A detection method of a dendrobium candidum composite extract

Abstract

The application discloses a Dendrobium candidum composite extract with anti-lung cancer activity and a preparation method thereof, wherein the mass ratio of the Dendrobium candidum binzyl extract and the Dendrobium candidum polysaccharide is 1:1-1:2. The composite extract prepared by the application can inhibit the growth of Lewis lung cancer, and the effect is better than that of the binzyl or polysaccharide alone, and the composite extract plays a better synergistic effect, and is expected to be prepared into an anti-lung cancer drug. The application also establishes a detection method of the Dendrobium candidum composite extract, and the quality of the composite extract can be well detected.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the preparation of a compound extract of Dendrobium officinale bibenzyl derivatives and Dendrobium officinale polysaccharides and its application in anti-lung cancer activity. Background Technology

[0002] Lung cancer, also known as bronchogenic carcinoma, originates from the bronchial mucosa or glands in the lungs. It is the malignant tumor with the highest incidence and mortality rate in my country, and is divided into small cell lung cancer and non-small cell lung cancer, the latter accounting for approximately 85% of all cancer cases. Traditional Chinese medicine (TCM) does not have a specific disease name for "lung cancer," but based on clinical and medical records, it is categorized under diseases such as "lung accumulation," "breath retention," "cough," and "hemoptysis," with phlegm stagnation, qi stagnation, and blood stasis as its main pathogenesis. Currently, the main treatments for lung cancer include chemotherapy, radiotherapy, surgery, and targeted therapy. However, these treatments often cause significant physical and psychological suffering and unavoidable side effects for patients. In recent years, with continuous exploration and practice based on ancient methods, more and more traditional Chinese medicines have been used to treat lung cancer with positive results. The general principle of TCM treatment for lung cancer is to strengthen the body's resistance and eliminate pathogenic factors, combining tonification and purgation. Treatment methods mainly include tonifying qi and strengthening the body's resistance, resolving phlegm and removing blood stasis, fighting cancer and detoxifying, and clearing heat and detoxifying.

[0003] Dendrobium officinale Kimura et Migo, the dried stem of Dendrobium officinale (family Orchidaceae), is also known as Tiepi Lan or Heijie Cao. The 2020 edition of the Chinese Pharmacopoeia states that Dendrobium officinale is sweet and slightly cold in nature, entering the stomach and kidney meridians, and possesses the effects of nourishing the stomach and promoting fluid production, nourishing yin and clearing heat. Its traditional applications are consistent with modern pharmacological research. Contemporary medicine has increasingly focused on its component analysis and corresponding biological activities, discovering a diverse range of chemical components in Dendrobium officinale. Polysaccharides are the main active ingredient, with the highest content, while bibenzyl compounds are its characteristic components. Modern pharmacological studies have shown that the effective components of Dendrobium officinale, such as polysaccharides, alkaloids, and bibenzyl compounds, possess various biological activities, including enhancing immunity, anti-tumor activity, and antioxidant activity. Polysaccharides, phenanthrene compounds, and bibenzyl compounds may be the main material basis for its anti-cancer effects, with bibenzyl compounds potentially exerting their anti-tumor effects by inhibiting tumor cell proliferation, invasion, migration, and metastasis, and promoting apoptosis.

[0004] Currently, there are no studies, either domestic or international, on the synergistic anti-lung cancer activity between Dendrobium officinale benzyl and polysaccharides, nor are there any reports on the combined use of Dendrobium officinale benzyl and polysaccharide components in the preparation of anti-lung cancer drugs.

[0005] This invention combines Dendrobium officinale bibenzyl extract and Dendrobium officinale polysaccharide to provide a Dendrobium officinale compound extract with anti-lung cancer activity and its application. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to provide a compound extract of Dendrobium officinale with anti-lung cancer activity and its application. This invention comprises a compound extract of Dendrobium officinale bibenzyl derivatives and Dendrobium officinale polysaccharides. This compound extract exhibits superior efficacy compared to either the polysaccharides or bibenzyl derivatives alone, and it demonstrates a better inhibitory effect on the growth of Lewis lung cancer tumors in mice.

[0007] Technical solution: The present invention is implemented using the following technical solution:

[0008] A compound extract of Dendrobium officinale with anti-lung cancer activity, the compound extract comprising Dendrobium officinale bibenzyl extract and Dendrobium officinale polysaccharide.

[0009] As a preferred embodiment, the Dendrobium officinale compound extract with anti-lung cancer activity described above has a mass ratio of Dendrobium officinale bibenzyl extract to Dendrobium officinale polysaccharide of 1:1 to 1:2.

[0010] The Dendrobium officinale compound extract provided by this invention is prepared by the following method:

[0011] Take the dried stems of Dendrobium officinale, crush them, add a certain volume ratio of ethanol and reflux extract 1 to 3 times, each time for 30 to 140 minutes, to obtain the product;

[0012] The Dendrobium officinale polysaccharide extract was prepared by the following method:

[0013] Take the residue after ethanol extraction of Dendrobium officinale, add water for extraction, combine the water extracts, add ethanol to the alcohol content to 80-90%, let stand, filter and collect the precipitate, and remove protein by Sevag method to obtain the final product.

[0014] As a preferred embodiment, the Dendrobium officinale compound extract described above, wherein the Dendrobium officinale bibenzyl extract is prepared by the following method:

[0015] Take dried stems of Dendrobium officinale, crush them, and extract them twice with 95% ethanol at a material-to-liquid ratio of 1:22, each extraction lasting 140 minutes.

[0016] The Dendrobium officinale polysaccharide extract was prepared by the following method:

[0017] Take the residue after ethanol extraction of Dendrobium officinale, add water at a material-to-liquid ratio of 1:50 and extract twice, each time for 1.5 hours. Combine the filtrates, add ethanol to the alcohol content to 80-90%, let stand at 4℃ for 24 hours, filter and collect the precipitate, and remove protein using the Sevag method to obtain the final product.

[0018] This invention prepares a traditional Chinese medicine preparation by combining a compound extract of Dendrobium officinale with a pharmaceutically acceptable carrier.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a compound extract of benzyl derivatives and polysaccharides from Dendrobium officinale. This compound extract has a better inhibitory effect on Lewis lung cancer tumor growth than either benzyl derivative or polysaccharide alone, and has the potential to be developed into an anti-lung cancer drug. Attached Figure Description

[0021] Figure 1 This is the standard curve for dendritic oxalool.

[0022] Figure 2 These are the results of a single-factor experiment on the bibenzyl extract of Dendrobium officinale.

[0023] Figure 3 This is the standard curve for glucose.

[0024] Figure 4 These are the results of a single-factor experiment on Dendrobium officinale polysaccharides.

[0025] Figure 5 The total ion chromatograms of Dendrobium officinale bibenzyl extract are shown in positive ion (A) and negative ion (B) modes.

[0026] Figure 6 This is an ion chromatogram of a monosaccharide standard.

[0027] Figure 7 This is an ion chromatogram of a Dendrobium officinale polysaccharide sample.

[0028] Figure 8 The curves showing the changes in tumor volume during the drug administration period for each group of tumor-bearing mice are shown.

[0029] Figure 9 Tumor images of each group 15 days after drug administration.

[0030] Figure 10 Tumor weight and tumor index of tumor-bearing mice in each group, *p<0.05 vs. model group.

[0031] Figure 11 The thymus and spleen indices of mice in each group are shown, *p<0.05 vs. model group; ## p<0.01, ### p<0.001 vs. blank group.

[0032] Figure 12 Serum cytokine levels in each group of mice, ****p<0.0001 vs. model group. Detailed Implementation

[0033] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0034] Example 1

[0035] 1. Single-factor experimental design was used to investigate the extraction process of benzyl derivatives from Dendrobium officinale.

[0036] 1.1 Screening for different extraction times

[0037] 3.0 g of Dendrobium officinale powder was weighed and soaked in a liquid-to-solid ratio of 1:20 for 1 hour. The effects of different extraction times (80, 100, 120, 140, 160, and 180 min) on the content of bibenzyl compounds were investigated. The mixture was extracted once by reflux in an 85℃ water bath, and the total bibenzyl content was determined. Under these conditions, each sample was measured in triplicate, and the average of the three measurements was taken. The highest content of bibenzyl compounds (0.829%) was observed at an extraction time of 160 min. Further increasing the extraction time decreased the content of bibenzyl compounds. This may be because, at a constant temperature, prolonged extraction time leads to the evaporation of ethanol in the solvent, increasing the polarity of the solvent. According to the principle of "like dissolves like," less polar bibenzyl compounds precipitate out, ultimately resulting in a decrease in the extraction rate of bibenzyl compounds. Simultaneously, impurities also dissolve over time, interfering with the extraction of bibenzyl compounds. Furthermore, prolonged extraction time may lead to the degradation and decomposition of compounds, reducing the extraction yield. Therefore, an extraction time of 140–180 min was selected for subsequent orthogonal experiments, and the results are shown in the appendix. Figure 2 A.

[0038] 1.2 Screening of different feed-liquid ratios

[0039] 3.0 g of *Dendrobium officinale* powder was weighed, and the effects of different material-to-liquid ratios (1:10, 1:16, 1:22, 1:28, 1:34, and 1:38) on the content of bibenzyl compounds were investigated. After soaking for 1 hour, the sample was extracted once by reflux in an 85℃ water bath for 160 minutes, and the total bibenzyl content was determined. Under the above conditions, each sample was measured in triplicate, and the average of the three measurements was taken. With the increase of the material-to-liquid ratio, the content of bibenzyl compounds gradually increased, reaching its highest value at a ratio of 1:28. Further increases in the material-to-liquid ratio led to a decrease in the content of bibenzyl compounds. This may be because an excessively high material-to-liquid ratio increases the dilution effect, and excess solvent dilutes the concentration of bibenzyl compounds in *Dendrobium officinale*. Simultaneously, other fat-soluble components also dissolve. Since the total energy during the reaction remains constant, insufficient energy supply may occur during the extraction of active ingredients, preventing complete disruption of plant cell walls and thus reducing the extraction rate. Furthermore, excessive reaction solution may cause the benzyl compounds in Dendrobium officinale to come into contact with other impurities (such as moisture, oxygen, and light). This contact may lead to oxidation, decomposition, or degradation of the compounds, thus affecting their relative content. When the material-to-liquid ratio is small, insufficient extraction solvent results in incomplete wetting of the raw materials and a reduced contact area between the medicinal material and the solvent, leading to a lower extraction rate. Considering solvent evaporation during the reaction, too little solvent will further incomplete the reaction. Therefore, a material-to-liquid ratio of 1:22 to 1:34 was selected for subsequent orthogonal experiments. The results are shown in the appendix. Figure 2 B.

[0040] 1.3 Filtering based on different extraction times

[0041] 3.0 g of Dendrobium officinale powder was accurately weighed, and the material-to-liquid ratio was 1:22. After soaking for 1 hour, the mixture was extracted by reflux in an 85℃ water bath. The effect of extraction times (1, 2, 3, and 4 times) on the content of bibenzyl derivatives was investigated. Each extraction lasted 160 minutes, and the total bibenzyl content was measured. Under the above conditions, each sample was measured in triplicate, and the average of the three measurements was taken. With the increase of extraction times, the content of bibenzyl derivatives gradually increased, reaching its highest value at 3 extraction times. Further increases in extraction times led to a decrease in the content of bibenzyl derivatives. Excessive extraction times increase time and cost. Each extraction requires the preparation of new solvent, and each process requires specific time and labor costs. Furthermore, with increasing extraction times, the content of effective components in the medicinal material decreases, and further increasing the number of extraction times may not yield more components from the material. Considering the operability in actual production, extraction times of 1 to 3 times were selected for subsequent orthogonal experiments. The results are shown in the appendix. Figure 2 C.

[0042] 1.4 Analysis of Orthogonal Experiment Results

[0043] Table 1 Factor Level Design Table

[0044]

[0045] Table 2 Results of the orthogonal experiment

[0046]

[0047] Table 3 Analysis of Variance

[0048] factor Sum of squared deviations Degrees of freedom F ratio F critical value Significance A 0.017 2 1.700 19.000 B 0.012 2 1.200 19.000 C 0.198 2 19.800 19.000 * D 0.010 2 1.000 19.000 error 0.01 2

[0049] Analysis of variance results showed that the number of extractions had the most significant impact on the content of bibenzyl compounds. According to the R-values ​​in Table 2, R... C >R A >R B The effects of the three factors on the extraction rate of bibenzyl derivatives, from largest to smallest, are: number of extractions > extraction time > material-liquid ratio. Based on the K value, the optimal levels for each factor were determined. Considering factors such as the time required for the experiment and the raw materials, the optimal extraction conditions for the bibenzyl derivatives of *Dendrobium officinale* were finally determined to be A1B1C2, namely, an extraction time of 140 min, a material-liquid ratio of 1:22, and two extractions.

[0050] 1.5 Verification Test

[0051] Three portions of 3.0 g of Dendrobium officinale were weighed and placed in 150 mL round-bottom flasks. 22 times the volume of 95% ethanol was added to each flask, and the mixture was refluxed at 85 °C for 140 min. The extractions were repeated twice. The extracts were filtered, combined, centrifuged at 10000 r / min for 15 min, diluted 8 times, and the absorbance was measured. The average content of bibenzyl derivatives was calculated to be 1.248%, with an RSD of 1.09% (n=3), indicating that the extraction process is stable and feasible.

[0052] 2. Preparation of Dendrobium officinale bibenzyl extract and determination of total bibenzyl content

[0053] Determination of total bibenzyl content in Dendrobium officinale

[0054] 2.1 Preparation of reference solution

[0055] Accurately weigh 10.11 mg of dendritic oxalis reference standard into a 10 mL brown volumetric flask, dissolve and dilute to the mark with an appropriate amount of 95% ethanol to prepare a solution containing 1.011 mg dendritic oxalis per mL, which is the reference solution.

[0056] 2.2 Preparation of the test solution

[0057] Accurately weigh 3.0g of Dendrobium officinale powder, extract it according to the single-factor experiment and orthogonal design scheme, filter it, and dilute the filtrate to a range of absorbance of 0.3 to 0.7. The diluted solution is the test solution.

[0058] 2.3 Determination of the measurement wavelength

[0059] Using the corresponding reagents as blank controls, the reference and test solutions were scanned in the full wavelength range (200–800 nm) using a UV-Vis spectrophotometer. The results showed that the reference solution had maximum absorption at 279 nm, and the test solution also had maximum absorption near 279 nm. Therefore, the detection wavelength was determined to be 279 nm.

[0060] 2.4 Preparation of Standard Curve

[0061] Accurately measure 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, and 1.3 mL of the dendroquinone reference solution and place them in separate 25 mL amber volumetric flasks. Add 95% ethanol to the mark to prepare a series of standard solutions. Using the corresponding reagents as blank controls, measure the absorbance at 279 nm. Plot a standard curve with absorbance as the ordinate (y) and dendroquinone concentration as the abscissa (x). (See attached diagram) Figure 1 The linear regression equation y = 12.432x + 0.0022 was obtained, with R². 2 =0.9997. The results show that the linear relationship is good in the range of 0.020-0.052 mg / mL for the concentration of dendriticin.

[0062] 2.5 Determination Method

[0063] The absorbance was determined according to the method in "2.4 Preparation of Standard Curve". The final result was then calculated by using the standard curve and combining it with the content of dandelion in the test solution.

[0064]

[0065] In the formula, Y is the content of bibenzyl compounds in Dendrobium officinale (%); m is the sample mass (g); C is the concentration parameter of bibenzyl compounds obtained from the standard curve (mg / mL); N is the dilution factor; and V is the volume of the filtrate after extraction (mL).

[0066] 2.6 Precision Test

[0067] The absorbance of the same test solution was measured six times consecutively. The results are shown in Table 4. Statistical analysis of the results showed an RSD of 0.12%, indicating good instrument precision.

[0068] Table 4 Precision Test Results

[0069] Serial Number 1 2 3 4 5 6 Absorbance (A) 0.437 0.437 0.438 0.437 0.438 0.437

[0070] 2.7 Stability Test

[0071] The absorbance of the same test solution (sealed, protected from light, and stored at room temperature) was measured at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, and 900 min. The results are shown in Table 5. The RSD was 0.14%, indicating that the absorbance value remained stable within 900 min. Therefore, the absorbance of the prepared test solution should be measured within 900 min.

[0072] Table 5. Stability Test Results

[0073]

[0074] 2.8 Repeatability Experiment

[0075] Six portions of 3.0g Dendrobium officinale powder were tested under the optimal conditions obtained from orthogonal experiments, and their absorbance was measured. The results are shown in Table 6. The RSD was 1.78%, indicating that the method has good repeatability.

[0076] Table 6 Results of Repeatability Experiments

[0077]

[0078]

[0079] 2.9 Spiking and Recovery Experiment

[0080] Six portions of Dendrobium officinale powder (containing 1.2% bibenzyl group) were accurately weighed, each approximately 0.20 g. 2.5 mL of reference standard (concentration 1.008 mg / mL) was accurately added to each portion. The test solution was prepared according to the optimal orthogonal experimental procedure. The total bibenzyl content in Dendrobium officinale was determined, and the recovery rate was calculated. The results are shown in Table 7. The average recovery rate was 92.93%, and the RSD was 1.75%.

[0081] Table 7 Results of the recovery experiment

[0082] serial number Sample content / mg Reference standard added amount / mg Total measured / mg Recovery rate / % 1 2.4300 2.50 4.78 93.86 2 2.4096 2.50 4.67 90.24 3 2.4204 2.50 4.72 92.03 4 2.4108 2.50 4.73 92.96 5 2.4600 2.50 4.83 94.87 6 2.4216 2.50 4.76 93.64 Example 2: Single-factor experimental method to investigate the extraction process of Dendrobium officinale polysaccharides.

[0083] 1.1 Investigation of different extraction times

[0084] 0.5 g of dried *Dendrobium officinale* residue after ethanol extraction was weighed. The effect of extraction time (1, 1.5, 2, and 2.5 h) on polysaccharide extraction rate was investigated. The material-to-liquid ratio was 1:40, and the extraction was performed once. The total polysaccharide content was determined. Under the above conditions, each sample was measured in triplicate, and the average of the three measurements was taken. The highest polysaccharide extraction rate (15.16%) was observed when the extraction time was 1.5 h. Further increasing the extraction time decreased the polysaccharide extraction rate. This may be because polysaccharides undergo degradation and denaturation at high temperatures, and prolonged extraction time exacerbates this irreversible loss of polysaccharide components, leading to a decrease in the polysaccharide extraction rate. In addition, excessively long extraction times may cause other compounds in the raw material to be extracted into the solvent, which could potentially interfere with the determination of polysaccharide content. Furthermore, some such impurity compounds may bind to polysaccharides, causing bias during analysis. However, since the transfer of polysaccharides from the cell wall of *Dendrobium officinale* from the solid phase to the liquid phase requires a certain amount of time, sufficient extraction time must be ensured to avoid insufficient extraction of effective components. Based on the above reasons, an extraction time of 1–2 hours was selected for subsequent orthogonal experiments. The results are shown in the appendix. Figure 4 A.

[0085] 1.2 Investigation of different feed-liquid ratios

[0086] 0.5 g of dried *Dendrobium officinale* residue after ethanol extraction was weighed, and the effect of solid-liquid ratios of 1:20, 1:30, 1:40, 1:50, 1:60, and 1:70 on the polysaccharide extraction rate was investigated. The extraction time was 1.5 h, and the extraction was performed once. The total polysaccharide content was measured. Under the above conditions, each sample was measured in triplicate, and the average of the three measurements was taken. The polysaccharide extraction rate gradually increased with increasing solid-liquid ratio, reaching a maximum of 37.27% at a ratio of 1:60. However, as the solid-liquid ratio increased further, the polysaccharide extraction rate of *Dendrobium officinale* showed a decreasing trend. This may be because, during mass transfer, the polysaccharide concentration in the solution is diluted and reduced with increasing solid-liquid ratio, thereby increasing the concentration difference of polysaccharides between the solid and liquid phases, increasing mass transfer, and accelerating the transfer rate. However, further increasing the solvent dosage actually decreased the extraction rate, possibly due to the dissolution of other impurities interfering with polysaccharide extraction and subsequent detection. Furthermore, an excessively high solid-liquid ratio can reduce energy transfer efficiency, preventing polysaccharides from effectively transitioning from the solid phase to the liquid phase, thus affecting the polysaccharide extraction rate. A solid-liquid ratio of 1:50–1:70 was selected for subsequent orthogonal experiments, and the results are shown in the appendix. Figure 4 B.

[0087] 1.3 Examination of different extraction times

[0088] 0.5g of dried *Dendrobium officinale* residue after ethanol extraction was weighed. The effect of 1, 2, 3, and 4 extraction times on polysaccharide extraction efficiency was investigated. The material-to-liquid ratio was 1:40, and the extraction time was 1.5 h. The total polysaccharide content was determined. Under the above conditions, each sample was measured in triplicate, and the average of the three measurements was taken. The polysaccharide extraction rate gradually increased with the number of extractions, reaching a maximum of 55.69% with 3 extractions. Further increases in the number of extractions led to a decrease in the polysaccharide extraction rate. Considering material and labor costs in actual production, subsequent orthogonal experiments were conducted with 1-3 extraction times. The results are shown in the appendix. Figure 4 C.

[0089] 1.4 Analysis of Orthogonal Experiment Results

[0090] Table 8 Factor Level Design Table

[0091]

[0092] Table 9 Results of the orthogonal experiment

[0093]

[0094]

[0095] Table 10. Analysis of Variance for Orthogonal Experiments

[0096] factor Sum of squared deviations Degrees of freedom F ratio F critical value Significance A 46.275 2 14.256 19.000 B 454.868 2 140.132 19.000 * C 297.118 2 91.534 19.000 * D 3.246 2 1.000 19.000 error 3.25 2

[0097] Analysis of variance results showed that the material-to-liquid ratio and the number of extractions had a significant impact on the polysaccharide extraction rate. According to the R-values ​​in Table 9, R0... B >R C >R A The effects of the three factors on the polysaccharide extraction rate, from largest to smallest, are: material-to-liquid ratio > number of extractions > extraction time. Based on the K value, the optimal levels for each factor were determined. Considering the time required for the experiment, raw materials, and other factors, the number of extractions was determined to be 2. Finally, the optimal extraction conditions for the unrefined extract of Dendrobium officinale polysaccharides were determined to be A2B1C2, namely, extraction time of 1.5 h, material-to-liquid ratio of 1:50, and number of extractions of 2.

[0098] 1.5 Verification Test

[0099] A verification experiment was conducted under the optimal process conditions (solid-to-liquid ratio 1:50, extraction time 1.5 h, extraction times 2). Under these conditions, the polysaccharide content was measured to be 48.78%, and the RSD was 2.23% (n=3). The results indicate that the process is stable and feasible.

[0100] Preparation of Dendrobium officinale polysaccharides and determination of total polysaccharide content

[0101] 2. Determination of total polysaccharide content in Dendrobium officinale

[0102] 2.1 Preparation of reference solution

[0103] Accurately weigh 18.03 mg of anhydrous glucose reference standard into a 10 mL volumetric flask, dissolve and dilute to the mark with an appropriate amount of ultrapure water, then take 0.5 mL to dilute to 10 mL, and prepare a solution containing 0.09015 mg of anhydrous glucose per mL, which is the reference standard solution.

[0104] 2.2 Preparation of the test solution

[0105] Weigh approximately 0.5g of Dendrobium officinale residue after ethanol extraction. Extract the residue according to single-factor experiments and orthogonal design schemes. Filter, combine the filtrates, transfer to a 250mL volumetric flask, add pure water to the mark, and shake well. Accurately measure 2mL of the diluted extract into a 15mL centrifuge tube, add 10mL of anhydrous ethanol, shake well, and refrigerate for 1 hour. Remove, centrifuge (4000r / min) for 20 minutes, discard the supernatant, wash the precipitate twice with 8mL of 80% ethanol each time, centrifuge, discard the supernatant, dissolve the precipitate in hot water, transfer to a 25mL volumetric flask, cool, add water to the mark, and shake well to obtain the final product.

[0106] 2.3 Preparation of Standard Curve

[0107] Accurately measure 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the reference solution into 10 mL stoppered test tubes, and add water to bring the volume to 1.0 mL. Accurately add 1 mL of 5% phenol solution (prepare immediately before use), shake well, then accurately add 5 mL of sulfuric acid, shake well, heat in a boiling water bath for 20 min, remove, and cool in an ice bath for 5 min. Using the corresponding reagent as a blank control, measure the absorbance at a wavelength of 488 nm. Plot a standard curve with absorbance as the ordinate (y) and glucose concentration as the abscissa (x). (See attached diagram) Figure 3 The linear regression equation was obtained as y = 9.1926x + 0.0075, with a correlation coefficient R0. 2 =0.9986. The results show that the linear relationship is good in the range of glucose concentration from 0.01803 to 0.09015 mg / ml.

[0108] 2.4 Determination Methods

[0109] Referring to the method for determining the polysaccharide content of Dendrobium officinale in the 2020 edition of the Pharmacopoeia of the People's Republic of China, 1 mL of the test sample solution was measured and placed in a 10 mL stoppered test tube. Following the method under "2.3 Preparation of Standard Curve", 1 mL of 5% phenol solution was added, and the absorbance was measured. Then, the final result was calculated by using the standard curve and combining it with the amount of anhydrous glucose in the test sample solution.

[0110]

[0111] In the formula, Y is the extraction rate of Dendrobium officinale polysaccharides (%); C is the polysaccharide concentration parameter obtained from the standard curve (mg / mL); and m is the sample mass (g).

[0112] 2.5 Precision Test

[0113] The absorbance of the same test solution was measured six times consecutively. The results are shown in Table 11. Statistical analysis of the results showed an RSD of 0.21%, indicating good instrument precision.

[0114] Table 11 Precision Test Results

[0115] Serial Number 1 2 3 4 5 6 Absorbance (A) 0.359 0.359 0.360 0.359 0.358 0.359

[0116] 2.6 Repeatability Experiment

[0117] Six portions of dried Dendrobium officinale residue (0.5g each) after ethanol extraction were tested under the optimal conditions obtained from orthogonal experiments, and their absorbance was measured. The results are shown in Table 12. The RSD was 2.03%, indicating that the method has good repeatability.

[0118] Table 12 Results of Repeatability Experiments

[0119]

[0120] 2.7 Colorimetric stability test

[0121] The absorbance of the same test solution (sealed, protected from light, and stored at room temperature) was measured at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min. The results are shown in Table 13. The RSD is 0.55%, indicating that the color development is stable within 120 min. Therefore, the absorbance of the prepared test solution should be measured within 120 min.

[0122] Table 13 Results of Color Development Stability Test

[0123]

[0124] 2.8 Spiking and Recovery Experiment

[0125] Six portions of dried *Dendrobium officinale* residue (polysaccharide concentration determined) after ethanol extraction were accurately weighed. Extraction was performed according to the optimal process determined by orthogonal experiments. The residues were filtered, combined, and transferred to a 250 mL volumetric flask. Pure water was added to the mark, and the mixture was shaken well. One mL of the diluted extract was accurately measured and placed in a 15 mL centrifuge tube. Five mL of anhydrous ethanol was accurately added, and the mixture was shaken well and refrigerated for 1 hour. The tube was then removed and centrifuged (4000 rpm) for 20 minutes. The supernatant was discarded. The precipitate was washed twice with 80% ethanol, centrifuged again, and the supernatant was discarded. The precipitate was dissolved in hot water and transferred to a 25 mL volumetric flask. The flask was cooled, and one mL of 0.951 mg / mL glucose standard solution was accurately added. Water was added to the mark, and the mixture was shaken well. The absorbance was measured, and the recovery rate was calculated. The results are shown in Table 14. The average recovery rate was 100.16%, and the RSD was 2.77%.

[0126] Table 14 Results of the recovery experiment

[0127]

[0128] Example 3 Qualitative Analysis of Bibenzyl Extract

[0129] 1. Preparation of mixed reference solution

[0130] Accurately weigh each reference standard (including arginine, arginine, protocatechuic aldehyde, caffeic acid, quercetin, protocatechuic acid, piperidine, echinacoside, chlorogenic acid, ferulic acid, gallic acid, aloe-emodin, and rutin) into a volumetric flask, dissolve in methanol and dilute to volume to prepare a 1 mg / mL reference standard stock solution, and store at 4°C for later use. Before use, take an appropriate amount of each reference standard stock solution to prepare a 5 μg / mL mixed reference standard solution, centrifuge at 13000 rpm for 10 min at 4°C, and filter through a 0.22 μm microporous membrane, collecting the filtrate.

[0131] 2. Preparation of the test solution

[0132] Weigh 20 mg of Dendrobium officinale bibenzyl extract, add 2 mL of methanol, sonicate to dissolve, centrifuge at 13000 rpm for 10 min, and filter through a 0.22 μm microporous membrane. Take the filtrate for analysis.

[0133] 3. Chromatographic-mass spectrometry conditions

[0134] 3.1 Chromatographic conditions

[0135] Adopting ACQUITY BEH C 18The chromatographic column (2.1 mm × 100 mm, 1.7 μm, Waters) was used at a column temperature of 40 °C. The mobile phase in both positive and negative ion modes was 0.1% formic acid (A) and acetonitrile (B). The flow rate was 0.3 mL / min, and the injection volume was 3 μL. The gradient elution program was as follows: 0–1.5 min, 2%–22% B; 1.5–8.5 min, 22%–35% B; 8.5–9.5 min, 35%–58% B; 9.5–12.5 min, 58%–63% B; 12.5–21 min, 63%–90% B; 21–22.5 min, 90% B; 22.5–23 min, 90%–2% B; 23–25.5 min, 2% B.

[0136] 3.2 Mass Spectrometry Conditions

[0137] The TOF MS-IDA-MS / MS mode was used, with an electrospray ionization (ESI) source, scanning in both positive and negative ion modes, and a mass scan range of m / z 100–1250. The following ESI parameters were used: electrospray voltage (ISVF) of -4500V (ESI). - ) / 5500V(ESI + The settings are as follows: curtain gas (CUR) 35psi, nebulizing gas (GS1) 55psi, auxiliary gas (GS2) 55psi, ion source temperature (TEM) 550℃, declustering voltage (DP) 80V, collision voltage set to 10V in TOF-MS mode, collision voltage set to 35V in IDA-MS / MS mode, and collision voltage difference set to 15V.

[0138] 4. Analysis Results

[0139] The experimental results are attached. Figure 5 According to Table 15, 40 bibenzyl compounds, including dendrobine and dendrobin, were identified.

[0140] Table 15. Identification results of benzyl derivatives in Dendrobium officinale.

[0141]

[0142]

[0143]

[0144] Example 4 Qualitative and quantitative analysis of polysaccharide extract

[0145] 1. Preparation of mixed reference solution

[0146] After accurately weighing each monosaccharide standard, add ultrapure water to prepare a 10 mg / mL monosaccharide standard solution. Then, measure an appropriate amount of the single standard stock solution and mix them to prepare a mixed standard solution with a maximum index concentration of 60, 50 or 40 μg / mL. Prepare a series of standard solutions required for monosaccharide component analysis according to the following concentration gradient.

[0147] Table 16 Information on Monosaccharide Mixed Standard Concentrations

[0148]

[0149] 2. Preparation of the test solution

[0150] Take 5 mg of Dendrobium officinale polysaccharide sample and place it in a pressure-resistant bottle. Add 2 mL of 2M trifluoroacetic acid solution and heat at 121℃ for 2 h. Purge with nitrogen using a nitrogen purging device and dry. Add 99.99% methanol to wash, then dry again. Repeat the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic bottle for analysis.

[0151] 3. HPAEC conditions

[0152] The chromatographic system was a Thermo ICS 5000. + Ion chromatography (ICS 5000) + Thermo Fisher Scientific (USA) used an electrochemical detector to analyze and detect monosaccharide components.

[0153] Using Dionex TM CarboPac TM PA20 (150×3.0mm, 10μm) liquid chromatography column; injection volume: 5μL. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5 mL / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0154] 4. Sample content calculation method

[0155]

[0156] In the formula, Y is the content of a certain monosaccharide component in the sample, with the unit (μg / mg); C is the concentration read by the instrument, with the unit (μg / mL); V is the volume of the sample extraction solution, with the unit (mL); F is the dilution factor; M is the total amount of the sample weighed, with the unit (mg).

[0157] 5. Results and Analysis

[0158] The standard product spectrum was compared with the sample spectrum, and the results are shown in the appendix Figure 6 and 7 , the polysaccharide of Dendrobium officinale is composed of mannose, glucose, rhamnose, arabinose, galactose, galacturonic acid and glucuronic acid; the external standard method was used for quantification. With the concentration of the standard product as the abscissa and the peak area of the standard product as the ordinate, a graph was plotted. According to the peak area of the corresponding compound in the sample, its content was calculated: the content of rhamnose was 0.206 μg / mg, the content of arabinose was 1.502 μg / mg, the content of galactose was 1.998 μg / mg, the content of glucose was 212.606 μg / mg, the content of mannose was 366.702 μg / mg, the content of galacturonic acid was 3.257 μg / mg, and the content of glucuronic acid was 2.383 μg / mg.

[0159] Example 5 Animal Experiment

[0160] 1. Cell Culture

[0161] Lewis lung adenocarcinoma (LLC) cells were cultured in DMEM (high glucose) medium containing 10% fetal bovine serum and 1% double antibody, and cultured in an incubator at 37°C and containing 5% CO2. When the cell growth density was 80 - 90%, after digestion with trypsin for 1 minute, subculture was carried out. After subculturing to the stable generation number of the cell line, cells in the logarithmic growth phase were taken for the experiment.

[0162] 2. Animal Modeling

[0163] The animal experiment of this invention was approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine, and the animal ethics approval number was 202309A026. BalB / c healthy male mice (3 - 5 weeks old), Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., certificate number: SCXK (Beijing) 2019 - 0004, were adaptively fed for about one week. During the experiment, the mice were in a feeding environment with 12h light and 12h darkness every day, a temperature of 18 - 22°C, and a relative humidity of about 60%. The sterilized bedding was changed two to three times a week, and drinking water and food were replenished in a timely manner. One to two days before modeling, the hair in the area near the right axilla of 48 mice was removed with hair removal cream. When the number of LLC cells reached the number required for modeling, they were digested, resuspended with pre-cooled PBS, counted, and the cell density was adjusted to 1.5×10 7Cells were kept at a density of 10 cells / mL and placed on an ice box to maintain cell condition. The cell suspension was gently mixed using a pipette, and then 0.2 mL (3 × 10⁻⁶ cells / mL) was drawn up using a 1 mL medical syringe and subcutaneously injected into the area near the right axilla of each mouse. 6 Cell inoculation (one cell per day) must be completed within 30 minutes to avoid affecting the accuracy of experimental results. After 4-5 days (during which time the mice's mental state, activity level, food intake, and weight changes should be observed and recorded daily), a palpable, round or oval mass (not exceeding 1 cm) under the skin is considered a successful model establishment. 3 Record the date of subcutaneous tumor formation in mice.

[0164] 3. Animal grouping and administration

[0165] Fifty-four mice were divided into nine groups: a control group, a model group, a positive control group (5-fluorouracil), a polysaccharide group, a bibenzyl group, and two groups (polysaccharide:bibenzyl = 1:1 and 2:1), with six mice in each group. Each drug was prepared using 0.5% sodium carboxymethyl cellulose. Each mouse was administered 0.1 mL / 10 g via gavage once daily for 15 days. During this period, the mice's mental state, activity level, and food intake were observed and recorded daily. Body weight and the longest and shortest diameters of the subcutaneous xenografts were measured every two days.

[0166] Table 17 Animal grouping and drug administration

[0167] label Group Dosage Number 1 Blank group Administer 0.5% sodium carboxymethyl cellulose by gavage. 6 2 Model group Administer 0.5% sodium carboxymethyl cellulose by gavage. 6 3 Positive drug group 15 mg / kg / day of 5-Fu administered orally 6 4 High-dose polysaccharide group 200 mg / kg / day of polysaccharide extract administered by gavage 6 5 low-dose polysaccharide group 100 mg / kg / day of polysaccharide extract administered by gavage 6 6 High-dose bibenzyl group Bibenzyl extract administered by gavage 200 mg / kg / day 6 7 Low-dose bibenzyl group Bibenzyl extract administered by gavage 100 mg / kg / day 6 8 Polysaccharide:Bibenzyl = 1:1 Gavage administration of 100 mg / kg polysaccharide + 100 mg / kg bibenzyl 6 9 Polysaccharide:Bibenzyl = 2:1 Gavage administration of 200 mg / kg polysaccharide + 100 mg / kg bibenzyl 6 .

[0168] 4. Results and Analysis

[0169] 4.1 Behavioral characteristics of mice

[0170] The mice in the blank control group were in good spirits, had shiny fur, and were active normally. After successful modeling, the mice in each group reacted slowly and their activity decreased. After drug intervention according to the groups, the mice in the polysaccharide, bibenzyl, and combined drug administration groups had normal diet, water intake, urination, and defecation, and their activity level increased compared to the model group. The positive drug group showed signs of lethargy, slow movement, rapid breathing, dull fur, loose stools around the anus, and weight loss in the later stages.

[0171] 4.2 Changes in tumor volume

[0172] During the drug administration period, the long and short diameters of the mouse tumors were measured every other day using vernier calipers. The formula was: Tumor volume = 0.5 × long diameter × short diameter. 2 The experimental results are attached. Figure 8 and 9 .

[0173] 4.3 Detection of tumor weight, tumor inhibition rate, and tumor index in mice

[0174] After the last administration, mice were fasted for 12 hours but allowed free access to water. Mice were anesthetized, weighed, and blood was collected from the eyeballs. The mice were then euthanized by cervical dislocation, and tumors were dissected. The tumors were cleaned in pre-cooled physiological saline to remove blood, and the surface moisture was blotted dry with filter paper. The tumor inhibition rate and tumor index were calculated using the following formulas: Tumor inhibition rate = 1 - (average tumor weight in the administered group (g) / average tumor weight in the control group (g) × 100%); Tumor index (mg / g) = tumor weight (mg) / body weight (g). Experimental results are attached. Figure 10 Compared with the model group, the tumor weight and tumor index of the positive control group, the high-dose bibenzyl group, and the polysaccharide:bibenzyl = 1:1 and 2:1 groups were significantly reduced, with statistically significant differences (p<0.05). However, there were no significant differences in tumor weight and tumor index among the low-dose polysaccharide, high-dose and low-dose bibenzyl groups, indicating that the combined administration of the two drugs was more effective and had a higher tumor inhibition rate than the single administration.

[0175] Table 18 Mean tumor weight and inhibition rate for each group (x±s, n=6)

[0176] Grouping Dosage (mg / kg) Average tumor weight (g) Inhibition rate (%) Model group — 0.94±0.35 — 5-Fu group 15 0.28±0.29* 70.17 low-dose polysaccharide group 100 0.59±0.45 37.33 High-dose polysaccharide group 200 0.44±0.38 53.66 Low-dose bibenzyl group 100 0.49±0.45 48.00 High-dose bibenzyl group 200 0.33±0.31* 64.67 Polysaccharide:Bibenzyl = 1:1 group 100 (polysaccharide) + 100 (bibenzyl) 0.35±0.35* 62.87 Polysaccharide:Bibenzyl = 2:1 group 200 (polysaccharide) + 100 (bibenzyl) 0.34±0.30* 63.51

[0177] Note: *p<0.05 compared to the model group.

[0178] 4.4 Organ Index Detection

[0179] After the last administration, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized, weighed, and blood was collected from the eyeballs. The mice were euthanized by cervical dislocation, and the thymus and spleen were dissected. The organs were cleaned in pre-cooled physiological saline to remove blood, and the surface moisture was blotted dry with filter paper. The organs were weighed separately, and the organ index of each immune organ was calculated. Formula: Organ index (mg / g) = Organ mass (mg) / Body weight (g). Experimental results are attached. Figure 11 Compared with the control group, the thymus index of mice in the model group was significantly reduced (p<0.001), and the spleen index was significantly increased (p<0.01), indicating that the attack of tumor cells severely damages the thymus and spleen of tumor-bearing mice. Compared with the model group, the thymus index of mice in the polysaccharide:bibenzyl = 2:1 group and the spleen index of mice in the high-dose bibenzyl group and the combined administration group were significantly improved (p<0.05), and were closer to the normal group, indicating that the drug can effectively protect the immune organs of mice from attack by LLC lung cancer cells.

[0180] 4.5 Determination of serum TNF-α, IL-1β and IFN-γ levels

[0181] After the last administration, mice were fasted for 12 hours but allowed free access to water. They were then anesthetized, weighed, and blood was collected from the eyeballs. After being left at room temperature for 30 minutes, the blood was centrifuged at 14,000 rpm for 10 minutes at 4°C, the supernatant was discarded, and the blood was stored at -80°C for later use. For assays, the blood was thawed at low temperature, and the levels of TNF-α, IL-1β, and IFN-γ were measured according to the kit instructions. The experimental results are attached. Figure 12The levels of inflammatory factors in the model group were significantly higher than those in the normal group (p<0.0001), while the levels of inflammatory factors in each drug-treated group were significantly lower than those in the model group (p<0.0001). This indicates that the large increase in tumor cells caused inflammatory and immune responses in the body, and that drug administration can downregulate the secretion level of inflammatory factors to achieve anti-tumor effects.

[0182] 4.6 Data Processing

[0183] Data results are presented in mean ± SEM format, and statistical analysis and plotting were performed using GraphPadPrism 9.0 software. One-way ANOVA was used if the sample data conformed to a normal distribution and had homogeneous variances; otherwise, the nonparametric Kruskal-Wallis test was used. A p-value < 0.05 was considered statistically significant.

Claims

1. A detection method of Dendrobium candidum composite extract, characterized in that, The Dendrobium officinale composite extract includes a Dendrobium officinale bibenzyl extract and a Dendrobium officinale polysaccharide extract; The Dendrobium officinale bibenzyl component detection method includes the following steps: (1) Preparation of mixed control solution Precisely weigh each control substance of maliandron, arginine, protocatechuic aldehyde, coffee acid, quercetin, protocatechuic acid, taxifolin, echinacoside, chlorogenic acid, ferulic acid, gallic acid, aloe-emodin and rutin in a volumetric flask, dissolve and dilute with methanol, prepare a control substance stock solution of 1 mg / mL, and store at 4 DEG C for standby; Before use, prepare a mixed control solution of 5 μg / mL from each control substance stock solution, centrifuge at 13000 rpm for 10 min at 4 DEG C, filter with a 0.22 μm microporous filter, and inject the filtrate into a liquid chromatograph-mass spectrometer for analysis; (2) Preparation of test solution Take 20 mg of the Dendrobium officinale bibenzyl extract, add 2 mL of methanol, ultrasonically dissolve, centrifuge at 13000 rpm for 10 min, filter with a 0.22 μm microporous filter, and inject the filtrate into a liquid chromatograph-mass spectrometer for analysis; (3) Chromatography-mass spectrometry conditions (3.1) Chromatography conditions ACQUITY UPLC ® BEH C 18 Column: 2.1 mm x 100 mm, 1.7 μm, Waters, Column temperature 40 ℃, mobile phase: 0.1% formic acid water A and acetonitrile B, flow rate 0.3 mL / min, injection volume 3 μL, gradient elution program: 0~1.5 min, 2%~22% B; 1.5~8.5 min, 22%~35% B; 8.5~9.5 min, 35%~58% B; 9.5~12.5 min, 58%~63% B; 12.5~21 min, 63%~90% B; 21~22.5 min, 90% B; 22.5~23 min, 90%~2% B; 23~25.5 min, 2% B; (3.2) Mass spectrometry conditions TOF MS-IDA-MS / MS mode, electrospray ion source ESI, scanning in positive and negative ion mode, mass scan range m / z 100-1250; using the following ESI parameters: electrospray voltage ISVF of -4500 V (ESI - ) / 5500 V (ESI + ), curtain gas CUR of 35 psi, nebulizing gas GS1 of 55 psi, auxiliary gas GS2 of 55 psi, ion source temperature of 550 °C, de-clustering voltage DP of 80 V, collision voltage of 10 V in TOF-MS mode, collision voltage of 35 V in IDA-MS / MS mode, collision voltage difference of 15 V; The Dendrobium officinale polysaccharide content determination includes the following steps: (1) Preparation of control solution Precisely weigh 18.03 mg of anhydrous glucose control substance in a 10 mL volumetric flask, dissolve and dilute with ultrapure water to the mark, and then take 0.5 mL and dilute to 10 mL to prepare a solution containing 0.09015 mg of anhydrous glucose per 1 mL, namely the control solution; (2) Preparation of test solution Take the Dendrobium officinale polysaccharide extract, transfer to a 250 mL volumetric flask, add pure water to the mark, and shake well; precisely take 2 mL of the extracted solution after dilution in a 15 mL centrifuge tube, add 10 mL of anhydrous ethanol, shake well, and refrigerate for 1 h, take out, centrifuge at 4000 r / min for 20 min, discard the supernatant, wash the precipitate with 80% ethanol for 2 times, 8 mL each time, centrifuge, discard the supernatant, dissolve the precipitate in hot water, transfer to a 25 mL volumetric flask, cool down, add water to the mark, and shake well to obtain the test solution; (3) Preparation of standard curve Precise metering glucose control sample solution 0.2, 0.4, 0.6, 0.8, 1.0 mL, respectively, in 10 mL stoppered test tube and add water to 1.0 mL, precise 5% phenol solution 1 mL, shake, then precise sulfuric acid 5 mL, shake, in boiling water bath for 20 min, take out, ice bath cooling 5 min, with the corresponding reagent as blank control, at 488 nm wavelength, the absorbance was determined, with absorbance as the ordinate y, glucose concentration as the abscissa x, standard curve was drawn, linear regression equation y = 9.1926x + 0.0075, correlation coefficient R 2 = 0.9986; the results showed that: glucose mass concentration in the range of 0.01803~0.09015mg / ml, good linear relationship; (4) Determination method Referring to the Dendrobium officinale polysaccharide content determination method in the 2020 edition of the People's Republic of China Pharmacopoeia, take 1 mL of the test solution in a 10 mL test tube with a stopper, add 1 mL of 5% phenol solution according to the method under "1.3 Preparation of standard curve", determine the absorbance, and then calculate the final result through the standard curve and the amount of anhydrous glucose in the test solution; ； In the formula, Y is the Dendrobium officinale polysaccharide extraction rate%; C is the polysaccharide concentration parameter mg / mL obtained from the standard curve; and m is the sample mass g; The Dendrobium officinale bibenzyl extract is prepared by the following method: Take Dendrobium officinale dry stems, crush, and extract twice by refluxing with 95% ethanol with a solid-liquid ratio of 1:22, and each extraction time is 140 min; The Dendrobium officinale polysaccharide extract is prepared by the following method: The residue after ethanol extraction of Dendrobium candidum Wall ex Lindl was added with water according to a solid-liquid ratio of 1:50, and extracted for 2 times, each time for 1.5 h; the filtrates were combined, and ethanol was added to the combined filtrate to a content of 80-90%, and then the mixture was placed at 4 DEG C for 24 h; the precipitate was collected by filtration, and deproteinized by the Sevag method, to obtain the product.

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