Preparation method and content determination method of uncaria-ganoderma lucidum bidirectional solid fermentation product

Abstract

The invention relates to the technical field of traditional Chinese medicine processing and analysis, in particular to an uncaria-ganoderma lucidum bidirectional solid fermentation method and a content determination method. Uncaria is fermented through ganoderma lucidum, the content of rhynchophylline and isorhynchophylline is increased after uncaria is fermented through ganoderma lucidum, the difference between uncaria before fermentation and uncaria after fermentation is obvious, it is indicated that the chemical composition characteristics of uncaria are changed in the fermentation process, and technical reference is provided for the processing technology or active ingredient strengthening of uncaria; the detection method established by the invention is strong in specificity, simple to operate and good in reproducibility, and can be used for determining the contents of the two components, namely rhynchophylline and isorhynchophylline before and after fermentation of the uncaria.

Description

Preparation method and content determination method of Uncaria rhynchophylla-Ganoderma lucidum dual solid fermentation product Technical Field

[0001] This invention relates to the field of traditional Chinese medicine processing and analysis technology, specifically to a method for preparing and determining the content of a two-way solid fermentation product of Uncaria rhynchophylla and Ganoderma lucidum. Background Technology

[0002] Uncaria rhynchophylla (Miq.) Miq. ex Havil., Uncaria macrophylla Wall., Uncaria hirsuta Havil., Uncaria sinensis (Oliv.) Havil., or Uncaria sessilifructus Roxb. (white uncaria) are all species belonging to the genus Uncaria in the Rubiaceae family. They are used for their effects of calming wind and relieving convulsions, clearing heat and soothing the liver; and are used for infantile convulsions, headaches, and dizziness. They are mainly distributed in Hubei, Guizhou, and Guangxi provinces. Studies have shown that Uncaria rhynchophylla is extremely rich in chemical components, including alkaloids, flavonoids, triterpenes, and glycosides, with more than 100 species. Alkaloids are the key active components in the pharmacological effects of Uncaria rhynchophylla, among which rhynchophylline and isorhynchophylline are the most important active components, accounting for more than 40% of the total alkaloids. With the continuous improvement of the planting area, yield, demand, planting technology and quality of Uncaria rhynchophylla, the Chinese medicine market for Uncaria rhynchophylla faces competition in terms of price, quality and market channels. Therefore, how to improve the medicinal value of Uncaria rhynchophylla and the sustainable development of the entire industrial chain is one of the current bottleneck issues. In addition, Uncaria rhynchophylla is cool in nature, and long-term and large-scale use may irritate the gastrointestinal tract and cause liver toxicity and other risks.

[0003] Microbial fermentation technology, with its advantages of being mild, efficient, and environmentally friendly, has become an important method for processing and modifying traditional Chinese medicine (TCM). Through enzymatic hydrolysis and transformation during microbial metabolism, the material basis of TCM can be optimized, increasing the content of active ingredients or generating new bioactive substances. Ganoderma lucidum, a fungus used in both medicine and food, produces various enzyme systems and secondary metabolites during its fermentation process. In TCM theory, Ganoderma lucidum is sweet and neutral in nature, and enters the heart, lung, liver, and kidney meridians. It can tonify kidney qi, calm the mind, relieve cough and asthma; it also possesses pharmacological activities in various aspects, including immune regulation and liver and kidney protection.

[0004] Fourier transform infrared spectroscopy (FT-IR) is characterized by its speed, non-destructive nature, and strong fingerprinting ability. It can comprehensively capture the overall characteristics of functional group types, chemical bond vibration modes, and chemical composition in samples, and has been widely used in research fields such as characterization of the material basis of traditional Chinese medicine, monitoring of processing procedures, and quality evaluation. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, this invention provides a method for two-way solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum and a method for content determination, comprising the following: a method for preparing a two-way solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum, the specific steps of which are as follows: edible lime and tryptophan are added to Uncaria rhynchophylla powder with a moisture content of 50-70%, the mixture is stirred evenly, sealed, sterilized, cooled, and then inoculated with activated Ganoderma lucidum inoculum, and fermented at a constant temperature of 20-30 ℃; when the mycelium has fully grown the culture medium, the mixture is dried, pulverized, and sieved to obtain the Uncaria rhynchophylla fermentation product.

[0006] Furthermore, the amount of edible lime added is sufficient to adjust the pH to 9-10; the mass ratio of Uncaria rhynchophylla to tryptophan is 100:9.

[0007] Furthermore, the Uncaria powder has a moisture content of 60%.

[0008] Furthermore, the activated Ganoderma lucidum strain is obtained by transferring the preserved Ganoderma lucidum mycelium to a PDA slant culture medium for activation and then incubating it at a constant temperature of 26 ℃ for 7 days for later use.

[0009] Furthermore, the constant temperature fermentation is carried out at a constant temperature of 26 ℃ for 15 days.

[0010] A dual solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum was prepared by the above method.

[0011] A method for determining the content of rhynchophylline and isorhynchophylline in a two-way solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum, with the following specific chromatographic conditions: The chromatographic column is a Wates reverse-phase column with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm; acetonitrile is used as mobile phase B, and 0.1% phosphoric acid solution is used as mobile phase A, with gradient elution: 0–10 min, 8%–14% B; 10–25 min, 14%–14% B; 25–26 min, 14%–90% B; 26–31 min, 90%–90% B; 31–32 min, 90%–8% B; flow rate is 0.3 ml / min; column temperature is 35 ℃; detection wavelength is 254 nm; injection volume is 1 μL.

[0012] Furthermore, the mixed reference solution used was a mixed reference solution with ethanol as the solvent and mass concentrations of rhododendronine 0.3036 mg / mL and isorhododendronine 0.6819 mg / mL, respectively.

[0013] Furthermore, the use of ethanol as a solvent refers to 70% ethanol.

[0014] Furthermore, the test solution used is prepared by the following method: Uncaria rhynchophylla or its products are added to 70% ethanol, weighed, heated under reflux, and after cooling, the weight loss is made up with 70% ethanol, filtered, and the test solution is obtained.

[0015] Furthermore, the blank solution used was a 70% ethanol solution.

[0016] A method based on Fourier transform infrared spectroscopy combined with chemometrics to analyze the component differences before and after two-way solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum is proposed. The Fourier transform infrared spectra of the samples are measured using a Fourier transform infrared spectrometer. The samples before and after fermentation are mixed with dried KBR (1:100, w / w) and compressed into tablets, with a frequency range of 4000-500 cm⁻¹. -1 (wavenumber); conducted at room temperature (25℃); and the following steps were used to analyze the products before and after the bidirectional solid fermentation of Uncaria rhynchophylla and Ganoderma lucidum: (1) infrared spectral comparison analysis; (2) analysis of the second derivative spectrum before and after Uncaria rhynchophylla fermentation; (3) cluster analysis (HCA); (4) principal component analysis (PCA); (5) partial least squares discriminant analysis (OPLS-DA) method.

[0017] Furthermore, the products obtained before and after the two-way solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum are the products obtained before and after fermentation using the method described in claim 1.

[0018] Furthermore, step (3) cluster analysis specifically involves preprocessing the original spectrum of the sample to be tested by second-order differentiation to obtain a data matrix, and then performing cluster analysis (HCA) using Euclidean distance.

[0019] Compared with the prior art, the technical effects created by the present invention are reflected in: (1) The present application uses Ganoderma lucidum fermentation technology to process Uncaria rhynchophylla, and studies the chemical composition of Uncaria rhynchophylla before and after fermentation, which provides a new basis for the further development and utilization of the Chinese medicine Uncaria rhynchophylla, and gives it certain advantages in the development of Uncaria rhynchophylla series products.

[0020] (2) In this application, the content of two components, rhynchophylline and isorhynchophylline, increases after the Rhynchophylla medicinal material is fermented by Ganoderma lucidum. As the amount of L-tryptophan added increases, the content of rhynchophylline and isorhynchophylline continues to rise, with the highest increases being 445.76% and 890.65%, respectively.

[0021] (3) Infrared spectroscopy combined with chemometric methods PCA, HCA and OPLS-DA analysis showed that there were significant differences between the Uncaria rhynchophylla before and after fermentation in this application, which could be effectively distinguished, indicating that the fermentation process changed the chemical composition characteristics of Uncaria rhynchophylla. The two-way solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum in this application is conducive to increasing the content of the two main components, rhynchophylline and isorhynchophylline, in Uncaria rhynchophylla, providing a technical reference for the processing technology or active ingredient enhancement of Uncaria rhynchophylla.

[0022] (4) The detection method established in this application is highly specific, simple to operate, and has good reproducibility. It can be used to determine the content of two components, rhododendronine and isorhododendronine, before and after fermentation of Uncaria rhynchophylla.

[0023] (5) Based on FT-IR and HPLC, combined with chemometric methods, this application studies the chemical composition and content of Uncaria rhynchophylla before and after fermentation, providing a new basis for the further development and utilization of Uncaria rhynchophylla preparations, giving it certain advantages in the development of Uncaria rhynchophylla series products. Attached Figure Description

[0024] Figure 1 shows the average infrared spectra of Uncaria rhynchophylla before and after fermentation. Note: GF: fermented; GN: unfermented.

[0025] Figure 2 shows the average infrared second derivative plot (A) and its magnified portion (B) of Uncaria rhynchophylla before and after fermentation. Note: GF: fermented; GN: unfermented.

[0026] Figure 3 shows the HCA values ​​of 16 batches of Uncaria rhynchophylla samples before and after fermentation. Note: GF: fermented; GN: unfermented.

[0027] Figure 4 is the PCA score chart. Note: GF: fermented; GN: unfermented.

[0028] Figure 5 shows the OPLS-DA diagram of Uncaria rhynchophylla before and after fermentation. Note: GF: fermented; GN: unfermented.

[0029] Figure 6 shows the HPLC chromatograms of the blank solution (A), the mixed reference solution (B), the unfermented test solution (C), and the fermented test solution (D). Note: 1. Isorhodin; 2. Rhizomorphine.

[0030] Figure 7 shows the results of the analysis of variance for fermented and unfermented Uncaria rhynchophylla. Note: GF: fermented; GN: unfermented. ****P<0.0001.

[0031] Figure 8 shows the effect of different amounts of tryptophan added on the content of rhodopsin and isorhodopsin. Note: GN: unfermented; GF: fermented. Detailed Implementation

[0032] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description.

[0033] Example: 1. Instruments and Materials 1.1 Instruments Fourier transform infrared spectrometer (INVENIO, Bruker Corporation, USA); Agilent ultra-high performance liquid chromatograph (1290 II, Agilent Technologies, USA); 1 / 100,000 electronic balance (S225D, Mettler Toledo Shanghai Co., Ltd.); 1 / 10,000 electronic analytical balance (AL104, Mettler Toledo Shanghai Co., Ltd.); autoclave (DGL-100G, Zhuode Instrument Equipment (Shanghai) Co., Ltd.); constant temperature incubator (RGX-300P, Tester); intelligent water bath reflux extractor (NAI-HL12S, Shanghai Nai Experimental Instrument Co., Ltd.).

[0034] 1.2 Materials The Uncaria rhynchophylla medicinal material was purchased from Guizhou Tongjitang Pharmaceutical Factory (batch number: 20181001). It was identified by Professor Sun Qingwen of Guizhou University of Traditional Chinese Medicine as Uncaria rhynchophylla (Miq.) Miq. ex Havil, a plant belonging to the genus Uncaria of the Rubiaceae family. Dried hooked stems and branches; reference standards rhynchophylline (batch number: DSTDG001502; mass fraction: 98%) and isorhynchophylline (batch number: DSTDM00901; mass fraction: 98%) were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.; Ganoderma lucidum (GL) was purchased from Chongqing Minyuan Fungi Industry; L-tryptophan (batch number: CT35141810, mass fraction: 98%, Beijing Kulaibo); potassium bromide (spectrally pure) was purchased from Maclean Biochemical Technology Co., Ltd.; food-grade lime was purchased from Leling Shengfatengda Food Co., Ltd.; acetonitrile (chromatographically pure, Fisher Scientific); phosphoric acid (analytical grade) and anhydrous ethanol (analytical grade) were purchased from Chongqing Chuandong Chemical Co., Ltd.; ultrapure water (self-made).

[0035] 2 Methods and Results 2.1 Preparation of Ganoderma lucidum-Uncaria rhynchophylla dual solid fermentation product 2.1.1 Activation of strain The Ganoderma lucidum mycelium preserved in test tubes was transferred to PDA test tube slant medium for activation and cultured at 26 ℃ for 7 days for later use.

[0036] 2.1.2 Preparation of Uncaria rhynchophylla fermentation product: Appropriate amounts of edible lime (used to adjust the pH to 9-10) and tryptophan were added to Uncaria rhynchophylla powder with a moisture content of approximately 60%. After thorough mixing, the mixture was sealed and sterilized at 121 ℃ for 20 min. After cooling, activated Ganoderma lucidum spawn was inoculated, and fermentation was carried out in a constant temperature incubator at 26 ℃. When the mycelium had fully colonized the culture medium, samples were taken, dried, pulverized, and passed through a 60-mesh sieve to obtain the Uncaria rhynchophylla fermentation product, denoted as GF. Unfermented Uncaria rhynchophylla was treated in the same way and labeled GN as a blank control.

[0037] 2.2 Infrared Spectroscopic Identification: Fourier transform infrared spectra of the samples were measured using an INVENIO, Bruker Corporation, USA. Samples before and after fermentation were mixed with dried KBR (1:100, w / w) and compressed into tablets. The frequency range was 4000-500 cm⁻¹. -1 (Wavenumber). Performed at room temperature (25°C).

[0038] 2.2.1 Infrared Spectral Comparison and Analysis The infrared spectrum is shown in Figure 1. At 3410 nm... -1 Near the 2921 nm peak, GN exhibits a broad and strong absorption peak, which can be attributed to the OH stretching vibration, primarily originating from the hydroxyl groups in polysaccharides or the phenolic hydroxyl groups in alkaloids; while at 2921 nm... -1 The nearby absorption peak corresponds to the CH stretching vibrations of the methyl (-CH3) and methylene (-CH2) groups; 1635 nm -1 The absorption peak at 1532 nm is due to the stretching vibration of the carbonyl group (C=O). -1 The absorption peak at 1250 nm may be a characteristic peak corresponding to the in-plane vibration of NH in aromatic secondary amines or the C=O stretching vibration of acyl halides; -1 The absorption peak at 1053 nm can be attributed to the CO stretching vibration; while the absorption peak at 1053 nm... -1 The nearby absorption peaks are due to the bending vibration of OH or the stretching vibration of CO.

[0039] GF at 3425 nm -1 The absorption peak at 2921 nm corresponds to the OH / NH stretching vibration. Compared with before fermentation, the peak position has shifted slightly and the peak shape has become broader. This phenomenon may be related to the mycelial proteins and microbial polysaccharides generated during the fermentation of Ganoderma lucidum. -1 The nearby absorption peak corresponds to the CH stretching vibration, and its peak intensity shows a relatively decreasing trend. This is presumably due to the aliphatic side chains and long-chain alkanes in Uncaria rhynchophylla being decomposed and utilized by microorganisms as carbon sources. (1635 nm) -1 The absorption peak at that point disappears, replaced by the peak at 1660-1575 nm. -1 A broader absorption peak appears within the range, suggesting that this phenomenon may originate from the formation of humic acids—in humic substances, the C=C skeleton vibration of aromatic rings, the C=O vibration of conjugated ketones, and the presence of carboxylate ions (-COO). - The asymmetric vibrations of 1251 nm undergo absorption and merging, leading to peak shift, intensity enhancement, and ultimately broadening. -1 The absorption peak at 1053 nm remains essentially unchanged, attributable to the stretching vibration peak of CO; -1The positions of the nearby absorption peaks remained basically unchanged, which were due to the bending vibration of OH or the stretching vibration of CO, but the peak intensity was relatively weakened. It is speculated that the structural polysaccharides in Uncaria rhynchophylla were effectively degraded by enzymes secreted by Ganoderma lucidum, serving as a source of microbial growth.

[0040] 2.2.2 Second-Derivative Spectral Analysis of Uncaria Rhizoma Before and After Fermentation Since the second derivative of infrared spectroscopy can provide higher resolution and clearer spectral profile changes than the original spectrum, while eliminating the influence of baseline drift or smooth background interference, this study performed second-derivative processing on the infrared spectral data based on overlapping spectra. The results are shown in Figure 2. It can be seen that after fermentation, Uncaria Rhizoma showed changes in spectral profiles at 400–500 cm⁻¹. −1 The number and intensity of the "serrated" absorption peaks at the site are significantly different from those before fermentation.

[0041] 2.2.3 Cluster Analysis The original spectra of 16 batches of samples were preprocessed by second-order differentiation to obtain a data matrix. Cluster analysis (HCA) was performed using Euclidean distance, and the results are shown in Figure 3. The results show that at a distance coefficient of 50, the 16 batches of Uncaria rhynchophylla before and after fermentation were clustered into two main groups. Unfermented Uncaria rhynchophylla (GN1-GN8) was clustered into one group (II), while fermented Uncaria rhynchophylla (GF1-GF8) was clustered into another group (I). This indicates a significant difference in the chemical composition of Uncaria rhynchophylla before and after fermentation. The fingerprint spectra of active components before and after fermentation were relatively consistent across different batches, suggesting a high correlation between the quality of Uncaria rhynchophylla and its processing technology.

[0042] 2.2.4 PCA is a statistical method that compresses multidimensional correlated data into a few independent data points. It achieves dimensionality reduction without losing key information, amplifies the differences between samples, and can solve the difficulty of band overlap analysis caused by the complexity of traditional Chinese medicine components. PCA was performed on 16 batches of pre-treated Uncaria rhynchophylla data before and after fermentation. The results are shown in Figure 4. From the PCA score plot, the model explanation rate parameter R was obtained. 2 X is 0.996, and the predictive ability parameter Q 2 R is 0.994. 2 X and Q 2 All values ​​were greater than 0.5, indicating good predictive reliability of the model. The PCA scatter plot showed that the 16 batches of samples clustered into two groups, with separate clusters before and after fermentation, demonstrating clear separation. The PCA results were consistent with the HCA results, indicating significant differences in the main active components of Uncaria rhynchophylla before and after fermentation.

[0043] 2.2.5 OPLS-DA To further verify the reliability of the analysis results, the supervised pattern recognition method OPLS-DA was used based on PCA. The infrared spectra of Uncaria rhynchophylla before and after 16 batches of fermentation were preprocessed, and the resulting matrix data were imported into the software for OPLS-DA analysis. The results are shown in Figure 5. The results show that the cumulative explanatory power parameter R of the prediction model is [missing information]. 2X is 0.957, R 2 Y is 0.917, and the predictive power Q is... 2 The values ​​were 0.871, all greater than 0.5, indicating that the model is stable, reliable, and has strong predictive ability. The results show that the Uncaria rhynchophylla vines before and after fermentation each clustered into distinct groups, demonstrating that the vines before and after fermentation can be effectively differentiated.

[0044] 2.3 HPLC Determination of Rhododendronine and Isorhododendronine Content 2.3.1 Preparation of Mixed Reference Solution Weigh appropriate amounts of rhododendronine and isorhododendronine reference standards, add 70% ethanol to make up to 50 mL, dissolve to prepare mixed reference solution with mass concentrations of 0.3036 and 0.6819 mg / mL, respectively, and store at 4 ℃ for later use.

[0045] 2.3.2 Preparation of test solution: Weigh 1 g of samples before and after fermentation of Uncaria rhynchophylla, add 70% ethanol, weigh, heat under reflux for 45 min, and after cooling, make up the weight loss with 70% ethanol. Filter to obtain Uncaria rhynchophylla test solution and fermented Uncaria rhynchophylla test solution.

[0046] 2.3.3 Preparation of blank solution: The 70% ethanol solution of the extraction solvent was used as the blank solution.

[0047] 2.3.4 Chromatographic conditions: The chromatographic column was a Wates reverse-phase column (150 mm length, 2.1 mm inner diameter, 1.7 μm particle size); acetonitrile was used as mobile phase B, and 0.1% phosphoric acid solution was used as mobile phase A. Gradient elution was used: 0–10 min, 8%–14% B; 10–25 min, 14%–14% B; 25–26 min, 14%–90% B; 26–31 min, 90%–90% B; 31–32 min, 90%–8% B; flow rate was 0.3 ml / min; column temperature was 35 ℃; detection wavelength was 254 nm; injection volume was 1 μL.

[0048] 2.4 Methodological Investigation 2.4.1 Specificity Investigation The mixed reference solution under section "2.3.1", the Uncaria rhynchophylla test solution and fermented Uncaria rhynchophylla test solution under section "2.3.2", and the blank solution under section "2.3.3" were analyzed and determined according to the chromatographic conditions under section "2.3.4", and the chromatograms were recorded. Results showed that under these chromatographic conditions, the resolution between the peaks of rhynchophylline and isorhynchophylline in the mixed reference solution and the test solution and their adjacent peaks was greater than 1.5, and the theoretical plate number for each component was above 5000. Furthermore, the blank solution did not interfere with the determination, indicating that this method has good specificity. The chromatogram is shown in Figure 6.

[0049] 2.4.2 Linearity Assessment The mixed reference solution prepared under section “2.3.1” was diluted with 70% ethanol to prepare a series of mixed reference solutions of varying mass concentrations. These solutions were then injected and analyzed under the chromatographic conditions described in section “2.3.4”, and the peak areas were recorded. A standard curve was plotted with the reference concentration as the abscissa (X) and the peak area as the ordinate (Y), and linear regression was performed. The results showed that the analyte exhibited a good linear relationship with the peak area within its corresponding concentration range (r ≥ 0.9990), as shown in Table 1.

[0050] Table 1. Regression equations, correlation coefficients, and linear ranges for each component to be tested.

[0051] 2.4.3 Precision Test: Take an appropriate amount of the Uncaria rhynchophylla test solution from section “2.3.2”, inject it according to the chromatographic conditions under section “2.3.4”, and perform the determination 6 times consecutively. Record the chromatogram and calculate the RSD value of the peak area. The results showed that the RSD of the Uncaria rhynchophylla peak area was 0.30% and 0.16%, indicating that the instrument precision was good.

[0052] 2.4.4 Stability Test: The fermented Uncaria rhynchophylla test solution from section “2.3.2” was taken and subjected to chromatographic analysis under the conditions described in section “2.3.4” at room temperature for 0, 2, 4, 8, 12, and 24 hours after preparation. The chromatograms were recorded. The results showed that the RSDs of the peak areas of rhynchophylline and isorhynchophylline were 0.70% and 0.18%, respectively, indicating that the test solution had good stability within 24 hours at room temperature.

[0053] 2.4.5 Repeatability Test: Six portions of the same batch of Uncaria rhynchophylla sample were accurately weighed and prepared according to the method described in section "2.3.2". The samples were then injected and analyzed under the chromatographic conditions described in section "2.3.4". The peak areas were recorded, and the RSDs of the mass fractions of the two components in the sample were calculated based on the peak areas. The results showed that the mass fractions of rhynchophylline and isorhynchophylline were 0.61 mg / g and 1.57 mg / g, respectively, with RSDs of 0.51% and 0.50%, respectively, indicating good repeatability of the method.

[0054] 2.4.6 Determination of Recovery Rate Six portions of 0.5 g of Uncaria rhynchophylla sample with known content were accurately weighed. Appropriate amounts of rhynchophylline and isorhynchophylline reference solutions were added quantitatively to each portion. The test solutions were prepared according to the method described in section "2.3.2". The solutions were then injected and analyzed under the chromatographic conditions described in section "2.3.4". The chromatograms were recorded, and the recovery rates of each component were calculated. The results showed that the average recoveries of rhynchophylline and isorhynchophylline were 101.35% and 102.75%, respectively, with RSDs of 1.48% and 1.31%, respectively, indicating good accuracy of the method. See Table 2 for details.

[0055] Table 2 Results of the sample recovery test

[0056] 2.5 Sample Content Determination 2.5.1 Content Determination of Fermented and Unfermented Uncaria Rhizome Samples Appropriate amounts of Uncaria Rhizome and fermented Uncaria Rhizome were taken and prepared into test solutions according to the method described in section “2.3.2”. The solutions were then injected and analyzed according to the chromatographic conditions described in section “2.3.4”. Chromatograms were recorded, and the content of each analyte was calculated based on the regression equation of the standard curve. Independent samples t-tests were performed on the two groups of samples. The contents of rhynchophylline and isorhynchophylline before and after fermentation were calculated based on the peak area, and a normality test was performed (P > 0.05, indicating normality). As shown in Figure 7, there were highly significant differences in the contents of rhynchophylline and isorhynchophylline before and after fermentation (P < 0.0001), indicating that fermentation has a strong promoting effect on the accumulation of the two key active ingredients, rhynchophylline and isorhynchophylline, in Uncaria Rhizome. The change rate of each component in fermented Uncaria Rhizome was calculated, taking the content of each component in unfermented Uncaria Rhizome as 100%. The results showed that the contents of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla after fermentation were significantly different from those before fermentation (P < 0.0001), with the contents of rhynchophylline and isorhynchophylline increasing significantly. The results are shown in Table 3.

[0057] Change rate (%) = (average content of fermented Uncaria rhynchophylla - average content of unfermented Uncaria rhynchophylla) / average content of unfermented Uncaria rhynchophylla × 100%.

[0058] The results showed that the contents of the two components in Uncaria rhynchophylla changed significantly after fermentation compared with those before fermentation (P<0.0001), and the contents of rhynchophylline and isorhynchophylline increased significantly. The results are shown in Table 3.

[0059] Table 3. Results of determination of the contents of two components before and after fermentation of Uncaria rhynchophylla.

[0060] Note: Compared with Uncaria rhynchophylla (unfermented), *****P < 0.0001 Note: Compared with Uncaria rhynchophylla (unfermented), ***P < 0.0001 2.5.2 Dynamic Detection of Rhynchophylline and Isorhynchophylline Content in Fermented Uncaria rhynchophylla Nitrogen source has an important influence on Ganoderma lucidum fermentation. An appropriate supply of nitrogen source helps the growth and synthesis of Ganoderma lucidum mycelium. Tryptophan is an amino acid that releases nitrogen through decomposition and metabolism, providing nitrogen raw materials for microbial growth, cell synthesis of proteins and other nitrogen-containing compounds (such as nucleic acids and alkaloids), and it is a precursor compound of indole alkaloids. Literature studies have shown that adding precursor amino acids can increase the active ingredients in the Ganoderma lucidum fermentation system. Based on the optimization of culture medium and fermentation conditions, the addition of different concentrations of ergothioneine precursors (histidine, cysteine, methionine) has an impact on ergothioneine production in Ganoderma lucidum, among which methionine has the most significant effect on increasing ergothioneine yield. After fermentation by Ganoderma lucidum, the contents of rhynchophylline and isorhynchophylline gradually increased with the increase of tryptophan addition. Before fermentation, the contents of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla were 0.1314 and 0.1229 mg / g, respectively. When Uncaria rhynchophylla was mixed with tryptophan in different ratios (100:1, 100:3, 100:5, 100:7, 100:9, and 100:11), the content of rhynchophylline increased by 445.76% and the content of isorhynchophylline increased by 890.65% when the ratio was 100:9. When the ratio was 100:11, the contents of rhynchophylline and isorhynchophylline tended to decrease slowly. The results are shown in Figure 8.

[0061] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for preparing a dual-phase solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum, characterized in that, The specific steps are as follows: Add edible lime and tryptophan to Uncaria rhynchophylla powder with a moisture content of 50-70%, stir evenly, seal, sterilize, cool, and then inoculate with activated Ganoderma lucidum spawn. Ferment at a constant temperature of 20-30 ℃. When the mycelium has fully grown the culture medium, dry, crush, and sieve to obtain the Uncaria rhynchophylla fermentation product.

2. The method for preparing the Uncaria rhynchophylla-Ganoderma lucidum dual-phase solid fermentation product according to claim 1, characterized in that, The amount of edible lime added is sufficient to adjust the pH to 9-10; the mass ratio of Uncaria rhynchophylla to tryptophan is 100:

9.

3. The method for preparing the Uncaria rhynchophylla-Ganoderma lucidum dual-phase solid fermentation product according to claim 1, characterized in that, The Uncaria powder has a moisture content of 60%.

4. The method for preparing the Uncaria rhynchophylla-Ganoderma lucidum dual-phase solid fermentation product according to claim 1, characterized in that, The activated Ganoderma lucidum strain is prepared by transferring the preserved Ganoderma lucidum mycelium to a PDA slant culture medium and culturing it at a constant temperature of 26 ℃ for 7 days before use.

5. The method for preparing the two-way solid-state fermentation product of Ganoderma lucidum and Ganoderma lucidum according to claim 1, characterized in that, The constant temperature fermentation is carried out at a constant temperature of 26 ℃ for 15 days.

6. A dual-phase solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum, characterized in that, It is prepared by the method described in any one of claims 1-5.

7. A method for determining the content of rhododendronine and isorhododendronine in a dual-phase solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum, characterized in that, The specific chromatographic conditions were as follows: A Wates reverse-phase column was used, with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm; acetonitrile was used as mobile phase B, and 0.1% phosphoric acid solution was used as mobile phase A, with gradient elution: 0–10 min, 8%–14% B; 10–25 min, 14%–14% B; 25–26 min, 14%–90% B; 26–31 min, 90%–90% B; 31–32 min, 90%–8% B; flow rate was 0.3 ml / min; column temperature was 35 ℃; detection wavelength was 254 nm; and injection volume was 1 μL.

8. The method for determining the content of rhynchophylline and isorhynchophylline in the dual solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum according to claim 7, characterized in that, The mixed reference solution used was a mixed reference solution with ethanol as the solvent and mass concentrations of rhododendronine 0.3036 mg / mL and isorhododendronine 0.6819 mg / mL, respectively.

9. The method for determining the content of rhynchophylline and isorhynchophylline in the dual solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum according to claim 8, characterized in that, The use of ethanol as a solvent refers to using 70% ethanol as a solvent.

10. The method for determining the content of rhynchophylline and isorhynchophylline in the dual solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum according to claim 7, characterized in that, The test solution was prepared by adding 70% ethanol to Uncaria rhynchophylla or its products, weighing, heating under reflux, and after cooling, making up the weight loss with 70% ethanol. The solution was then filtered to obtain the test solution.

11. The method for determining the content of rhynchophylline and isorhynchophylline in the dual solid-state fermentation product of Uncaria rhynchophylla and Ganoderma lucidum according to claim 7, characterized in that, The blank solution used was a 70% ethanol solution.

12. A method for analyzing the differences in components before and after two-way solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum based on Fourier transform infrared spectroscopy combined with chemometrics, characterized in that, Fourier transform infrared (FTIR) spectra of the samples were determined using a Fourier transform infrared spectroscopy analyzer. The samples before and after fermentation were mixed with dried KBR (1:100, w / w) and compressed into tablets. The frequency range was 4000-500 cm⁻¹. -1 (wavenumber); conducted at room temperature (25℃); and the following steps were used to analyze the products before and after the bidirectional solid fermentation of Uncaria rhynchophylla and Ganoderma lucidum: (1) infrared spectral comparison analysis; (2) analysis of the second derivative spectrum before and after Uncaria rhynchophylla fermentation; (3) cluster analysis (HCA); (4) principal component analysis (PCA); (5) partial least squares discriminant analysis (OPLS-DA) method.

13. The method for analyzing the component differences before and after two-way solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum based on Fourier transform infrared spectroscopy combined with chemometrics, as described in claim 12, is characterized in that... The products obtained before and after the bidirectional solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum are products obtained before and after fermentation using the method described in claim 1.

14. The method for analyzing the component differences before and after two-way solid-state fermentation of Uncaria rhynchophylla and Ganoderma lucidum based on Fourier transform infrared spectroscopy combined with chemometrics, as described in claim 12, is characterized in that... The step (3) cluster analysis specifically involves preprocessing the original spectrum of the sample to be tested by second-order differentiation to obtain a data matrix, and then performing cluster analysis (HCA) using Euclidean distance.

Images