Ultrasonic treatment method for regulating and controlling molecular weight of dendrobium officinale polysaccharide and enhancing anti-tumor activity of dendrobium officinale polysaccharide and application

Abstract

The invention relates to the technical field of regulation and ultrasonic treatment of dendrobium officinale polysaccharides, in particular to an ultrasonic treatment method for regulating the molecular weight of the dendrobium officinale polysaccharides and enhancing the anti-tumor activity of the dendrobium officinale polysaccharides and application of the method. The method comprises the following steps: preparing dendrobium officinale polysaccharide into a solution, treating for 2-100 minutes under the condition of ice bath by controlling the ultrasonic frequency to be 20 kHz and the intensity to be 113-226 W / cm, and then post-treating to obtain a degradation product. According to the method disclosed by the invention, the difference of degradation kinetic models under different ultrasonic intensities is found and utilized, so that the accurate regulation and control of the polysaccharide molecular weight in a range of 150-1,000 kDa is realized. More importantly, the optimal molecular weight window of anti-tumor activity is determined, and about 697 kDa polysaccharide obtained by treating for 20 minutes under 113 W / cm has the highest inhibition rates on A431 and MDA-MB-231 tumor cells, and the inhibition rates respectively reach 85.8% and 88.6%. The method disclosed by the invention is green and controllable, and provides a new way for developing high-activity dendrobium polysaccharide antitumor drugs or health-care foods.

Description

Technical Field

[0001] This invention relates to the field of regulation and ultrasonic treatment technology of Dendrobium officinale polysaccharides, specifically to ultrasonic treatment methods and applications for regulating the molecular weight of Dendrobium officinale polysaccharides and enhancing their antitumor activity. Background Technology

[0002] Dendrobium officinale is a traditional and precious Chinese medicinal herb. The polysaccharides contained in its stems are one of its main bioactive components, and modern pharmacological studies have confirmed that they possess various physiological functions, including immunomodulation, antioxidation, and antitumor activity. However, the molecular weight of naturally extracted Dendrobium officinale polysaccharides is typically high (reaching over 1200 kDa), resulting in high viscosity, poor solubility, and low bioavailability in its aqueous solutions. This severely limits its development and application in pharmaceuticals, functional foods, and other fields.

[0003] Degradation methods are commonly used to improve the physicochemical properties of macromolecular polysaccharides. Existing degradation technologies include chemical degradation methods (such as acid hydrolysis and oxidative degradation) and enzymatic degradation methods. However, chemical methods may introduce toxic reagent residues, damage the active structure of polysaccharides, and cause environmental pollution; enzymatic hydrolysis is costly, requires harsh conditions, and is difficult to control precisely, resulting in poor reproducibility. Ultrasonic degradation, as a physical modification method, has advantages such as being green, safe, efficient, and easy to control. The enormous energy generated by its cavitation effect can effectively break polysaccharide molecular chains, thereby reducing their molecular weight and viscosity. Currently, although there are reports of applying ultrasound to polysaccharide extraction, its application in the controlled degradation of Dendrobium officinale polysaccharides, systematic research on its degradation kinetics, and clarification of the structure-activity relationship between the molecular weight of degradation products and antitumor activity are still lacking. Existing technologies have not provided a standardized process method that can precisely control the molecular weight of Dendrobium officinale polysaccharides while ensuring or even enhancing their specific biological activities. Summary of the Invention

[0004] This invention provides an ultrasonic treatment method and its application for regulating the molecular weight of Dendrobium officinale polysaccharides and enhancing their antitumor activity, in order to solve the problems mentioned in the background art.

[0005] To address the existing problems, the present invention provides the following technical solution to achieve the above objectives:

[0006] This invention provides an ultrasonic treatment method for regulating the molecular weight of Dendrobium officinale polysaccharides and enhancing their antitumor activity, comprising the following steps:

[0007] (1) Solution preparation: Dissolve the purified Dendrobium officinale polysaccharide (DOPS) in distilled water or deionized water to prepare a solution with a concentration of 0.5–2.0 mg / mL. The preferred concentration is 1.0 mg / mL, at which the solution has good fluidity, which is conducive to the transfer of ultrasonic energy and has high degradation efficiency.

[0008] (2) Ultrasonic treatment: The solution obtained in step (1) was placed in an ice-water bath for temperature-controlled ultrasonic treatment. The ultrasonic frequency was fixed at 20 kHz, and the ultrasonic intensity was controlled within the range of 113–226 W / cm² by adjusting the amplitude of the ultrasonic instrument. The treatment time was 2–100 minutes. The ice bath condition is crucial and can effectively avoid the local high temperature generated during the ultrasonic process, which could lead to the inactivation of the polysaccharide structure.

[0009] (3) Post-treatment: After the initial treatment, the solution is post-treated to terminate the reaction and obtain the product. The post-treatment includes heating the solution in a boiling water bath for 5-10 minutes to completely terminate the degradation, followed by freeze drying to obtain white flocculent or powdery degraded Dendrobium officinale polysaccharide.

[0010] In a preferred embodiment, the ultrasonic intensity in step (2) is 113 W / cm², and the treatment time is 20–60 minutes. Experiments have shown that under these conditions, the polysaccharide degradation process is mild, and products with significantly enhanced antitumor activity can be obtained.

[0011] In a more preferred embodiment, the processing time in step (2) is 20 minutes. Under these specific conditions, the molecular weight of Dendrobium officinale polysaccharide can be precisely degraded from an initial 1207 kDa to approximately 697 kDa, at which point the inhibitory rate of the product against human epidermal cancer cells A431 and human breast cancer cells MDA-MB-231 reaches its peak.

[0012] A key finding of this invention is that the degradation kinetics model of the ultrasonic treatment process varies with the intensity. When the ultrasonic intensity is 113 W / cm², the degradation kinetics conform to the first-order reaction kinetic model ln(Mt / M0) = k•t; while when the ultrasonic intensity is 170 W / cm² or 226 W / cm², the degradation kinetics conform more closely to the second-order reaction kinetic model (1 / Mt - 1 / M0) = k•t. This finding provides a theoretical basis for the accurate prediction and regulation of polysaccharide molecular weight.

[0013] Furthermore, the present invention provides a Dendrobium officinale polysaccharide prepared by the above method, the weight-average molecular weight of which is adjustable in the range of 150 kDa to 1000 kDa. Preferably, its weight-average molecular weight is about 697 kDa, and the polysaccharide with this specific molecular weight exhibits optimal antitumor activity.

[0014] Furthermore, this invention provides the application of the above-mentioned Dendrobium officinale polysaccharide in the preparation of drugs or health foods for the prevention and / or treatment of tumors. The tumors include, but are not limited to, human epidermal carcinoma and human breast cancer.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1. By controlling the intensity and duration of ultrasound, this invention enables the directional and predictable regulation of the molecular weight of Dendrobium officinale polysaccharides, overcoming the blindness of traditional degradation methods.

[0017] 2. This invention does not simply reduce the molecular weight, but rather discovers the nonlinear relationship between molecular weight and antitumor activity, and finds the optimal molecular weight window for activity (approximately 697 kDa), which significantly enhances the antitumor activity of the degradation product compared to the untreated polysaccharide.

[0018] 3. Under the preferred low-intensity ultrasound conditions, the primary structure (functional groups, glycosidic bond type) of polysaccharides is maintained, ensuring that the basis of their biological activity is not destroyed.

[0019] 4. The entire process of this invention uses water as a solvent, requires no chemical reagents, leaves no toxic residues, and is safe and environmentally friendly.

[0020] 5. This invention is the first to systematically elucidate the degradation kinetics of Dendrobium officinale polysaccharides under different ultrasonic intensities, providing solid theoretical support for the scale-up and optimization of the process. Attached Figure Description

[0021] Figure 1 This is a curve showing the change of DOPS intrinsic viscosity over time under different ultrasonic intensities according to the present invention.

[0022] Figure 2 This is a curve showing the change of DOPS weight-average molecular weight (Mw) over time under different ultrasonic intensities according to the present invention.

[0023] Figure 3 The Fourier transform infrared spectra are of the control sample and polysaccharide samples under different ultrasonic treatment conditions in this invention.

[0024] Figure 4 The figures show the fitted curves of DOPS ultrasonic degradation kinetics under different ultrasonic intensities according to the present invention.

[0025] Figure 5 The inhibition rate of different concentrations of ultrasound-treated DOPS samples on A431 and MDA-MB-231 tumor cells is shown in this invention.

[0026] Figure 6 This is a correlation analysis diagram showing the relationship between the molecular weight of DOPS and its antitumor activity in this invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Please see Figure 1-6 Example 1: Powdered dried stems of *Dendrobium officinale* were defatted successively by reflux with acetone and methanol until the extract was colorless. The residue was extracted three times with distilled water at 90°C. The aqueous extracts were combined, concentrated, and then four times their volume of 95% ethanol were added. The mixture was allowed to settle overnight at 4°C. The precipitate was collected by centrifugation, washed with anhydrous ethanol, redissolved in water, and proteins were removed using the Sevag method. After dialysis, the solution was freeze-dried to obtain purified *Dendrobium officinale* polysaccharides (DOPS) for later use.

[0029] Example 2: Weigh the DOPS obtained in Example 1 and prepare a 1.0 mg / mL solution with distilled water. Take 20 mL of this solution into a 50 mL beaker and place the beaker in an ice-water bath. Use a SonicVCX-750 ultrasonic cell disruptor (frequency 20 kHz) with amplitudes of 20% (intensity 113 W / cm²), 30% (intensity 170 W / cm²), and 40% (intensity 226 W / cm²) for a total treatment time of 100 minutes. Take 5 mL samples at 0, 2, 5, 10, 20, 30, 40, 60, and 100 minutes.

[0030] Example 3: The sample taken in Example 2 was immediately placed in a boiling water bath and heated for 5 minutes, then cooled and freeze-dried to obtain a series of degraded DOPS samples. The weight-average molecular weight of each sample was determined by the HPGFC method, and the results are as follows. Figure 2 As shown. Treatment at 113 W / cm² intensity for 20 minutes yielded the target product with a molecular weight of approximately 697 kDa.

[0031] Example 4: The antitumor activity of the polysaccharide samples obtained in Example 3 was evaluated using the MTT assay. Human epidermal cancer cells A431 and human breast cancer cells MDA-MB-231 were used as models, and three polysaccharide concentrations of 0.5, 1.0, and 1.5 mg / mL were set. The results are as follows: Figure 5 As shown, the sample with a molecular weight of approximately 697 kDa exhibited inhibition rates of up to 85.8% and 88.6% against two types of cancer cells at a concentration of 1.5 mg / mL, respectively, demonstrating significantly better activity than the untreated sample and samples under other treatment conditions.

[0032] Comparative Example 1: Except for setting the ultrasound intensity to 226 W / cm² and the treatment time to 5 minutes, the remaining steps were the same as in Examples 2 and 3. The molecular weight of the obtained polysaccharide decreased rapidly, but its antitumor activity did not reach the optimal level, proving that lower molecular weight does not necessarily mean stronger activity.

[0033] Comparative Example 2: Except for the absence of an ice-water bath for temperature control, ultrasonic treatment was performed at room temperature (113 W / cm², 20 minutes), with the remaining steps identical to Examples 2 and 3. FTIR spectra showed slight changes in the absorption peaks of some functional groups, and the antitumor activity of the final product was relatively unstable, indicating that temperature control is crucial for maintaining the integrity of the polysaccharide structure.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the above description. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, utilizing the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. An ultrasonic treatment method for regulating the molecular weight of Dendrobium candidum polysaccharide and enhancing its anti-tumor activity, characterized in that, The method comprises the following steps: (1) dissolving the Dendrobium polysaccharide in water to prepare a solution with a concentration of 0.5-2.0 mg / mL; (2) subjecting the solution obtained in step (1) to ultrasonic treatment under ice-bath conditions, wherein the ultrasonic frequency is 20 kHz, the ultrasonic intensity is 113-226 W / cm2, and the treatment time is 2-100 minutes; (3) post-treating the solution treated in step (2) to obtain degraded Dendrobium polysaccharide.

2. The ultrasonic treatment method for regulating the molecular weight of Dendrobium candidum polysaccharides and enhancing the anti-tumor activity according to claim 1, characterized in that, In step (2), the ultrasonic intensity is 113 W / cm2, and the treatment time is 20-60 minutes. 3.The ultrasonic treatment method for regulating the molecular weight of Dendrobium candidum polysaccharide and enhancing its anti-tumor activity according to claim 2, characterized in that, In step (2), the treatment time is 20 minutes, and the molecular weight of the Dendrobium polysaccharide is degraded to about 697 kDa. 4.The method for controlling the molecular weight of Dendrobium officinale polysaccharide and enhancing its anti-tumor activity by ultrasonic treatment according to claim 1, characterized in that, The degradation kinetics of the ultrasonic treatment process in step (2) conforms to a first-order reaction kinetic model or a second-order reaction kinetic model. Preferably, when the ultrasonic intensity is 113 W / cm2, the degradation kinetics conforms to the first-order reaction kinetic model; and when the ultrasonic intensity is 170 W / cm2 or 226 W / cm2, the degradation kinetics conforms to the second-order reaction kinetic model. 5.The method of claim 1, wherein the ultrasonic treatment is performed at a frequency of 20-40 kHz, a power of 100-200 W, and a temperature of 20-30 ℃. In step (1), the concentration of the Dendrobium polysaccharide solution is 1.0 mg / mL. 6.The method for controlling the molecular weight of Dendrobium officinale polysaccharide and enhancing its anti-tumor activity by ultrasonic treatment according to claim 1, characterized in that, In step (3), the post-treatment comprises freeze-drying to obtain a solid product.

7. The Dendrobium officinale polysaccharide prepared by the method of claim 1-6, wherein the Dendrobium officinale polysaccharide has a molecular weight of 1.0×105-1.0×106, and the Dendrobium officinale polysaccharide has an anti-tumor activity. The weight-average molecular weight thereof is controllable in the range of 150 kDa to 1000 kDa.

8. The Dendrobium candidum polysaccharide of claim 7, characterized in that, The weight-average molecular weight thereof is about 697 kDa.

9. Use of the Dendrobium polysaccharide according to claim 7 or 8 in the preparation of a drug or health food for preventing and / or treating tumors.

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