Preparation method and application of dendrobium flexicaule extract

Abstract

The invention relates to the technical field of traditional Chinese medicine extraction, in particular to a preparation method and application of a dendrobium flexicaule extract. The preparation method comprises the following steps: firstly, accurately weighing a rutin standard substance, dissolving the rutin standard substance with ethanol, preparing contrast solutions with different concentrations, drawing a standard curve through a chromogenic reaction and absorbance determination, then, cleaning stems of dendrobium flexicaule with tap water, and drying the cleaned stems of dendrobium flexicaule to obtain the dendrobium flexicaule extract. The preparation method comprises the following steps: drying in a drying oven at 50 DEG C for 5 days, crushing by using a pulverizer, sieving to obtain uniform powder for later use, extracting by adopting an alcohol extraction method or a water extraction method in combination with ultrasonic-assisted extraction to obtain an extract, and finally obtaining the optimized preparation method through a single factor experiment, a response surface test, optimization item verification and specific condition verification according to a drawn standard curve. By adopting the method, the extraction conditions of the water extraction method and the alcohol extraction method are optimized, so that the extraction efficiency of the flavonoid compounds in the dendrobium flexicaule is improved, and the retaining effect of active ingredients is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a preparation method and application of a Dendrobium curcumae extract. Background Art

[0002] Dendrobium curcumae (Dendrobium candidum) is a plant of the genus Dendrobium in the Orchidaceae family. It boasts benefits such as nourishing the stomach, promoting fluid production, and nourishing yin and clearing heat. Its main chemical components include polysaccharides, alkaloids, flavonoids, sesquiterpenoid glycosides, amino acids, and compounds such as phenolic acids, bibenzyls, and phenanthrenes. Flavonoids are a class of secondary synthetic products widely found in plants in nature. These compounds typically exist in a specific form: in a few specialized species, they are present independently as free glycoside groups. In recent years, intensive scientific research has revealed a rich array of physiological activities. These compounds exhibit significant anti-aging effects, effectively combat cellular hypoxia, alleviate fatigue, and significantly enhance immunity. Flavonoids also demonstrate remarkable efficacy in extending lifespan and combating cancer, bringing numerous benefits to human health. Furthermore, total flavonoids play a key role in protecting the human cardiovascular system, regulating inflammatory cytokines and inhibiting inflammatory bacteria. These compounds also exhibit multiple pharmacological actions, including anti-hepatorenal, antibacterial, antiviral, and antioxidant properties, and effectively enhance immunity. These unique physiological activities and functions have been widely recognized and applied in scientific research and clinical practice, demonstrating their important medical value and potential.

[0003] Currently, research on Dendrobium plants primarily focuses on tissue culture and the extraction of selected components. However, the extraction process and efficacy of flavonoids from stem segments of Dendrobium curcumae have yet to be reported. Traditional plant extraction methods primarily include aqueous extraction and alcohol extraction. Aqueous extraction typically involves decoction or soaking, while alcohol extraction utilizes organic solvents such as ethanol, with reflux or ultrasound-assisted extraction. However, these methods have limitations in extraction efficiency, active ingredient retention, and process optimization.

[0004] In summary, the traditional plant extraction method has problems in extraction efficiency, active ingredient retention and process optimization when extracting flavonoids from Dendrobium curcumae. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of a Dendrobium curcumae extract, so as to solve the problems of insufficient extraction efficiency, active ingredient retention and process optimization in traditional plant extraction methods when extracting flavonoids from Dendrobium curcumae.

[0006] To achieve the above object, the present invention provides a method for preparing a Dendrobium curcumae extract, the method comprising the following steps:

[0007] Determination of flavonoid standard curve: Accurately weigh rutin standard, dissolve it in ethanol and prepare control solutions of different concentrations, and draw a standard curve through color reaction and absorbance measurement;

[0008] Raw material pretreatment: clean the stem segments of Dendrobium candidum with tap water, dry them in an oven at 50°C for 5 days, grind them with a powder grinder, and sieve them to obtain a uniform powder for later use;

[0009] Raw material extraction: alcohol extraction or water extraction combined with ultrasound-assisted extraction to obtain the extract;

[0010] Verification and optimization: Based on the drawn standard curve, the optimized preparation method is obtained after single-factor experiments, response surface experiments, preferred option verification and specific condition verification.

[0011] Among them, the specific steps of the step "determination of flavonoid standard curve" are:

[0012] Accurately weigh a certain amount of rutin standard, dissolve it in 80% ethanol, transfer it to a 100 mL volumetric flask, dilute to the mark, and shake well to obtain a rutin control solution.

[0013] Accurately pipette different volumes of rutin reference solution into 10 mL volumetric flasks, fill to the 3.0 mL mark with ethanol, shake well, add 0.5 mL of 5% sodium nitrite solution, shake well and let stand for 6 minutes, add 0.5 mL of 10% aluminum nitrate solution, shake well again and let stand for 6 minutes, add 4 mL of 1 mol / L sodium hydroxide solution, and fill to the mark with 80% ethanol, shake well and let stand for 15-20 minutes;

[0014] With distilled water as a blank control, the absorbance of each solution was measured at a wavelength of 510 nm. A standard curve was drawn with rutin concentration as the abscissa and absorbance as the ordinate, and the regression equation was obtained.

[0015] Among them, in the step of "raw material pretreatment", when sieving after crushing with a powder grinder, a 1 mm sieve is used.

[0016] Among them, in the step of "raw material extraction", water extraction combined with ultrasound-assisted extraction is used to obtain the specific content of the extract:

[0017] The Dendrobium officinale was crushed with a grinder, and about 10 g of the crushed Dendrobium officinale sample was weighed into a 400 ml glass bottle. 300 ml of distilled water was added to the glass bottle and covered. The extraction was carried out at 20°C to 100°C and an ultrasonic power of 100W to 300W for 20 to 60 minutes. The residual liquid was collected after filtering with gauze, centrifuged, and placed in a refrigerator at 4°C for 12 hours, and then freeze-dried.

[0018] Among them, in the step of "raw material extraction", the specific content of the extract obtained by using alcohol extraction combined with ultrasound-assisted extraction is:

[0019] The Dendrobium officinale was crushed with a grinder, and about 10 g of the crushed Dendrobium officinale sample was weighed into a 400 ml glass bottle. 200 ml of methanol was added to the glass bottle and covered. The extraction was carried out at a temperature of 20°C to 100°C and an ultrasonic power of 100W to 300W for 20 to 60 minutes. The residual liquid was collected by centrifugation after filtering with gauze, rotary evaporation, and freeze-drying.

[0020] Among them, in the step "Verification and Optimization", the single-factor experiment specifically refers to investigating the effects of different extraction temperatures, times and ultrasonic powers on the flavonoid extraction amount;

[0021] Response surface experiment specifically refers to selecting appropriate factor levels for three-factor three-level experimental design based on the results of single-factor experiments, using Design-Expert software for data analysis, and optimizing extraction conditions;

[0022] Optimal option verification specifically refers to conducting alcohol extraction and water extraction experiments under optimal extraction conditions to compare the extraction efficiency;

[0023] Specific condition verification specifically refers to further verifying the effects of different extraction temperatures, times and ultrasonic powers on flavonoid extraction under optimal alcohol extraction conditions.

[0024] The present invention also provides an application of a Dendrobium curcumae extract, and uses the Dendrobium curcumae extract prepared by the above-mentioned method for preparing the Dendrobium curcumae extract in preparing a drug for improving immunity and hematopoietic function.

[0025] The invention provides a preparation method and application of a Dendrobium curcumae stem extract. First, a rutin standard is accurately weighed, dissolved in ethanol and prepared into control solutions of different concentrations, and a standard curve is drawn through color development reaction and absorbance measurement. Then, the stem segments of the Dendrobium curcumae stem are cleaned with tap water, dried in an oven at 50°C for 5 days, crushed with a powder grinder and sieved to obtain a uniform powder for standby use, and an alcohol extraction method or a water extraction method combined with ultrasound-assisted extraction is used to obtain an extract. Finally, based on the drawn standard curve, an optimized preparation method is obtained after single-factor experiments, response surface experiments, preferred option verification and specific condition verification. By adopting the above method, the extraction conditions of the water extraction method and the alcohol extraction method are optimized, thereby improving the extraction efficiency of flavonoid compounds in the Dendrobium curcumae stem and improving the active ingredient retention effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The present invention provides a flowchart of the steps of the preparation method of the Dendrobium curcumae extract.

[0028] Figure 2 This is the rutin standard curve provided by the present invention.

[0029] Figure 3 It is a schematic diagram of the influence of different temperatures on the extraction effect provided by the present invention.

[0030] Figure 4 It is a schematic diagram of the influence of different times on the extraction effect provided by the present invention.

[0031] Figure 5 It is a schematic diagram of the influence of different ultrasonic powers on the extraction effect provided by the present invention.

[0032] Figure 6 It is a contour map and a response surface map of the interaction effect between the three factors on the flavonoid content provided by the present invention.

[0033] Figure 7 Schematic diagram of the effect of DFE provided by the present invention on hematological parameters of immunosuppressed mice.

[0034] Figure 8 This is a schematic diagram of the effect of DFE provided by the present invention on hematopoietic-related cytokines in immunosuppressive mice.

[0035] Figure 9 This is a schematic diagram of the effect of DFE provided by the present invention on inflammatory cytokines in immunosuppressed mice.

[0036] Figure 10 This is a schematic diagram showing the effects of DFE provided by the present invention on serum biochemical indicators in immunosuppressed mice. DETAILED DESCRIPTION

[0037] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0038] See also Figures 1 to 6The present invention provides a method for preparing a Dendrobium curcumae extract, which comprises the following steps:

[0039] S1. Determination of flavonoid standard curve: Accurately weigh rutin standard, dissolve it in ethanol and prepare control solutions of different concentrations. Draw a standard curve through color reaction and absorbance measurement;

[0040] S2. Raw material pretreatment: clean the stem segments of Dendrobium curcumae with tap water, dry them in an oven at 50°C for 5 days, grind them with a powder grinder, and sieve them to obtain a uniform powder for later use;

[0041] S3. Raw material extraction: using alcohol extraction or water extraction combined with ultrasound-assisted extraction to obtain the extract;

[0042] S4. Verification and optimization: Based on the drawn standard curve, the optimized preparation method is obtained after single-factor experiments, response surface experiments, preferred option verification and specific condition verification.

[0043] In this embodiment, first, the rutin standard is accurately weighed, dissolved in ethanol and prepared into control solutions of different concentrations, and a standard curve is drawn by color reaction and absorbance measurement. Then, the stem segments of the curvatured stem of Dendrobium officinale are cleaned with tap water, dried in an oven at 50°C for 5 days, crushed with a powder grinder and sieved to obtain a uniform powder for standby use, and an alcohol extraction method or a water extraction method combined with ultrasound-assisted extraction is used to obtain an extract. Finally, based on the drawn standard curve, after single-factor experiments, response surface experiments, preferred option verification and specific condition verification, an optimized preparation method is obtained. The above method is used to optimize the extraction conditions of the water extraction method and the alcohol extraction method, thereby improving the extraction efficiency of flavonoid compounds in the curvatured stem of Dendrobium officinale and improving the retention effect of the active ingredients.

[0044] Furthermore, the specific steps of the step "determination of flavonoid standard curve" are:

[0045] Accurately weigh a certain amount of rutin standard (e.g., 0.009 g), dissolve it in 80% ethanol, transfer it to a 100 mL volumetric flask, dilute to the mark, and shake well to obtain a rutin control solution.

[0046] Accurately pipette different volumes of rutin reference solution (such as 0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL) into 10 mL volumetric flasks, fill to the 3.0 mL mark with ethanol, shake well, add 0.5 mL of 5% sodium nitrite solution in sequence, shake well and let stand for 6 minutes, add 0.5 mL of 10% aluminum nitrate solution, shake well again and let stand for 6 minutes, add 4 mL of 1 mol / L sodium hydroxide solution, and fill to the mark with 80% ethanol, shake well and let stand for 15-20 minutes;

[0047] With distilled water as a blank control, the absorbance of each solution was measured at a wavelength of 510 nm. A standard curve was drawn with rutin concentration as the abscissa and absorbance as the ordinate, and the regression equation was obtained.

[0048] In this embodiment, if Figure 2 As shown, the regression equation is y = 3.0595x + 0.0123, and the correlation coefficient R 2 =0.9933. Within the set concentration range, it can be observed that there is an excellent linear correlation between the concentration of the solution and its absorbance value. This result proves the stable linear relationship between concentration and absorbance. In subsequent experiments, the concentration of flavonoids in the sample can be calculated by measuring the absorbance of the sample and substituting it into the regression equation.

[0049] Furthermore, in the step of "raw material pretreatment", when sieving after crushing with a powder grinder, a 1 mm sieve is used.

[0050] Furthermore, in the step of "raw material extraction", water extraction combined with ultrasound-assisted extraction was used to obtain the following extract:

[0051] The Dendrobium officinale was crushed with a grinder, and about 10 g of the crushed Dendrobium officinale sample was weighed into a 400 ml glass bottle. 300 ml of distilled water was added to the glass bottle and covered. The extraction was carried out at 20°C to 100°C and an ultrasonic power of 100W to 300W for 20 to 60 minutes. The residual liquid was collected after filtering with gauze, centrifuged, and placed in a refrigerator at 4°C for 12 hours, and then freeze-dried.

[0052] Furthermore, in the step of "raw material extraction", the specific contents of the extract obtained by using alcohol extraction combined with ultrasound-assisted extraction are:

[0053] The Dendrobium officinale was crushed with a grinder, and about 10 g of the crushed Dendrobium officinale sample was weighed into a 400 ml glass bottle. 200 ml of methanol was added to the glass bottle and covered. The extraction was carried out at a temperature of 20°C to 100°C and an ultrasonic power of 100W to 300W for 20 to 60 minutes. The residual liquid was collected by centrifugation after filtering with gauze, rotary evaporation, and freeze-drying.

[0054] Furthermore, in the step "Verification and Optimization", the single-factor experiment specifically refers to investigating the effects of different extraction temperatures (such as 20°C, 40°C, 60°C, 80°C, 100°C), time (such as 20min, 30min, 40min, 50min, 60min) and ultrasonic power (such as 100W, 150W, 200W, 250W, 300W) on the flavonoid extraction amount, and conducting alcohol extraction and water extraction experiments respectively to compare the extraction efficiency.

[0055] In this embodiment, if Figure 3It can be seen that the flavonoid content first increases and then decreases with the increase of extraction temperature, reaching a peak at 60°C. This shows that too high a temperature may cause the flavonoids to be destroyed, which in turn reduces the extraction rate. Therefore, the optimal temperature is 60°C. In addition, the highest flavonoid content value of the water extraction method is 17.7 mg / g, while the highest value of the alcohol extraction method is 19.3 mg / g. It can be seen that at the same temperature, the efficiency of the alcohol extraction method is better than that of the water extraction method.

[0056] like Figure 4 It can be seen that the efficiency of the alcohol extraction method reached its peak at 19.5 mg / g at 30 minutes, while the water extraction method reached its peak at 18.5 mg / g at 40 minutes. The flavonoid content first increased and then decreased with time. It can be seen that too long heating time may cause the decomposition of active ingredients and reduce the extraction rate. Therefore, the optimal heating time is 30 minutes for alcohol extraction.

[0057] Depend on Figure 5 As shown, the flavonoid content in Dendrobium curcumae first increases and then decreases with increasing microwave ultrasonic power, reaching a maximum at 200 W and gradually decreasing thereafter. This suggests that increasing microwave extraction power may damage the structure of the active ingredients, reducing the extraction yield. Therefore, the optimal microwave extraction power is 200 W. Furthermore, at the same ultrasonic power, alcohol extraction is superior to water extraction.

[0058] Furthermore, in the step “Verification and Optimization”, the response surface experiment specifically refers to selecting appropriate factor levels for a three-factor three-level experimental design based on the results of the single-factor experiment, using Design-Expert software for data analysis, and optimizing the extraction conditions.

[0059] In this embodiment, on the basis of a single-factor experiment, extraction temperature (A), extraction time (B), and ultrasonic power (C) were selected as independent variables, and flavonoid content was used as the observation value. The Design-expert10 software was used to perform a three-factor three-level experimental design. The experimental factors and levels are shown in Table 1.

[0060] Table 1. Response surface experiment factor level table

[0061]

[0062] The response surface design and results are shown in Table 2, and the variance analysis results are shown in Table 3. According to the software analysis, the quadratic polynomial regression equation model with total flavonoid content as the objective function is: Y = 19.97-0.1575A+0.0387B+0.2362C-0.03AB-0.3850AC-0.0225BC-1.39A 2 -0.4327B 2 -0.5228C 2 .

[0063] Table 2. Response surface experimental design and results

[0064]

[0065] Table 3. Analysis of variance of response surface experiment

[0066]

[0067]

[0068] Note: * indicates statistical difference (P<0.05); ** indicates significant statistical difference (P<0.01); *** indicates extremely significant statistical difference (P<0.001).

[0069] From Table 2, we can see that the P value of the model is less than 0.0001, which indicates that the model is extremely significant. 2 =0.9905, indicating that the actual value and the predicted value have a high correlation. A has a significant effect on the total flavonoid content (P=0.0135), B has a significant effect on the total flavonoid content (P=0.4461), and C has a significant effect on the total flavonoid content (P=0.0017). From the F value in Table 3, it can be seen that the importance of each factor is C>A>B, that is, ultrasonic temperature>ultrasonic time>ultrasonic power.

[0070] Design-Expert 13 software was used to draw contour lines and response surface diagrams of the interaction effects of various factors on the flavonoid content in the extract of Dendrobium curcumae. Figure 6 . The horizontal projection of the response surface graph is the contour line. If the contour line is elliptical, it means that the two factors have a very significant interaction, and if it is circular, it means that it is not significant. And the steepness of the slope of the response surface of each factor reflects the degree of influence of the factor on the flavonoid content. The steeper the slope, the greater the influence. Therefore, based on the above analysis of the contour lines and response surface graphs, it can be concluded that: the interaction between temperature and ultrasonic power is extremely significant (P<0.001), and the interactions of other factors are not significant. The single factor temperature has the most obvious effect on flavonoid content, followed by ultrasonic time, and the least influence is ultrasonic power.

[0071] Furthermore, in the step "Verification and Optimization", the specific condition verification specifically refers to further verifying the effects of different extraction temperatures (such as 55°C, 60°C, 65°C), time (such as 25min, 30min, 35min) and ultrasonic power (such as 180W, 200W, 220W) on the flavonoid extraction amount under the optimal alcohol extraction conditions.

[0072] In this embodiment, the optimal temperature is 57.683 ° C, the time is 30.4324 min, and the ultrasonic power is 202.487 W through the response surface test. At this time, the predicted value of the total flavonoid content is 19.981 mg / g. Combined with the actual operation, the temperature is 60 ° C, the time is 30 min, and the ultrasonic power is 200 W. The total flavonoid content is measured to be 19.950 ± 0.2 mg / g, which is close to the model prediction value, indicating that the established model has high reliability.

[0073] See also Figures 7 to 10 The present invention also provides an application of a Dendrobium curcumae extract, and uses the Dendrobium curcumae extract prepared by the method for preparing the Dendrobium curcumae extract as described above in the preparation of drugs for enhancing immunity and improving hematopoietic function.

[0074] Furthermore, in the application of the Dendrobium curcumae extract, the extracted Dendrobium curcumae extract was applied to animal experiments, and its effect on the immune function of immunosuppressed mice was observed by gavage administration.

[0075] Specific, animal models and groups:

[0076] Fifty male KM mice, weighing 17-21 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. and housed in a barrier environment at the Hunan Drug Safety Evaluation and Research Center. The room temperature was controlled at 22°C ± 2°C with a 12-h light-dark cycle. The rats were further randomly divided into five groups (n = 10 per group): a control group (Control), a model group (Model), a positive drug group (levamisole hydrochloride group), a low-dose D. curcumae extract group (LDFE), and a high-dose D. curcumae extract group (HDFE). The model group was an immunocompromised model induced by cyclophosphamide. All rats in the groups were given a normal maintenance diet.

[0077] During the experiment, all mice had free access to clean water and food, and their mental state, activity, hair gloss, appetite, and excretion were closely observed. On days 1-14, mice were intragastrically administered 0.75g / kg and 1.50g / kg of Dendrobium curcumae extract, respectively. The control and cyclophosphamide groups were gavaged with an equal volume of water. On days 1-7, mice in the positive drug group were gavaged with an equal volume of water. On days 8-14, mice in the positive drug group were gavaged with 20mg / kg of levamisole hydrochloride (LH). On days 12-14, mice in the cyclophosphamide, positive drug, and Dendrobium curcumae extract groups were intraperitoneally injected with 80mg / kg of cyclophosphamide. The blank control group was intraperitoneally injected with an equal volume of normal saline. On the afternoon of day 14, mice were fasted and anesthetized with 4% sodium pentobarbital via intraperitoneal (IP) injection.

[0078] Routine blood test:

[0079] After anesthesia, blood was collected from the retro-orbital vein of the mice using EDTA K2 blood collection tubes. The number of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), reticulocytes (Ret), and platelets (PLT) was measured using an XT-2000i automated hematology analyzer.

[0080] Detection of hematopoietic-related cytokines in serum:

[0081] Use the blood collected above at 3000 r·min -1 Centrifuge for 15 minutes and remove the supernatant. Analyze erythropoietin (EPO), thrombopoietin (TPO), and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels strictly according to the kit instructions. Add 50 μL of the diluted standard solution to the standard wells, 10 μL of the serum sample to be tested and 40 μL of the sample diluent to the sample wells, and gently shake the plate to mix. Add 100 μL of HRP-conjugated antibody and react at 37°C for 60 minutes. Wash five times, add 50 μL each of colorimetric solutions A and B, and develop at 37°C in the dark for 15 minutes. Add 50 μL of stop solution, read the absorbance at 450 nm within 15 minutes, and perform calculations and analysis.

[0082] Detection of inflammatory cytokines in serum:

[0083] According to the kit instructions, the experimental method was the same as described above. The mouse serum supernatant was used to detect the levels of IL-1β, IL-6, IL-10, IFN-γ, and TNF-α in the serum using enzyme-linked immunosorbent assay (ELISA) kits.

[0084] Serum biochemical index detection:

[0085] After anesthesia, blood was collected from the retro-orbital vein of the mice, placed in a coagulant tube, and centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected for serum biochemical analysis. AST, ALP, and LDH levels were measured using a multifunctional microplate reader, strictly following the kit instructions and selecting the appropriate wavelength.

[0086] Data processing and statistical analysis:

[0087] Data are expressed as mean ± standard deviation (x ± s). One-way analysis of variance was performed using SPSS 20 (IBM) to assess mean differences between groups, followed by least significant difference (LSD) tests. Significant differences were indicated when p < 0.05, p < 0.01, and p < 0.005, respectively. Model, LH, LDFE, HDFE vs control, p < 0.05, #; p < 0.01, ##; p < 0.005, ###; control, SIM, LDFE, HDFE vs Model, p < 0.05, *; p < 0.01, **; p < 0.005, ***

[0088] In this embodiment, the effect of the extract of Dendrobium curcumae on the blood parameters of immunosuppressive mice: Figure 7 As can be seen, after modeling, the number of WBC, RBC, HGB, Ret, and PLT in the model group mice was significantly reduced compared with the blank group (P<0.01); the results showed that CP had a significant effect on the hematological indices of mice. The blood cell content of mice in the LDFE and HDFE groups was higher than that of mice in the model group (P<0.05). DFE significantly inhibited the CP-induced decrease in RBC, HGB, Ret, and PLT in immunosuppressive mice (P<0.05 or P<0.001); however, LDFE had no significant effect on the WBC level of model mice (P>0.05). HDFE had a stronger preventive effect on WBC and Ret in immunosuppressive mice than LDFE (P<0.05). These results show that DFE can effectively prevent and improve the CP-induced decrease in some hematological indices in immunosuppressive mice.

[0089] Effects of Dendrobium curcumae extract on hematopoietic-related cytokines in immunosuppressive mice: Figure 8 The results showed that the levels of TPO, EPO, and GM-CSF in the model group were significantly lower than those in the control group (P<0.001). In the treatment group, LDFE and HDFE significantly improved the reduction of TPO, EPO, and GM-CSF levels in CP-induced immunosuppressive mice (P<0.01). At the same time, HDFE was superior to LDFE in the effects on TPO, EPO, and GM-CSF. Figure 8 C, P < 0.05). The above results indicate that CP severely impairs the hematopoietic function of mice and reduces the levels of hematopoietic-related cytokines; however, DFE can effectively prevent the side effects of CP, and HDFE is more effective than LDFE.

[0090] Effects of Dendrobium curcumae extract on inflammatory cytokines in immunosuppressed mice: 5 inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). Figure 9As shown, the levels of various inflammatory factors in the model group were significantly lower than those in the control group (P < 0.05), while those in the HDFE group were significantly higher than those in the model group (P < 0.05). This indicates that HDFE can inhibit the reduction of various inflammatory factors in the model mice (P < 0.05). Compared with the model group, the levels of IL-6 and IFN-γ in the LDFE group were significantly increased (P < 0.05); the IL-10 level in the LDFE group was slightly higher than that in the model group, but the difference was not statistically significant (P > 0.05). Furthermore, HDFE significantly improved the levels of IL-1β, IL-6, IL-10, TNF-α, and IFN-γ compared with LDFE (P < 0.05). These results indicate that both LDFE and HDFE can significantly prevent and improve the CP-induced decrease in most inflammatory cytokines in immunosuppressed mice, with HDFE being more effective than LDFE.

[0091] Effects of Dendrobium curcumae extract on serum biochemical parameters in immunosuppressive mice Figure 10 Results showed that the LDH levels of mice in the model group were significantly lower than those in the control group (P < 0.01). In the drug-treated group, HDFE significantly prevented and improved the decreased LDH levels in the model mice (P < 0.05). LDFE also had a modest improvement effect on the decreased LDH levels in the model mice, but the difference was not statistically significant (P > 0.05). AST and ALP levels in the model group were significantly higher than those in the control group (P < 0.01). In the drug-treated group, HDFE significantly prevented and improved the increased AST and ALP levels in the model mice (P < 0.05). These results indicate that CP severely impairs the blood function of mice and disrupts the levels of serum biochemical markers. DFE, however, effectively prevents and improves the side effects of CP, and HDFE is more effective than LDFE in improving LDH levels.

[0092] In summary, in this technical solution, traditional water extraction and alcohol extraction are used to extract Dendrobium curcumae. Under the ultrasound-assisted extraction method, the extraction rate of alcohol-extracted flavonoids in Dendrobium curcumae is greater than that of water extraction. Animal studies have found that the Dendrobium curcumae extract obtained by extraction can inhibit bone marrow cell apoptosis, improve bone marrow cell S phase arrest, protect bone marrow hematopoietic function, and restore the overall immune response ability by regulating the levels of inflammatory factors (IL-1β, IL-6, IL-10, TNF-α and IFN-γ), improve the ability of macrophages to recognize and eliminate pathogens, and improve the overall immune ability level of the body.

[0093] The above disclosure is only a preferred embodiment of the present invention, and certainly cannot be used to limit the scope of the rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a Dendrobium curcumae extract, characterized in that: The steps include: Determination of flavonoid standard curve: Accurately weigh rutin standard, dissolve it in ethanol and prepare control solutions of different concentrations, and draw a standard curve through color reaction and absorbance measurement; Raw material pretreatment: clean the stem segments of Dendrobium candidum with tap water, dry them in an oven at 50°C for 5 days, grind them with a powder grinder, and sieve them to obtain a uniform powder for later use; Raw material extraction: alcohol extraction or water extraction combined with ultrasound-assisted extraction to obtain the extract; Verification and optimization: Based on the drawn standard curve, the optimized preparation method is obtained after single-factor experiments, response surface experiments, preferred option verification and specific condition verification.

2. The method for preparing the Dendrobium curcumae extract according to claim 1, wherein The specific steps of the step "determination of flavonoid standard curve" are: Accurately weigh a certain amount of rutin standard, dissolve it in 80% ethanol, transfer it to a 100 mL volumetric flask, dilute to the mark, and shake well to obtain a rutin control solution. Accurately pipette different volumes of rutin reference solution into 10 mL volumetric flasks, fill to the 3.0 mL mark with ethanol, shake well, add 0.5 mL of 5% sodium nitrite solution, shake well and let stand for 6 minutes, add 0.5 mL of 10% aluminum nitrate solution, shake well again and let stand for 6 minutes, add 4 mL of 1 mol / L sodium hydroxide solution, and fill to the mark with 80% ethanol, shake well and let stand for 15-20 minutes; With distilled water as a blank control, the absorbance of each solution was measured at a wavelength of 510 nm. A standard curve was drawn with rutin concentration as the abscissa and absorbance as the ordinate, and the regression equation was obtained.

3. The method for preparing the Dendrobium curcumae extract according to claim 2, wherein: In the step "Raw Material Pretreatment", when sieving after crushing with a pulverizer, a 1 mm sieve is used.

4. The method for preparing the Dendrobium curcumae extract according to claim 1, wherein In the step "raw material extraction", water extraction combined with ultrasound-assisted extraction was used to obtain the following extract: The Dendrobium officinale was crushed with a grinder, and about 10 g of the crushed Dendrobium officinale sample was weighed into a 400 ml glass bottle. 300 ml of distilled water was added to the glass bottle and covered. The extraction was carried out at 20°C to 100°C and an ultrasonic power of 100W to 300W for 20 to 60 minutes. The residual liquid was collected after filtering with gauze, centrifuged, and placed in a refrigerator at 4°C for 12 hours, and then freeze-dried.

5. The method for preparing the Dendrobium curcumae extract according to claim 4, wherein: In the step "raw material extraction", the specific contents of the extract obtained by using alcohol extraction combined with ultrasound-assisted extraction are: The Dendrobium officinale was crushed with a grinder, and about 10 g of the crushed Dendrobium officinale sample was weighed into a 400 ml glass bottle. 200 ml of methanol was added to the glass bottle and covered. The extraction was carried out at a temperature of 20°C to 100°C and an ultrasonic power of 100W to 300W for 20 to 60 minutes. The residual liquid was collected by centrifugation after filtering with gauze, rotary evaporation, and freeze-drying.

6. The method for preparing the Dendrobium curcumae extract according to claim 5, wherein: In the step "Verification and Optimization", the single-factor experiment specifically refers to the investigation of the effects of different extraction temperatures, times, and ultrasonic powers on the flavonoid extraction yield; Response surface experiment specifically refers to selecting appropriate factor levels for three-factor three-level experimental design based on the results of single-factor experiments, using Design-Expert software for data analysis, and optimizing extraction conditions; Optimal option verification specifically refers to conducting alcohol extraction and water extraction experiments under optimal extraction conditions to compare the extraction efficiency; Specific condition verification specifically refers to further verifying the effects of different extraction temperatures, times and ultrasonic powers on flavonoid extraction under optimal alcohol extraction conditions.

7. An application of a Dendrobium curcumae extract, characterized in that: Use of the Dendrobium curcuma extract prepared by the method for preparing the Dendrobium curcuma extract according to claim 1 in preparing drugs for enhancing immunity and improving hematopoietic function.

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