, extraction solvent dosage, and extraction time used in the preparation of the test solution in Example 5 on the results of high-performance liquid chromatography detection. Except for the above differences, the remaining steps and parameters are the same as those in Example 5.

[0113] 1. Investigation on Extraction Solvent

[0114] The extraction effects of four solvents, namely 50% methanol, 50% ethanol, water, and absolute ethanol, were investigated separately. Take 0.5 g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of each of the above five solvents respectively, stopper tightly, weigh, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with 50% methanol (50% ethanol, water, absolute ethanol), shake well, filter, and take the subsequent filtrate to obtain the solution. The determination results are shown in Table 4 and Figure 14 - 15 as follows. It can be seen that the extraction efficiencies of the remaining three solvents are quite similar. Since the 50% ethanol solution is more environmentally friendly and the samples are not prone to mildew when stored for a long time, 50% ethanol was selected as the extraction solvent.

[0115] Table 4 Effects of Extraction Solvent on High-Performance Liquid Chromatography Detection Results

[0116]

[0117]

[0118] 2. Investigation on Extraction Method

[0119] The extraction effects of the 50% ethanol extraction methods (ultrasonic for 30 minutes, heating under reflux for 30 minutes) were investigated. The results are shown in Table 5. It can be seen that the contents of other components except isoschaftoside are higher when extracted by heating under reflux. Therefore, heating under reflux was selected as the experimental method.

[0120] Table 5 Effects of Extraction Method on High-Performance Liquid Chromatography Detection Results

[0121] Extraction Method Reflux for 30 minutes Ultrasonic for 30 minutes Content of Vicenin - 2 (mg / g) 0.1687 0.1336 Content of Vicenin - 1 (mg / g) 0.0953 0.0581 Content of Schaftoside (mg / g) 0.2770 0.2584 Content of Isoschaftoside (mg / g) 0.0902 0.0901

[0122] 3. Investigation on Extraction Solvent Dosage

[0123] The extraction effects with different amounts of 50% ethanol (10 mL, 20 mL, 50 mL, 100 mL) were investigated. The results are shown in Table 6. It can be seen that when the amount of 50% ethanol is 10 mL, the components to be measured are basically completely extracted. To reduce reagent waste, the amount of 50% ethanol was selected as 10 mL.

[0124] Table 6 Influence of the amount of extraction solvent on the results of high performance liquid chromatography detection

[0125] Extraction Solvent 10 mL 20 mL 50 mL 100 mL Content of Vicenin - 2 (mg / g) 0.1936 0.1877 0.1686 0.1619 Content of Vicenin - 1 (mg / g) 0.0997 0.0930 0.0953 0.0920 Content of Schaftoside (mg / g) 0.2843 0.2869 0.2768 0.2815 Content of Isoschaftoside (mg / g) 0.0911 0.0878 0.0902 0.0882

[0126] 4. Investigation of extraction time

[0127] The extraction effects with different reflux times of 50% ethanol (30 minutes, 60 minutes) were investigated. The results are shown in Table 7. It can be seen that when the reflux extraction time is 30 minutes, the components to be measured can be basically completely extracted. To save time, the extraction time was selected as 30 minutes.

[0128] Table 7 Influence of extraction time on the results of high performance liquid chromatography detection

[0129]

[0130]

[0131] 5. Optimal preparation conditions of the test solution

[0132] The preparation method of the test solution was determined as follows: Take this product, grind it finely, take 0.5 g, place it in a stoppered conical flask, accurately add 10 mL of 50% ethanol, weigh it, reflux for 30 minutes, cool it, weigh it again, make up the weight with 50% ethanol, shake well, filter, and take the subsequent filtrate to obtain. The results of high performance liquid chromatography detection are as Figure 16 shown.

[0133] Methodology investigation and determination of samples in Example 8

[0134] 1. Stability of the test solution

[0135] For the same test solution (GGC017), it was measured at regular intervals for a total of 6 times. The RSDs of the peak areas of wicaenin-2, wicaenin-1,schaftoside, and isoschaftoside in the 6 measurements met the requirements (see Table 8). The test showed that the test solution was stable within 24 hours.

[0136] Table 8 Results of the stability experiment

[0137]

[0138] 2. Precision test

[0139] For the same test solution (GGC017), under the chromatographic conditions described in Example 5, it was continuously determined 6 times. The RSDs of the peak areas of wicaenin-2, wicaenin-1,schaftoside, and isoschaftoside in the 6 determinations met the requirements (see Table 9). The test shows that the precision of the method of the present invention is good.

[0140] Table 9 Results of Instrument Precision Determination

[0141]

[0142]

[0143] 3. Repeatability Test

[0144] Take the same batch of test samples (GGC017), about 0.5 g, accurately weighed, and determined in parallel 6 times under the chromatographic conditions described in Example 5. Calculate the contents of wicaenin-2, wicaenin-1,schaftoside, and isoschaftoside respectively. The RSDs of the contents of wicaenin-2, wicaenin-1,schaftoside, and isoschaftoside in the 6 test solutions all met the requirements (see Table 10). The test shows that the repeatability of this method is good.

[0145] Table 10 Results of Repeatability Experiment

[0146]

[0147] 4. Determination of Accuracy (Spiking Recovery Rate)

[0148] Take an appropriate amount of Dicliptera chinensis sample with known contents of wiesenin-2 (0.1726 mg / g), wiesenin-1 (0.0883 mg / g),schaftoside (0.2416 mg / g), and isoschaftoside (0.0853 mg / g). Crush it and sieve through a No. 4 sieve. Weigh 6 portions in parallel, each about 0.25 g, accurately weigh, place in a stoppered conical flask, accurately add 10 mL of the recovery test mixed reference substance solution (wiesenin-2 3.8988 μg / mL, wiesenin-1 2.1188 μg / mL, schaftoside 5.9172 μg / mL, isoschaftoside 2.0149 μg / mL), stopper tightly, weigh, heat under reflux for 30 minutes, cool, weigh again, make up the lost weight with 50% ethanol, shake well, filter, and take the subsequent filtrate to obtain the solution. Determine according to the chromatographic conditions described in Example 5, calculate the recovery rate of added samples, and the results are shown in Table 11. The average recovery rate of wiesenin-2 is 101.50%, RSD = 1.15% (n = 6); the average recovery rate of wiesenin-1 is 95.96%, RSD = 1.18% (n = 6); the average recovery rate of schaftoside is 100.97%, RSD = 0.57% (n = 6); the average recovery rate of isoschaftoside is 98.23%, RSD = 1.82% (n = 6). All meet the requirements of quantitative analysis. The experiment shows that the recovery rate of the method of the present invention is good.

[0149] Table 11 Experimental results of recovery rate of added samples

[0150]

[0151] 5. Investigation of linear relationship

[0152] Accurately weigh 4.973 mg of wiesenin-2 reference substance, 5.297 mg of wiesenin-1 reference substance, 5.196 mg of schaftoside reference substance, and 5.140 mg of isoschaftoside reference substance, respectively place them in 10 mL volumetric flasks, add 50% ethanol to dissolve and dilute to the scale, shake well, and use as the stock solution of the reference substance solution. Accurately measure 1 mL of each of the stock solutions of wiesenin-2, wiesenin-1, schaftoside, and isoschaftoside reference substances into a 25 mL volumetric flask, dilute to the scale with 50% ethanol, and shake well to obtain the linear solution (1). Then accurately measure 800 μL, 400 μL, 1200 μL, and 400 μL of the stock solutions of wiesenin-2, wiesenin-1, schaftoside, and isoschaftoside reference substances into a 100 mL volumetric flask respectively, dilute to the scale with 50% ethanol, and shake well to obtain the linear solution (2).

[0153] Accurately aspirate 5, 10, 25 μL of the linear solution (2) and 5, 10, 15 μL of the linear solution (1), inject them into the liquid chromatograph, and determine according to the chromatographic conditions described in Example 5. Plot the standard curve with the injection amount of the reference substance as the abscissa (x) and the peak area integral value as the ordinate (y) (asFigure 17 and Figure 18 as shown), the regression equations are as follows:

[0154] Regression equation of vicenin-2: y = 2180x + 5609.7 (n = 6, R = 0.9999)

[0155] Regression equation of vicenin-1: y = 2592.7x - 4483.7 (n = 6, R = 0.9995)

[0156] Regression equation ofschaftoside: y = 2744.5x - 4054.1 (n = 6, R = 0.9998)

[0157] Regression equation of isoschaftoside: y = 2652.8x - 8275.2 (n = 6, R = 0.9997)

[0158] The results of the standard curve determination are shown in Table 12. The results show that when the injection volume of vicenin-2 is in the range of 19.4942 - 292.4124 μg, when the injection volume of vicenin-1 is in the range of 10.594 - 317.820 μg, when the injection volume ofschaftoside is in the range of 29.586 - 95.860 μg, and when the injection volume of isoschaftoside is in the range of 10.074 - 302.232 μg, there is a good linear relationship between the injection volume and the peak area.

[0159] Table 12 Results of the standard curve determination

[0160]

[0161]

[0162] 6. Specificity

[0163] Take 50% ethanol as the solvent and determine it according to the method described in Example 5. The results show that at the detection wavelength of 335 nm, there are no absorption peaks at the retention times corresponding to vicenin-2, vicenin-1,schaftoside, and isoschaftoside, indicating that the negative sample has no interference and the method of the present invention has good specificity ( Figure 19 ).

[0164] 7. Sample detection

[0165] Perform high performance liquid chromatography detection on the Dicliptera chinensis samples in Table 1 according to the method described in Example 5. The results are shown in Table 13.

[0166] Table 13 Detection results of Dicliptera chinensis samples

[0167]

[0168]

[0169] Example 9: Methodology Investigation and Sample Determination

[0170] 1. Instrument Precision Test

[0171] For the same test solution (GGC017), inject it continuously 6 times under the chromatographic conditions described in Example 5, and record the characteristic chromatogram. Calculate the retention time of the characteristic peaks. The results are shown in Table 14. It can be seen that the RSD of the relative retention time of each characteristic peak is < 3.0%, indicating that the instrument precision is good.

[0172] Table 14 Results of Instrument Precision Determination

[0173] Peak Number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S Peak) Peak 6 1 0.39 0.65 0.75 0.89 1.00 1.29 2 0.39 0.65 0.74 0.88 1.00 1.29 3 0.38 0.65 0.75 0.88 1.00 1.29 4 0.38 0.65 0.74 0.88 1.00 1.29 5 0.39 0.65 0.74 0.88 1.00 1.29 6 0.39 0.65 0.74 0.89 1.00 1.30 Average 0.39 0.65 0.74 0.88 1.00 1.29 RSD 1.51％ 0.36％ 0.38％ 0.43％ 0.00％ 0.29％

[0174] 2. Method Repeatability Test

[0175] For the same test solution (GGC017), prepare 6 parallel test solutions according to the method described in Example 5, and inject the test solutions into the high-performance liquid chromatograph under the chromatographic conditions described in Example 5, record the characteristic chromatogram, and calculate the retention time of the characteristic peaks. The results are shown in Table 15. It can be seen that the RSD of the relative retention time of each characteristic peak is < 3.0%, indicating that the repeatability of this method is good.

[0176] Table 15 Results of Method Repeatability Determination

[0177]

[0178]

[0179] 3. Method Stability Test

[0180] For the same test solution (GGC017), under the chromatographic conditions described in Example 5, measure it at 0h, 4h, 8h, 12h, 16h, and 24h respectively, record the characteristic chromatogram and calculate the retention time of the characteristic peaks. The results are shown in Table 16. The RSD of the relative retention time of each characteristic peak is < 3.0%, indicating that the stability of this method is good.

[0181] Table 16 Results of Method Stability Experiment

[0182] Peak Number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S Peak) Peak 6 1 0.39 0.65 0.75 0.89 1.00 1.29 2 0.39 0.65 0.74 0.88 1.00 1.30 3 0.39 0.66 0.74 0.89 1.00 1.30 4 0.39 0.66 0.74 0.88 1.00 1.29 5 0.39 0.65 0.75 0.88 1.00 1.25 6 0.40 0.65 0.74 0.89 1.00 1.25 Average 0.39 0.65 0.75 0.88 1.00 1.28 RSD 0.47％ 0.25％ 0.18％ 0.16％ 0.00％ 1.90％

[0183] 4. Column Durability Test

[0184] For the same test solution (GGC017), under the chromatographic conditions described in Example 5, the samples were determined using CAPCELL PAK C18 MGⅡ (5μm, 4.6×250mm), Kromasil 100-5-C18 (5μm, 4.6×250mm) and CAPCELL PAK C18 MGⅢ (5μm, 4.6×250mm) chromatographic columns respectively. The results are shown in Table 17. The RSD of the relative retention time of each characteristic peak < 3.0%, indicating that the chromatographic column durability of this method is good.

[0185] Table 17 Results of Determination of Chromatographic Column Durability

[0186] Peak Number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S Peak) Peak 6 CAPCELL PAK C18 MGⅡ 0.39 0.65 0.75 0.89 1.00 1.29 Kromasil 100 - 5 - C18 0.38 0.65 0.75 0.88 1.00 1.29 CAPCELL PAK C18 MGⅢ 0.39 0.66 0.74 0.89 1.00 1.30 Average 0.39 0.65 0.75 0.89 1.00 1.30 RSD 1.43％ 0.97％ 0.85％ 0.50％ 0.00％ 0.41％

[0187] 5. Instrument Durability Test

[0188] For the same test solution (GGC017), under the chromatographic conditions described in Example 5, the samples were determined using Agilent1260, Agilent1260 Infinity II, and Waters2690 respectively. The results are shown in Table 18. The RSD of the relative retention time of each characteristic peak < 3.0%, indicating that the instrument durability of this method is good.

[0189] Table 18 Results of Instrument Durability Experiment

[0190] Peak Number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 (S Peak) Peak 6 Waters 2690 0.39 0.65 0.75 0.89 1.00 1.29 Agilent 1260 Infinity II 0.39 0.65 0.74 0.89 1.00 1.29 Agilent 1260 0.39 0.64 0.74 0.87 1.00 1.29 Average 0.39 0.65 0.74 0.88 1.00 1.29 RSD 0.93％ 0.85％ 0.85％ 0.81％ 0.00％ 0.34％

[0191] 6. Determination of Dicliptera chinensis Samples

[0192] According to the method described in Example 5, the Dicliptera chinensis samples in Table 1 were determined, and the characteristic chromatograms obtained are as Figure 20 shown, and the relative retention times are shown in Table 19.

[0193] Table 19 Relative Retention Times of Characteristic Chromatograms of 18 Batches of Dicliptera chinensis Samples

[0194]

[0195]

[0196] 7. Comparison between Dicliptera chinensis Samples and False Dicliptera polycephala Samples

[0197] According to the method described in Example 5, the Dicliptera chinensis samples in Table 1 and Dicliptera polycephala in Table 2 were determined, and the characteristic chromatograms obtained are as Figure 21 shown. It can be seen that the chromatographic behaviors of Dicliptera chinensis samples and false Dicliptera polycephala are not exactly the same, indicating that the method of the present invention can better distinguish between the two.

[0198] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for detecting wormwood, characterized in that: The detection method includes a thin layer chromatography identification method, and the thin layer chromatography identification method includes the following steps: Preparation of thin layer test solution: extract the sample to be tested with an organic solvent to obtain an organic solvent extract, evaporate to dryness to obtain a residue, dissolve the residue with an organic solvent to obtain a thin layer test solution; Preparation of thin layer control medicinal material solution: extracting the dog liver vegetable control medicinal material with an organic solvent to obtain an organic solvent extract, evaporating to dryness to obtain a residue, and dissolving the residue with an organic solvent to obtain a thin layer control medicinal material solution; Preparation of thin layer reference solution: Dissolve the dog liver vegetable reference substance in methanol to obtain a thin layer reference substance solution; Thin layer chromatography analysis: the thin layer test solution, the thin layer reference medicinal material solution and the thin layer reference substance solution are spotted on the same thin layer plate, developed with a developing agent, taken out, dried and inspected; the developing agent is ethyl acetate-butanone-formic acid-water, and the volume ratio of ethyl acetate, butanone, formic acid and water is 4 to 6: 0.8～1.2：0.8～1.2：0.8～1.2。 2. The method according to claim 1, characterized in that The Herba Lysimachiae reference substance is schaftoside.

3. The method according to claim 1, characterized in that The organic solvent is methanol with a concentration of 40-60%, and the mass volume ratio of the sample to be tested or the dog liver herb to the organic solvent during extraction is 0.8-1.2 g: 15-25 mL; The extraction method is heating reflux, and the heating reflux time is 20 to 40 minutes; When the residue is dissolved by an organic solvent, the mass volume ratio of the organic solvent to the sample to be tested or the Herba Lysimachiae control medicinal material is 0.8-1.2 mL: 0.8-1.2 g.

4. The method according to claim 1, characterized in that Dissolve the Glechoma longituba reference substance in methanol to a solution with a concentration of 0.4-0.6 mg / mL; The thin layer plate is a silica gel G thin layer plate, and the amount of each solution used in thin layer chromatography analysis is 2 to 15 μL; The inspection method is as follows: spraying aluminum chloride solution on the thin layer plate, drying it with hot air, and inspecting it under an ultraviolet lamp.

5. The method according to claim 1, characterized in that The detection method also includes a fingerprint detection method, and the fingerprint detection method includes the following steps: Preparation of liquid test solution: extract the sample to be tested with an organic solvent, filter, and take the filtrate to obtain a liquid test solution; Preparation of liquid phase reference medicinal material reference solution: extracting the dog liver vegetable reference medicinal material with an organic solvent, filtering, and taking the filtrate to obtain a liquid phase reference medicinal material reference solution; Preparation of liquid phase reference substance solution: dissolve the dog liver vegetable reference substance in methanol to obtain a liquid phase reference substance solution; High performance liquid chromatography detection: inject the liquid phase test sample solution, the liquid phase reference medicinal material reference solution, and the liquid phase reference material reference solution into a liquid chromatograph for determination.

6. The method according to claim 5, characterized in that The Herba Lysimachiae reference substance is schaftoside.

7. The method according to claim 5, characterized in that The organic solvent is ethanol with a concentration of 40-60%, and the mass volume ratio of the sample to be tested or the dog liver herb to the organic solvent during extraction is 0.4-0.6 g: 15-25 mL; The extraction method is heating reflux, and the heating reflux time is 20 to 40 minutes; The Herba Lysimachiae reference substance is dissolved in methanol to form a solution with a concentration of 15 to 25 μg / mL.

8. The method according to claim 5, characterized in that The chromatographic column used in the high performance liquid chromatography detection is CAPCELLPAK MGⅡ, 5.0μm, 4.6×250mm; the filler is octadecylsilane bonded silica gel; the mobile phase A is acetonitrile, and the mobile phase B is 0.08-0.12% phosphoric acid solution; a gradient elution method is adopted; the detection wavelength is 335nm, the flow rate is 0.7-0.9mL / min, and the column temperature is 23-27°C.

9. The method according to claim 8, characterized in that The conditions of the gradient elution are: From 0 to 20 min, the volume percentage of mobile phase A changed from 13% to 15%; From 20 to 35 min, the volume percentage of mobile phase A was maintained at 15%; From 35 to 40 min, the volume percentage of mobile phase A changed from 15% to 16%.

10. Use of the method according to any one of claims 1 to 9 in the detection of Glechoma longituba.

Images