GC method for rapidly identifying rhizoma dryopteris crassirhizomae and counterfeit species thereof

Abstract

The invention belongs to the technical field of medicine detection, particularly provides a GC method for rapidly identifying rhizoma dryopteris crassirhizomae and fake rhizoma dryopteris crassirhizomae, and particularly discloses application of trans-nerolidol in identification or auxiliary identification of rhizoma dryopteris crassirhizomae and fake rhizoma dryopteris crassirhizomae. Compared with the prior art, the method provided by the invention has the advantages that the content in the male fern rhizome is lower, the content in other male fern rhizome fake products is lower, the male fern rhizome can be used as the marker, and a male fern rhizome identification method is established, has a better linear relation, has specificity and is good in instrument precision, stability, repeatability and reproducibility; the quantitation limit of the trans-nerolidol is 0.139 mu g / ml, and the detection limit is 0.046 mu g / ml; the trans-nerolidol content of 32 batches of cyrtomium rhizome samples is 0.11%-1.39%, the trans-nerolidol content of rhizoma dryopteris crassirhizomae should not be less than 0.25%, and a more accurate method is provided for identification and quality control of rhizoma dryopteris crassirhizomae.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a rapid GC method for identifying Dryopteris crassirhizoma and its counterfeits. Background Technology

[0002] Dryopteris crassirhizoma, commonly known as Chinese Dryopteris crassirhizoma, is the dried rhizome and petiole remnants of Dryopteris crassirhizoma Nakai, a plant belonging to the Dryopteris family. It is a traditional Chinese medicine with a long history, wide application, and unique characteristics. While Dryopteris crassirhizoma is a common source plant for the Chinese medicinal herb Dryopteris, the composition of its source plants is quite complex. According to literature reports, there are no fewer than 35 species of source plants for Dryopteris crassirhizoma in my country, belonging to 6 families and 12 genera. The *Dictionary of Traditional Chinese Medicine* records 7 species as source plants for Dryopteris crassirhizoma. Among them, Dryopteris crassirhizoma Nakai is the most important source plant. The *Chinese Pharmacopoeia* lists two species of Dryopteris crassirhizoma: Dryopteris crassirhizoma and Dryopteris crassirhizoma var. crassirhizoma. In the 1977 edition of the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Pharmacopoeia"), the medicinal plants closely related to Dryopteris crassirhizoma were recorded as two species: Dryopteris crassirhizoma Nakai. and Osmunda japonica Thunb. Since the 1995 edition of the Pharmacopoeia, in order to distinguish Dryopteris crassirhizoma from other original plants, Dryopteris crassirhizoma has been listed separately.

[0003] As is well known to those in the field, traditional Chinese medicine (TCM) has complex components and is easily affected by its place of origin, processing methods, and harvesting time. To ensure the safety and efficacy of clinical medication, establishing a scientific and reasonable quality evaluation model is of great significance for the overall quality control of TCM materials. Dryopteris crassirhizoma is the most widely used and best-selling variety on the market; however, quality control is limited to routine items such as appearance, thin-layer chromatography identification, and extractive content determination, and quality control standards are relatively lagging. Literature indicates that not all clinically used *Dryopteris crassirhizoma* is the same as *Dryopteris japonica*. After long-term verification and market regulation, the rhizomes of at least five other plants are still being used as a substitute for *Dryopteris crassirhizoma*. These include the commonly used *Woodwardia unigemmata* Naka (family Apocynaceae), *Woodwardia japonica* Sm (family Apocynaceae), *Matteuccia truthipteris* Todaro (family Apocynaceae), *Lunathyriuma crostichoides* Ching (family Apocynaceae), and the adulterant *Braainia insignis* J.Sm (family Apocynaceae). They are very similar in appearance to *Dryopteris crassirhizoma*, making accurate sensory identification difficult for those without extensive experience. Therefore, establishing rapid and accurate supplementary testing methods to differentiate *Dryopteris crassirhizoma* from other commonly used and adulterated products is of significant clinical importance. Summary of the Invention

[0004] The primary objective of this invention is to provide the application of trans-nerolidol in the identification or auxiliary identification of Dryopteris crassirhizoma and its adulterants.

[0005] The second objective of this invention is to provide the application of trans-nerolidol in the preparation of products for the identification or auxiliary identification of Dryopteris crassirhizoma and its counterfeit products.

[0006] A third objective of this invention is to provide the application of trans-nerolidol in identifying or assisting in the identification of whether Dryopteris crassirhizoma contains adulterants.

[0007] The fourth objective of this invention is to provide the application of trans-nerolidol in the preparation of products for identifying or assisting in the identification of whether a sample of Dryopteris crassirhizoma contains counterfeit Dryopteris crassirhizoma.

[0008] The fifth objective of this invention is to provide the application of trans-nerolidol in the preparation of products for identifying or assisting in the identification of adulterated Dryopteris crassirhizoma.

[0009] Preferably, the adulterants of Dryopteris crassirhizoma include one or more of Cycas revoluta, Osmunda crassirhizoma, Dryopteris crassirhizoma, and Cibotium barometz.

[0010] The sixth objective of this invention is to provide a method for identifying Dryopteris crassirhizoma, the method comprising the following steps:

[0011] (1) Preparation of reference solution:

[0012] Take an appropriate amount of trans-nerolidol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 0.09 mg per ml.

[0013] (2) Preparation of the test solution:

[0014] Take the sample powder, weigh it accurately, place it in a stoppered conical flask, add 25-35 ml of extraction solvent accurately, stopper tightly, weigh it, heat and reflux for extraction or ultrasonic extraction, cool and weigh it again, make up the weight loss with ethyl acetate, shake well, filter, and take the filtrate to obtain the sample.

[0015] (3) Detection

[0016] Take the reference solution prepared in step (1) and the test solution prepared in step (2) respectively, and perform the determination. The chromatographic conditions are as follows: programmed temperature rise: initial temperature 60 ℃, hold for 5 min, increase to 90 ℃ at a rate of 10 ℃ / min, hold for 10 min, then increase to 160 ℃ at a rate of 10 ℃ / min, hold for 20 min, then increase to 280 ℃ at a rate of 5 ℃ / min, hold for 10 min, then increase to 300 ℃ at a rate of 20 ℃ / min, hold for 20 min; or

[0017] Programmed temperature rise: Initial temperature 60 ℃, hold for 5 min, increase to 90 ℃ at a rate of 10 ℃ / min, hold for 10 min, then increase to 160 ℃ at a rate of 10 ℃ / min, hold for 20 min, then increase to 250 ℃ at a rate of 10 ℃ / min, hold for 5 min; or

[0018] Programmed temperature rise: Initial temperature 70℃, hold for 2 minutes, then increase to 180℃ at a rate of 15℃ per minute, hold for 25 minutes;

[0019] A capillary column with polyethylene glycol as the stationary phase; an injection port temperature of 230℃ and a detector temperature of 250℃; a split ratio of 5:1; and a column flow rate of 1.5 ml / min.

[0020] Preferably, the extraction solvent in step (2) is one or more of ethyl acetate, cyclohexane, dichloromethane, and n-hexane.

[0021] Preferably, the chromatographic conditions in step (3) are: programmed temperature rise: initial temperature 70°C, hold for 2 minutes, increase to 180°C at a rate of 15°C per minute, hold for 25 minutes;

[0022] Preferably, the extraction in step (2) is ultrasonic extraction, and the ultrasonic extraction time is 90 min.

[0023] The beneficial effects of the present invention are: (1) The present invention provides the application of trans-nerolidol in the identification or auxiliary identification of Dryopteris crassirhizoma and its counterfeit products. The trans-nerolidol content is extremely high in Dryopteris crassirhizoma samples, while the content is low in other counterfeit products of Dryopteris crassirhizoma. It can be used as a marker of Dryopteris crassirhizoma.

[0024] (2) This invention establishes an identification method for *Dryopteris crassirhizoma* specifically for trans-nerolidol, and optimizes the conditions of the method. It was found that when ethyl acetate is used as the extraction solvent, the solid-liquid ratio is 1:25, ultrasonic extraction is performed for 30 min, and the chromatographic conditions are: programmed temperature rise: initial temperature 70℃, hold for 2 min, then increase to 180℃ at a rate of 15℃ per minute, hold for 25 min; injection port temperature is 230℃, detector temperature is 250℃; split ratio is 5:1; column flow rate is 1.5 ml / min. The results are satisfactory.

[0025] (3) When the identification method described above is used to identify Dryopteris crassirhizoma and its adulterants, trans-nerolidol shows good linearity in the range of 3.1345 μg / ml ~ 282.1011 μg / ml, exhibiting good specificity, instrument precision, stability, and repeatability. Different instruments and chromatographic columns have minimal impact on this test method, resulting in good reproducibility. The limit of quantification for trans-nerolidol is 0.139 μg / ml, and the limit of detection is 0.046 μg / ml. The trans-nerolidol content in 32 batches of Dryopteris crassirhizoma ranged from 0.11% to 1.39%, indicating that Dryopteris crassirhizoma contains trans-nerolidol (C... 15 H 26 O) should not be less than 0.25%. Attached Figure Description

[0026] Figure 1 Different forms of the genealogical chart of Mianma Guanzhong

[0027] Note: A: Solvent blank; B: Dryopteris crassirhizoma (raw material); C: Dryopteris crassirhizoma (processed slices); D: Dryopteris crassirhizoma (reference material);

[0028] Figure 2 Isolation and identification of characteristic components of Dryopteris crassirhizoma.

[0029] Note: A: NIST spectral library search image; B: trans-nerolidol

[0030] Figure 3 Chromatograms of different extraction methods

[0031] Note: A: Heating and reflux extraction; B: Ultrasonic extraction;

[0032] Figure 4 Chromatograms of different extraction solvents

[0033] Note: A: n-hexane; B: dichloromethane; C: ethyl acetate; D: cyclohexane;

[0034] Figure 5 Chromatograms of different extraction solid-liquid ratios

[0035] Note: A: 1:25; B: 1:30; C: 1:35;

[0036] Figure 6 Chromatograms of different extraction times

[0037] Note: A: 30 min; B: 60 min; C: 90 min;

[0038] Figure 7 Chromatograms under different chromatographic conditions

[0039] Note: A: Chromatographic condition 1; B: Chromatographic condition 2; C: Chromatographic condition 3;

[0040] Figure 8 Exclusivity Examination

[0041] Note: A: Dryopteris crassirhizoma; B: Fern pods; C: Single-budded Cynodon dactylon; D: Cycas revoluta; E: Dryopteris crassirhizoma; F: trans-nerolidol

[0042] Figure 9 linear relationship

[0043] Figure 10 Durability test of different instruments

[0044] Note: A: Agilent 8890 blank solution; B: Agilent 8890 reference solution; C: Agilent 8890 sample solution;

[0045] D: Agilent 7890 blank solution; E: Agilent 7890 reference solution; F: Agilent 7890 sample solution;

[0046] Figure 11 Durability Study of Different Chromatographic Columns

[0047] Note: A: Blank solution for column 1; B: Reference solution for column 1; C: Sample solution for column 1; D: Blank solution for column 2; E: Reference solution for column 2; F: Sample solution for column 2; G: Blank solution for column 3; H: Reference solution for column 3; I: Sample solution for column 3; Detailed Implementation

[0048] The scope of protection of the present invention will be described below with reference to specific embodiments. It should be noted that the scope of protection of the present invention is not limited by the following embodiments.

[0049] It should be noted that, unless otherwise specified, the methods described in the following embodiments can be obtained from the literature, and the reagents can be purchased commercially.

[0050] Example 1: Screening, separation and identification of characteristic compounds of Dryopteris crassirhizoma.

[0051] 1. Reagents and reagents

[0052] Trans-nerolidol (manufacturer: Lemeitian Pharmaceutical / Dest Biotechnology; batch number: DST230915-148; purity: 95.04%); Dryopteris crassirhizoma sample (batch of commercially available Dryopteris crassirhizoma sample) is shown in Table 1, cyclohexane (analytical grade, Sinopharm Group Co., Ltd.), dichloromethane (analytical grade, Sinopharm Group Co., Ltd.), ethyl acetate (analytical grade, chromatographic grade, Sinopharm Group Co., Ltd.), n-hexane (analytical grade, Sinopharm Group Co., Ltd.).

[0053] 2. Methods

[0054] 2.1 Preparation of test solution

[0055] Accurately weigh approximately 1 g of the fine powder and place it in a stoppered conical flask. Accurately add 25 ml of ethyl acetate, seal tightly, and weigh. Sonicate (500 W, 40 kHz) for 30 minutes, cool, and weigh again. Make up the weight loss with ethyl acetate, shake well, and filter. Collect the filtrate to obtain the test sample mother liquor. Alternatively, take 50 μl of the test sample mother liquor, add ethyl acetate to a final volume of 1 ml, filter through a 0.45 μm organic filter membrane, and collect the filtrate.

[0056] 2.2 Chromatographic conditions and system suitability test

[0057] Agilent DB-17MS capillary column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness) with 5% phenylmethyl polysiloxane crosslinked as the stationary phase was used. The system operated under constant pressure (146 kPa). The temperature program was as follows: initial temperature 60 °C, held for 10 minutes, increased to 140 °C at a rate of 10 °C / min, held for 10 minutes, then increased to 200 °C at a rate of 10 °C / min, held for 10 minutes. The injection port temperature was 250 °C, the transfer line temperature was 250 °C, and the ion source temperature was 260 °C. The residence time was 2.5 min, the solvent delay time was 6 min, and the acquisition parameters were SCAN (50-500).

[0058] 3. Results

[0059] like Figure 1 As shown in AD, the mass spectra of *Dryopteris crassirhizoma* samples and its control materials were analyzed using a triple quadrupole GC-MS in full scan mode. The results showed that the *Dryopteris crassirhizoma* samples contained an extremely high level of an unknown component. Therefore, the NIST library search system built into the GC-MS was used to retrieve the mass spectra and compare them with the library (see [link to database]). Figure 2 A) The results showed that the mass spectrum of the unknown component had a 99% similarity to that of trans-nerolidol in the spectral library. Therefore, trans-nerolidol reference standard was purchased for comparison, and the comparison results are shown in [reference needed]. Figure 2 B indicates that the unknown component is trans-nerolidol; C 15 H 26 O), is a characteristic component of the Chinese herbal medicine formula.

[0060] Based on the above research, trans-nerolidol was used as the differentiating component to distinguish Dryopteris crassirhizoma and its adulterants, and a rapid gas chromatography method was established to identify Dryopteris crassirhizoma and its adulterants.

[0061] Example 2: A rapid GC method for identifying Dryopteris crassirhizoma and its counterfeits.

[0062] 1. Instruments, reagents and reagents

[0063] 1.1 Instruments

[0064] The equipment includes an Agilent 8890 gas chromatograph, a flame ionization detector (FID), a Mettler analytical balance (model: SECURA125-1CN) and a Mettler analytical balance (model: QUINTIX224-1CN), and a CNC ultrasonic cleaner (manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.; model: KQ-300DB).

[0065] 1.2 Reagents and Drugs

[0066] Trans-nerolidol (manufacturer: Lemeitian Pharmaceutical / Dest Biotechnology; batch number: DST230915-148; purity: 95.04%); Dryopteris crassirhizoma sample, cyclohexane (analytical grade, Sinopharm Group Co., Ltd.), dichloromethane (analytical grade, Sinopharm Group Co., Ltd.), ethyl acetate (analytical grade, chromatographic grade, Sinopharm Group Co., Ltd.), n-hexane (analytical grade, Sinopharm Group Co., Ltd.).

[0067] 2. Extraction method selection

[0068] Chromatographic conditions: Capillary column with polyethylene glycol as the stationary phase (60 m length, 0.32 mm inner diameter, 0.25 μm film thickness); Temperature program: initial temperature 70 °C, hold for 2 minutes, then increase to 180 °C at a rate of 15 °C per minute, hold for 25 minutes; Injector temperature 230 °C, detector temperature 250 °C; Split ratio 5:1; Column flow rate: 1.5 ml / min.

[0069] Preparation of the test solution:

[0070] (1) Hot reflux extraction: Weigh about 1 g of the fine powder of this product accurately, place it in a stoppered conical flask, add 25 ml of ethyl acetate accurately, stopper tightly, weigh, heat and reflux for 45 min, cool and weigh again. Make up the weight loss with ethyl acetate, shake well, filter, and take the filtrate to obtain the product.

[0071] (2) Ultrasonic extraction: Take about 1 g of the fine powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of ethyl acetate, seal tightly, weigh it, sonicate (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh it, make up the weight loss with ethyl acetate, shake well, filter, and take the filtrate to obtain the product.

[0072] like Figure 3 As shown, the chromatographic behavior of the test solutions prepared by the two extraction methods is very similar. Considering the peak shape and resolution, as well as the ease of operation, ultrasonic extraction is ultimately the better choice.

[0073] 3. Selection of extraction solvent

[0074] Chromatographic conditions: Capillary column with polyethylene glycol as the stationary phase (60 m length, 0.32 mm inner diameter, 0.25 μm film thickness); Temperature program: initial temperature 70 °C, hold for 2 minutes, then increase to 180 °C at a rate of 15 °C per minute, hold for 25 minutes; Injector temperature 230 °C, detector temperature 250 °C; Split ratio 5:1; Column flow rate: 1.5 ml / min.

[0075] Preparation of the test solution: Weigh approximately 1 g of the fine powder of this product accurately. Weigh four parallel samples and place them in 150 ml stoppered conical flasks respectively. Accurately add 25 ml of ethyl acetate, cyclohexane, dichloromethane, and n-hexane solvent to each flask. Seal tightly, weigh, and let stand for 1 hour, shaking occasionally during the standing process. Sonicate for 30 minutes, cool, weigh, and replenish the lost weight with ethyl acetate. Shake well, filter, and collect the filtrate to obtain the test solution.

[0076] like Figure 4As shown, dichloromethane produces poor peak shapes when used as a solvent, while n-hexane results in fewer and lower concentrations of dissolved components. After comparison, the differences between the two are minimal. Ethyl acetate and cyclohexane exhibit the best peak elution effects. Based on these comparisons, ethyl acetate demonstrates the best separation effect and is more environmentally friendly as a solvent. Therefore, ethyl acetate was selected as the extraction solvent.

[0077] 4. Selection of extraction material-liquid ratio

[0078] Chromatographic conditions: Capillary column with polyethylene glycol as the stationary phase (60 m length, 0.32 mm inner diameter, 0.25 μm film thickness); Temperature program: initial temperature 70 °C, hold for 2 minutes, then increase to 180 °C at a rate of 15 °C per minute, hold for 25 minutes; Injector temperature 230 °C, detector temperature 250 °C; Split ratio 5:1; Column flow rate: 1.5 ml / min.

[0079] Preparation of the test solution: Weigh approximately 1 g of the fine powder of this product accurately. Weigh three parallel samples and place them in 150 ml stoppered conical flasks respectively. Accurately add 25 ml, 30 ml, and 35 ml of ethyl acetate solvent to each flask, seal tightly, weigh, and let stand for 1 hour, shaking occasionally during the standing process. Sonicate for 30 minutes, cool, weigh, and replenish the lost weight with ethyl acetate. Shake well, filter, and collect the filtrate to obtain the test solution.

[0080] like Figure 5 As shown, it was found that the analyte exhibited better peak shape and separation when the material-to-liquid ratio was 1:25, therefore, a material-to-liquid ratio of 1:25 was selected.

[0081] 5. Selection of extraction time

[0082] Chromatographic conditions: Capillary column with polyethylene glycol as the stationary phase (60 m length, 0.32 mm inner diameter, 0.25 μm film thickness); Temperature program: initial temperature 70 °C, hold for 2 minutes, then increase to 180 °C at a rate of 15 °C per minute, hold for 25 minutes; Injector temperature 230 °C, detector temperature 250 °C; Split ratio 5:1; Column flow rate: 1.5 ml / min.

[0083] Preparation of the test solution: Weigh approximately 1 g of the fine powder of this product accurately. Weigh three parallel samples and place them in 150 ml stoppered conical flasks. Accurately add 25 ml of ethyl acetate solvent to each flask, seal tightly, weigh, and let stand for 1 hour, shaking occasionally during the standing process. Sonicate for 30 min, 60 min, and 90 min respectively, cool, weigh, and replenish the lost weight with ethyl acetate. Shake well, filter, and collect the filtrate to obtain the test solution.

[0084] like Figure 6As shown, there was no significant difference in the volatile components dissolved at 30 min and 60 min, and ethyl acetate volatilized significantly at 90 min. Therefore, sonication for 30 min was chosen, and the lost mass was replenished after sonication.

[0085] 6. Selection of chromatographic conditions

[0086] Preparation of the test solution: Weigh about 1 g of the fine powder of this product accurately, place it in a stoppered conical flask, add 25 ml of ethyl acetate accurately, stopper tightly, weigh, sonicate (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh, make up the weight loss with ethyl acetate, shake well, filter, and take the filtrate to obtain the test solution.

[0087] Chromatographic conditions 1: Capillary column with polyethylene glycol as the stationary phase (column length 60 m, inner diameter 0.32 mm, membrane thickness 0.25 μm); Temperature program: initial temperature 60 ℃, hold for 5 min, increase to 90 ℃ at a rate of 10 ℃ / min, hold for 10 min, then increase to 160 ℃ at a rate of 10 ℃ / min, hold for 20 min, then increase to 280 ℃ at a rate of 5 ℃ / min, hold for 10 min, then increase to 300 ℃ at a rate of 20 ℃ / min, hold for 20 min. Injector temperature: 250 ℃; detector temperature: 250 ℃; split ratio: 5:1. Column flow rate: 1.5 ml / min.

[0088] Chromatographic conditions 2: Capillary column with polyethylene glycol as the stationary phase (column length 60 m, inner diameter 0.32 mm, membrane thickness 0.25 μm); Temperature program: initial temperature 60 ℃, hold for 5 min, increase to 90 ℃ at a rate of 10 ℃ / min, hold for 10 min, then increase to 160 ℃ at a rate of 10 ℃ / min, hold for 20 min, then increase to 250 ℃ at a rate of 10 ℃ / min, hold for 5 min. Injector temperature: 250 ℃; detector temperature: 250 ℃; split ratio: 5:1. Column flow rate: 1.5 ml / min.

[0089] Chromatographic conditions 3: Capillary column with polyethylene glycol as the stationary phase (column length 60 m, inner diameter 0.32 mm, film thickness 0.25 μm); Temperature program: initial temperature 70 °C, hold for 2 minutes, increase to 180 °C at a rate of 15 °C per minute, hold for 25 minutes; Injector temperature 230 °C, detector temperature 250 °C; Split ratio 5:1. Column flow rate: 1.5 ml / min.

[0090] like Figure 7As shown, under chromatographic condition 1, the analytes generally elute after 60 minutes, which is too long; under chromatographic condition 2, the analytes generally elute within 40 minutes, and the content of the analytes is low after 40 minutes, so the low-content components can be ignored, reducing the measurement time; under chromatographic condition 3, the measurement time is short, the chromatographic peaks are well separated, and the sample is eluted more completely, so chromatographic condition 3 is selected as the optimal chromatographic condition.

[0091] Example 3: Gas Chromatography Study

[0092] 1. Instruments, reagents and reagents

[0093] 1.1 Instruments

[0094] The equipment includes an Agilent 8890 gas chromatograph, a flame ionization detector (FID), a Mettler analytical balance (model: SECURA125-1CN) and a Mettler analytical balance (model: QUINTIX224-1CN), and a CNC ultrasonic cleaner (manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.; model: KQ-300DB).

[0095] 1.2 Reagents and Drugs

[0096] Trans-nerolidol (manufacturer: Lemeitian Pharmaceutical / Dest Biotechnology; batch number: DST230915-148; purity: 95.04%); Dryopteris crassirhizoma sample (batch of commercially available Dryopteris crassirhizoma sample) is shown in Table 1, cyclohexane (analytical grade, Sinopharm Group Co., Ltd.), dichloromethane (analytical grade, Sinopharm Group Co., Ltd.), ethyl acetate (analytical grade, chromatographic grade, Sinopharm Group Co., Ltd.), n-hexane (analytical grade, Sinopharm Group Co., Ltd.).

[0097] Table 1 Information on 42 batches of Dryopteris crassirhizoma samples

[0098] 2. Methods and Results

[0099] 2.1 Chromatographic conditions

[0100] A capillary column with polyethylene glycol as the stationary phase (60 m in length, 0.32 mm in inner diameter, and 0.25 μm in film thickness) was used. The temperature program was as follows: initial temperature 70 °C, hold for 2 minutes, then ramp to 180 °C at a rate of 15 °C per minute and hold for 25 minutes. The injection port temperature was 230 °C, and the detector temperature was 250 °C. The split ratio was 5:1. The column flow rate was 1.5 ml / min.

[0101] 2.2 Solution Preparation

[0102] 2.2.1 Preparation of the reference solution

[0103] Take an appropriate amount of trans-nerolidol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 0.09 mg per ml.

[0104] 2.2.2 Preparation of the test solution

[0105] Take about 1 g of the fine powder of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of ethyl acetate, seal tightly, weigh it, sonicate (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh it, make up the weight loss with ethyl acetate, shake well, filter, and take the filtrate to obtain the product.

[0106] 2.3 Methodological Examination

[0107] 2.3.1 Specificity Examination

[0108] Take the trans-nerolidol reference solution from section “2.2.1”, the test solution from section “2.2.2”, and the blank solution, and inject them for analysis according to the chromatographic conditions from section “2.1”.

[0109] See results Figure 8 Therefore, the theoretical plate number of volatile components, calculated as trans-nerolidol, is greater than 10,000, and there is no interference from the blank, indicating that the method has good specificity.

[0110] 2.3.2 Examination of Linear Relationships

[0111] Accurately pipette an appropriate amount of the reference solution from section "2.2.1", and add ethyl acetate to prepare a series of standard curve solutions with concentrations of 3.1345 μg / ml, 4.7017 μg / ml, 9.4034 μg / ml, 94.0337 μg / ml, 188.0674 μg / ml, and 282.1011 μg / ml. Inject these solutions under the chromatographic conditions described in section "2.1". Perform linear regression with the reference concentration as the abscissa (X) and the peak area as the ordinate (Y) to obtain the regression equation.

[0112] See results Figure 9 Table 2 shows the linear regression equation as y = 3.6083x + 7.2494; R = 0.9999. The results indicate that trans-nerolidol exhibits good linearity in the range of 3.1345 μg / ml ~ 282.1011 μg / ml.

[0113] Table 2 Results of linear relationship examination

[0114] 2.3.3 Precision Test

[0115] Take an appropriate amount of Dryopteris crassirhizoma sample Y3, prepare the test solution according to the method in section “2.2.2”, and inject 1 μL into the sample 6 times under the chromatographic conditions in section “2.1”.

[0116] The results are shown in Table 3. The peak area RSD of trans-nerolidol was measured to be 0.91%. The results indicate that the instrument has good precision.

[0117] Table 3 Precision test results

[0118] 2.3.4 Stability Test

[0119] Take an appropriate amount of Dryopteris crassirhizoma sample Y3, prepare the test solution according to the method in section “2.2.2”, place it at room temperature, and inject it for determination under the chromatographic conditions in section “2.1” at 0 h, 2 h, 4 h, 8 h, 16 h, 24 h and 48 h respectively.

[0120] The peak area RSD of the *Dryopteris crassirhizoma* sample was measured to be 1.06%, indicating that the test solution had good stability within 48 h. The results are shown in Table 4.

[0121] Table 4 Stability test results

[0122] 2.3.5 Repeatability Test

[0123] Take an appropriate amount of Dryopteris crassirhizoma sample Y3, prepare the test solution according to the method in section "2.2.2", prepare 6 samples in parallel, and inject and determine them under the chromatographic conditions in section "2.1".

[0124] The RSD of trans-nerolidol content was 1.23%, indicating that the experiment had good repeatability. The results are shown in Table 5.

[0125] Table 5 Repeatability Test Results

[0126] 2.3.6 Recovery Rate Test

[0127] Take six portions of Dryopteris crassirhizoma sample Y3 powder, each approximately 0.5 g, accurately weigh them, place them in stoppered conical flasks, accurately add 12.5 ml of trans-nerolidol reference solution (concentration 0.094 mg / ml) to each flask, and then accurately add 12.5 ml of ethyl acetate. Determine the recovery rate according to the detection conditions under section “2.1”.

[0128] The results are shown in Table 6. The average recovery rate of trans-nerolidol was 109.89%, and the RSD was 0.80%, indicating that the method has good accuracy.

[0129] Table 6 Results of Recovery Determination

[0130] 2.3.8 Detection Limit

[0131] Take an appropriate amount of the reference solution under section “2.2.1”, dilute it to different concentrations, and inject it under the chromatographic conditions under section “2.1”. The concentration corresponding to the trans-nerolidol chromatographic peak signal-to-noise ratio (S / N) of 10:1 is the limit of quantitation, and the concentration corresponding to the signal-to-noise ratio (S / N) of 3:1 is the limit of detection.

[0132] The results showed that the limit of quantification for trans-nerolidol was 0.139 μg / ml and the limit of detection was 0.046 μg / ml.

[0133] 2.3.9 Durability Test

[0134] 2.3.9.1 Instrument durability test

[0135] The effects of Agilent 8890 and Agilent 7890 gas chromatographic systems on the above experimental methods were investigated using a DB-WAX capillary column (60 m × 0.32 mm × 0.25 μm) with polyethylene glycol as the stationary phase. The reference solution (section 2.2.1), the test solution (Dryopteris crassirhizoma sample Y3) (section 2.2.2), and the blank solution were injected and analyzed according to the chromatographic conditions in section 2.1.

[0136] The results are as follows. Figure 10 The blank solution showed no interference under different instruments, and the relative average deviation of the trans-nerolidol content between the two instruments was 0.97%, indicating that the different instruments had little impact on the experimental method and the reproducibility was good.

[0137] 2.3.9.2 Column robustness test

[0138] The effects of three capillary columns with identical stationary phases on the above experimental methods were investigated using an Agilent 8890 gas chromatograph: column 1 (Agilent DB-WAX, 60 m × 0.32 mm × 0.25 μm, catalog number 123-7062), column 2 (Agilent DB-WAX, 30 m × 0.32 mm × 0.25 μm, catalog number 123-7032), and column 3 (Thermo Fisher Scientific TG-WAXMS, 30 m × 0.25 mm × 0.25 μm, catalog number 26088-1420). The reference solution (section 2.2.1), the test solution (Dryopteris crassirhizoma sample Y3) (section 2.2.2), and the blank solution were injected and analyzed according to the chromatographic conditions described in section 2.1.

[0139] See results Figure 11 The blank solution showed no interference under different chromatographic columns, and the relative average deviation of trans-nerolidol content under different chromatographic columns was 1.11%, indicating that different chromatographic columns had little impact on the experimental method and good reproducibility.

[0140] 2.4 Sample Determination

[0141] The test solutions for 32 batches of samples were prepared according to section “2.2.2”, and the chromatographic conditions under section “2.1” were used for injection and determination. The results showed that the content of trans-nerolidol in the 32 batches of Dryopteris crassirhizoma ranged from 0.11% to 1.39%, as shown in Table 7.

[0142] Table 7. Content of trans-nerolidol in Dryopteris crassirhizoma samples

[0143] 3. Setting limits

[0144] The content of trans-nerolidol in 32 batches of Dryopteris crassirhizoma samples was determined (Table 7). The results showed that the content of trans-nerolidol ranged from 0.11% to 1.39%. Taking into account the origin, planting years, growing environment, chemical composition, and other factors of the medicinal material, and combined with the determination results, the preliminary limit for the determination of trans-nerolidol in Dryopteris crassirhizoma is: This product contains trans-nerolidol (C... 15 H 26 O) should not be less than 0.25%.

[0145] In summary, this invention provides the application of trans-nerolidol in the identification or auxiliary identification of Dryopteris crassirhizoma and its adulterants. The trans-nerolidol is present in extremely high concentrations in Dryopteris crassirhizoma samples, while its concentration is low in other adulterants, thus it can serve as a marker for Dryopteris crassirhizoma. This invention establishes an identification method for Dryopteris crassirhizoma using trans-nerolidol, and optimizes the method conditions. It was found that when ethyl acetate is used as the extraction solvent, the solid-liquid ratio is 1:25, ultrasonic extraction is performed for 30 min, and the chromatographic conditions are: programmed temperature rise: initial temperature 70℃, hold for 2 min, increase to 180℃ at a rate of 15℃ per minute, hold for 25 min; injection port temperature 230℃, detector temperature 250℃; split ratio 5:1; column flow rate 1.5 ml / min, the results are satisfactory. When the aforementioned identification method is used to distinguish between Dryopteris crassirhizoma and its adulterants, trans-nerolidol exhibits good linearity in the range of 3.1345 μg / ml to 282.1011 μg / ml, demonstrating good specificity, instrument precision, stability, and repeatability. Different instruments and chromatographic columns have minimal impact on this method, resulting in good reproducibility. The limit of quantification for trans-nerolidol is 0.139 μg / ml, and the limit of detection is 0.046 μg / ml. The trans-nerolidol content in 32 batches of Dryopteris crassirhizoma ranged from 0.11% to 1.39%, indicating that Dryopteris crassirhizoma contains trans-nerolidol (C...). 15 H 26 O) should not be less than 0.25%, providing a more accurate method for identifying Dryopteris crassirhizoma.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of trans-nerolidol in the identification or auxiliary identification of Dryopteris crassirhizoma and its adulterants.

2. Application of trans-nerolidol in the preparation of products for identification or auxiliary identification of Dryopteris crassirhizoma and its adulterants.

3. Application of trans-nerolidol in identifying or assisting in the identification of whether Dryopteris crassirhizoma contains adulterants.

4. Application of trans-nerolidol in the preparation of products for the identification or auxiliary identification of whether Dryopteris crassirhizoma samples contain counterfeit Dryopteris crassirhizoma products.

5. Application of trans-nerolidol in the preparation of identification or auxiliary identification of adulterated Dryopteris crassirhizoma.

6. The application as described in any one of claims 1-5, characterized in that, The aforementioned counterfeit Dryopteris crassirhizoma includes one or more of Cycas revoluta, Osmunda crassirhizoma, Dryopteris crassirhizoma, and Cibotium barometz.

7. A method for identifying Dryopteris crassirhizoma, characterized in that, The method includes the following steps: (1) Preparation of reference solution: Take an appropriate amount of trans-nerolidol reference standard, accurately weigh it, and add ethyl acetate to prepare a solution containing 0.09 mg per ml. (2) Preparation of the test solution: Take the sample powder, weigh it accurately, place it in a stoppered conical flask, add 25-35 ml of extraction solvent accurately, stopper tightly, weigh it, heat and reflux for extraction or ultrasonic extraction, cool and weigh it again, make up the weight loss with ethyl acetate, shake well, filter, and take the filtrate to obtain the sample. (3) Detection Take the reference solution prepared in step (1) and the test solution prepared in step (2) respectively, and perform the determination. The chromatographic conditions are as follows: Programmed temperature rise: Initial temperature 60 ℃, hold for 5 min, increase to 90 ℃ at a rate of 10 ℃ / min, hold for 10 min, then increase to 160 ℃ at a rate of 10 ℃ / min, hold for 20 min, then increase to 280 ℃ at a rate of 5 ℃ / min, hold for 10 min, then increase to 300 ℃ at a rate of 20 ℃ / min, hold for 20 min; or Programmed temperature rise: Initial temperature 60 ℃, hold for 5 min, increase to 90 ℃ at a rate of 10 ℃ / min, hold for 10 min, then increase to 160 ℃ at a rate of 10 ℃ / min, hold for 20 min, then increase to 250 ℃ at a rate of 10 ℃ / min, hold for 5 min; or Programmed temperature rise: Initial temperature 70℃, hold for 2 minutes, then increase to 180℃ at a rate of 15℃ per minute, hold for 25 minutes; A capillary column with polyethylene glycol as the stationary phase; the injection port temperature is 230℃, and the detector temperature is 250℃; The split ratio was 5:1; the column flow rate was 1.5 ml / min.

8. The identification method as described in claim 7, characterized in that, The extraction solvent in step (2) is one or more of ethyl acetate, cyclohexane, dichloromethane, and n-hexane.

9. The identification method as described in claim 7, characterized in that, The chromatographic conditions described in step (3) are as follows: programmed temperature rise: initial temperature 70°C, hold for 2 minutes, then increase to 180°C at a rate of 15°C per minute, and hold for 25 minutes.

10. The identification method as described in claim 7, characterized in that, The extraction described in step (2) is ultrasonic extraction, and the ultrasonic extraction time is 90 min.

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