Method for extracting and evaluating antioxidant activity of oreorchis patens polysaccharide
Patent Information
- Application Number
- CN202611392222.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-09
- Publication Date
- 2026-11-03
AI Technical Summary
There is a lack of systematic research on Heliotropium polysaccharide in existing technologies, the efficient and stable polysaccharide extraction process system is not perfect, and the material basis of polysaccharide's antioxidant efficacy lacks systematic experimental evidence, which affects the quality control and modern development of this ethnic medicinal material.
An ultrasonic-assisted extraction method was adopted, combined with the phenol-sulfuric acid method and the DPPH/ABTS free radical scavenging method. The extraction process parameters, including the material-liquid ratio, extraction time, ultrasonic power and extraction times, were optimized to establish an extraction method and an antioxidant activity evaluation method for Helianthus angustifolia polysaccharide.
Efficient and stable polysaccharide extraction was achieved, and the optimal extraction conditions were determined. The polysaccharide content of *Heliotropium indicum* reached 11.54 mg/g, exhibiting good antioxidant activity. This provides a basis for the selection of *Heliotropium indicum* polysaccharide varieties and provides technical support for the research and development of functional foods and natural medicinal products.
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Abstract
Description
Technical Field
[0001] This invention relates to the technical field of methods for extracting and evaluating polysaccharides from *Heliotropium indicum*, specifically to methods for extracting polysaccharides from *Heliotropium indicum* and evaluating their antioxidant activity. Background Technology
[0002] *Ludisia discolor* (Ker-GaWl) A.Rich, belonging to the genus *Ludisia* in the family Orchidaceae, is a plant with horizontally creeping rhizomes, clearly defined stem segments, and plump, fleshy tissue, resembling the shape of a lotus root, hence its alternative name, "rock lotus root." This medicinal herb has a sweet taste and cooling properties, with significant medicinal value. It possesses various conditioning effects, including nourishing yin and moistening the lungs, calming the mind and strengthening the spleen, cooling the blood and clearing heat. Clinically, it is often used to improve various symptoms such as hemoptysis from pulmonary tuberculosis, neurasthenia, and poor appetite. Modern research shows that *Ludisia discolor* mainly contains compounds such as lignans, cyclic dipeptides, terpenes, phenolic acids, steroids, fatty acids, and polysaccharides. Some components have been reported to possess antioxidant and anti-inflammatory biological activities.
[0003] Bloodleaf orchid has a long history of medicinal use, but systematic research on its core active polysaccharide components is still lacking. The differences in polysaccharide quality among different germplasm medicinal materials are not yet clear, the efficient and stable polysaccharide extraction process system has not yet been perfected, and the material basis of the antioxidant efficacy of polysaccharides also lacks systematic experimental evidence, which seriously restricts the quality control and modern development of this ethnic medicinal material. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides, comprising the following steps:
[0005] S1. Take the raw material of Blood Leaf Orchid, dry it, pulverize it, and sieve it to obtain Blood Leaf Orchid powder;
[0006] S2. Add the *Hypericum erythrorhizon* powder to a solvent and extract using ultrasound-assisted extraction to obtain an extract;
[0007] S3. The extract is centrifuged, the supernatant is collected, and the volume is adjusted to obtain the *Heliotropium indicum* polysaccharide extract;
[0008] S4. The polysaccharide content in the *Heliotropium indicum* polysaccharide extract was determined using the phenol-sulfuric acid method;
[0009] S5. The antioxidant activity of the *Heliotropium indicum* polysaccharide extract was evaluated using the DPPH radical scavenging method and / or the ABTS radical scavenging method.
[0010] Preferably, in step S1, the drying temperature is 65°C; after pulverization, the material is passed through a No. 5 sieve.
[0011] Preferably, in step S2, the extraction solvent is ultrapure water, the material-to-liquid ratio is 1:40 g:mL, the ultrasonic-assisted extraction temperature is 60℃, the ultrasonic power is 300 W, the extraction time is 60~80 min, and the extraction is performed 3 times.
[0012] Preferably, in step S2, the material-to-liquid ratio is 1:40 g:mL, the ultrasonic power is 300 W, the extraction time is 80 min, and the extraction is performed 3 times.
[0013] Preferably, in step S3, the centrifugation speed is 4200 r / min and the centrifugation time is 10 min.
[0014] Preferably, in step S4, the specific operation of the phenol-sulfuric acid method is as follows: take 1 mL of the supernatant after volume adjustment, add 1 mL of phenol solution with a mass fraction of 5%, shake well, slowly add 5 mL of concentrated sulfuric acid, let stand for 5 min, heat in a boiling water bath for 15 min, cool, and then measure the absorbance at a wavelength of 490 nm.
[0015] Preferably, in step S5, the DPPH free radical scavenging system is as follows: 150 μL of DPPH solution and 50 μL of *Heliotropium indicum* polysaccharide extract are reacted at 37°C for 30 min, and the absorbance is measured at a wavelength of 517 nm. The scavenging rate is calculated according to the formula scavenging rate (%) = (1-(A1-A2) / A0)×100%, where A0 is the absorbance of the blank group, A1 is the absorbance of the experimental group, and A2 is the absorbance of the zeroing group.
[0016] Preferably, in step S5, the system for the ABTS free radical scavenging method is as follows: 200 μL of ABTS working solution and 50 μL of *Hemiberlesia javanica* polysaccharide extract are reacted at 37°C for 30 min, and the absorbance is measured at a wavelength of 734 nm. The scavenging rate is calculated according to the formula scavenging rate (%) = (1-(A1-A2) / A0)×100%, where A0 is the absorbance of the blank group, A1 is the absorbance of the experimental group, and A2 is the absorbance of the zeroing group.
[0017] Beneficial effects
[0018] This invention provides a method for extracting polysaccharides from *Heliotropium indicum* and evaluating their antioxidant activity. It has the following beneficial effects:
[0019] 1. This invention provides a method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharide. The process parameters for ultrasonic-assisted extraction of *Heliotropium indicum* polysaccharide were optimized through orthogonal experimental design, and the optimal extraction conditions were determined: material-to-liquid ratio of 1:40 g:mL, extraction time of 80 min, ultrasonic power of 300 W, and extraction times of 3 times. Under these conditions, the polysaccharide content of *Heliotropium indicum* can reach 11.54 mg / g, with high extraction efficiency, stable process, and good reproducibility.
[0020] 2. This invention provides a method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides. The antioxidant activity of polysaccharides from different *Heliotropium indicum* varieties was evaluated using two in vitro free radical scavenging systems: DPPH and ABTS. It was found that the IC50 of *Heliotropium indicum* polysaccharides from the *Yinlong* variety against DPPH free radicals was significantly higher. 50 The value was 0.3191 mg / mL, and the IC50 value for ABTS radicals was [missing value]. 50 The value was 0.4780 mg / mL, which was superior to other varieties, providing a scientific basis for the selection of superior varieties of Bloodleaf Orchid.
[0021] 3. This invention provides a method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides. The extraction process established in this invention can efficiently extract *Heliotropium indicum* polysaccharides while better preserving their active structure. The prepared *Heliotropium indicum* polysaccharides exhibit good in vitro antioxidant activity, confirming their development potential as a natural antioxidant. This provides technical support and theoretical reference for the research and development and transformation of *Heliotropium indicum* in functional foods and natural medicinal products. Attached Figure Description
[0022] Figure 1 This is a schematic diagram illustrating the DPPH free radical scavenging rate of polysaccharide extracts from different varieties of *Hymenoplastia pulcherrima* according to the present invention.
[0023] Figure 2 This is a schematic diagram illustrating the ABTS free radical scavenging rate of polysaccharide extracts from different varieties of *Hylocereus undatus* according to the present invention. Detailed Implementation
[0024] 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.
[0025] Example 1
[0026] The extraction steps for Heliotropium indicum polysaccharides are as follows: The washed aerial parts of Heliotropium indicum were dried in a 65℃ forced-air drying oven, pulverized using a 100g handheld high-speed pulverizer, and passed through a No. 5 sieve to obtain Heliotropium indicum powder. 0.1g of the Heliotropium indicum powder was accurately weighed and placed in a container. Ultrapure water was added at a material-to-liquid ratio of 1:40 (g:mL). Extraction was performed at a constant temperature water bath of 60℃ using 300W ultrasonic power for 80 min, and this extraction was repeated three times. The extracts from each extraction were combined and centrifuged at 4200 r / min for 10 min. The supernatant was collected, transferred to a volumetric flask, and diluted to volume to obtain the Heliotropium indicum polysaccharide extract.
[0027] Accurately pipette 1 mL of the diluted supernatant into a colorimetric tube, add 1 mL of 5% (w / w) phenol solution, and shake well; slowly add 5 mL of concentrated sulfuric acid, shake well, and let stand at room temperature for 5 min; place the colorimetric tube in a boiling water bath for 15 min, then remove and allow to cool naturally to room temperature. Use ultrapure water as a blank control instead of the sample, and measure the absorbance at 490 nm. Simultaneously, plot a standard curve using D(+)-anhydrous glucose standard solutions of different concentrations. Calculate the polysaccharide concentration in the extract based on the standard curve, and then calculate the *Heliotropium indicum* polysaccharide content using the following formula:
[0028] Polysaccharide extraction yield (mg / g) = C × D / m
[0029] Where C is the polysaccharide concentration in the extract (mg / mL) calculated based on the standard curve, D is the dilution factor, and m is the sample mass of *Hymenoplastrus orientalis* powder (g).
[0030] The polysaccharide content of *Hemiberleinii* under the conditions of this embodiment was determined to be 11.54 mg / g.
[0031] Example 2
[0032] The optimization of the polysaccharide extraction process of *Heliotropium indicum* is as follows:
[0033] (1) Single-factor experiment:
[0034] ① Material-liquid ratio experiment: Accurately weigh 0.1 g of *Hypericum erythrorhizon* powder, use ultrapure water as the extraction solvent, and set the material-liquid ratio to be 1:10, 1:20, 1:30, 1:40, and 1:50 g:mL respectively. Control the extraction temperature at 60℃, the extraction time at 60 min, and the ultrasonic power at 300W. After extraction, centrifuge at 4200 r / min for 10 min, take the supernatant and make up the volume, and determine the polysaccharide content according to the method in Example 1.
[0035] ② Ultrasonic power experiment: Accurately weigh 0.1 g of *Hypericum esculentum* powder, add ultrapure water at a material-to-liquid ratio of 1:40 g:mL, control the extraction temperature at 60℃ and the extraction time at 60 min, and set the ultrasonic power to 100 W, 200 W, 300 W, 400 W and 500 W respectively. After extraction, centrifuge at 4200 r / min for 10 min, take the supernatant and dilute to volume, and determine the polysaccharide content.
[0036] ③ Extraction time experiment: Accurately weigh 0.1 g of *Hypericum erythrorhizon* powder, add ultrapure water at a material-to-liquid ratio of 1:40 g:mL, control the extraction temperature at 60℃, and the ultrasonic power at 300 W. Set the extraction time to 20 min, 40 min, 60 min, 80 min, and 100 min respectively. After extraction, centrifuge at 4200 r / min for 10 min, take the supernatant and dilute to volume, and determine the polysaccharide content.
[0037] ④ Extraction number experiment: Accurately weigh 0.1 g of *Hypericum erythrorhizon* powder, add ultrapure water at a material-to-liquid ratio of 1:40 g:mL, control the extraction temperature at 60℃, ultrasonic power at 300 W, and extraction time at 60 min, and set the extraction number to 1, 2, 3, 4 and 5 times respectively. After extraction, centrifuge at 4200 r / min for 10 min, combine the extracts, take the supernatant and make up to volume, and determine the polysaccharide content.
[0038] (2) Orthogonal experiment:
[0039] Based on the results of the single-factor experiments, the material-to-liquid ratio (A), extraction time (B), ultrasonic power (C), and extraction times (D) were selected as the factors to be investigated, with polysaccharide content as the evaluation index. The L9(3) model was adopted. 4 An orthogonal array design was used to conduct a four-factor, three-level orthogonal experiment. The factor levels are shown in the table below:
[0040] level A. Material-to-liquid ratio (g:mL) B Extraction time (min) C Extraction power (W) Number of D extractions (times) 1 1:30 40 200 3 2 1:40 60 300 4 3 1:50 80 400 5
[0041] Press L9 (3) 4 Nine sets of experiments were conducted using an orthogonal array, with each set measured in parallel. The measurement methods were the same as in Example 1. Range analysis and variance analysis were performed on the orthogonal experimental results to screen the optimal combination of extraction process parameters.
[0042] Orthogonal experimental results showed that the influence of each factor on the polysaccharide extraction rate was in the following order: extraction power > solid-liquid ratio > extraction time > number of extractions. The optimal process conditions were: solid-liquid ratio 1:40 (g:mL), extraction time 80 min, ultrasonic power 300 W, and 3 extractions.
[0043] (3) Confirmatory experiments:
[0044] Three parallel extracts of *Heliotropium indicum* polysaccharide were prepared under the optimal process conditions described above: a material-to-liquid ratio of 1:40 (g:mL), an extraction time of 80 min, an ultrasonic power of 300 W, and three extractions. The polysaccharide content was determined according to the method in Example 1. The average value of the three results was 10.38 mg / g, with an RSD of 0.9968%, indicating that the extraction process conditions were stable and reproducible.
[0045] Example 3
[0046] The comparison of polysaccharide content among different varieties of *Hylocereus undatus* is conducted using the following steps:
[0047] Five varieties of *Hypericum erythrophyllum*, namely Saigon Red, Danxia, Ruyi, Yuanshuai, and Yinlong, were selected. Polysaccharide extracts of each variety were prepared according to the optimal process conditions optimized in Example 2 (material-liquid ratio 1:40 (g:mL), extraction time 80 min, ultrasonic power 300 W, and extraction times 3 times). The polysaccharide content of each variety was determined according to the method in Example 1.
[0048] The results showed that the polysaccharide content varied among different varieties of *Hydrocotyle sibthorpioides*, ranging from 9.94 to 11.93 mg / g. The Danxia variety had the highest polysaccharide content (11.93 mg / g), followed by Saigon Red (11.71 mg / g), Ruyi (11.14 mg / g), and Yuanshuai (10.66 mg / g), while the Yinlong variety had the lowest polysaccharide content (9.94 mg / g).
[0049] Example 4
[0050] The in vitro DPPH free radical scavenging activity of *Heliotropium indicum* polysaccharides was evaluated using the following steps:
[0051] Accurately weigh 19.72 mg DPPH, dissolve it in 5 mL of anhydrous ethanol, then take 400 μL of the above solution and dilute it to 40 mL with anhydrous ethanol to prepare a 0.1 mmol / L DPPH stock solution, and store it under cold for later use.
[0052] Polysaccharide extracts from various *Heliotropium indicum* varieties were prepared into test sample solutions with different concentration gradients (concentration range: 0.1–2.0 mg / mL) using ultrapure water. Experiments were conducted according to the following reaction systems: Experimental group (A1) consisted of 150 μL DPPH solution plus 50 μL sample solution; Zeroing group (A2) consisted of 50 μL sample solution plus 150 μL sample solvent; Blank group (A0) consisted of 150 μL DPPH solution plus 50 μL sample solvent. After reacting at 37℃ in the dark for 30 min, the absorbance of each well was measured at 517 nm. Vitamin C (Vc) at the same concentration gradient was used as a positive control. The DPPH free radical scavenging rate was calculated using the following formula:
[0053] Clearance rate (%) = (1 - (A1 - A2) / A0) × 100%
[0054] Calculate the half-maximal inhibitory concentration (IC50) of each polysaccharide extract. 50 Value. For example... Figure 1 The results showed that the polysaccharide from the Yinlong variety of *Hymenoplastia pulcherrima* exhibited the strongest DPPH free radical scavenging activity, with an IC50 value of [missing value]. 50 The value was 0.3191 mg / mL; the IC50 values for Saigon Red, Danxia, Ruyi, and Yuanshuai were... 50The values were 0.4016 mg / mL, 0.5552 mg / mL, 0.8759 mg / mL, and 0.6406 mg / mL, respectively; the IC50 of the positive control vitamin C was... 50 The value was 0.00838 mg / mL. The DPPH free radical scavenging ability of polysaccharides from all varieties of *Heliotropium indicum* increased with increasing concentration.
[0055] Example 5
[0056] The in vitro ABTS free radical scavenging activity of *Heliotropium indicum* polysaccharide was evaluated using the following steps:
[0057] Accurately weigh 38.4 mg of ABTS, dissolve it in distilled water, and bring the volume to 10 mL. Separately, accurately weigh 6.6 mg of potassium persulfate, dissolve it in distilled water, and bring the volume to 10 mL. Mix the two solutions at a volume ratio of 1:1 and let them stand overnight (12–16 h) at room temperature in the dark to obtain the ABTS stock solution. Dilute the ABTS stock solution with ultrapure water to obtain a diluent with an absorbance of 0.70 ± 0.02 at 734 nm, which will be used as the ABTS working solution.
[0058] Polysaccharide extracts from various *Heliotropium indicum* varieties were prepared into test sample solutions with different concentration gradients (concentration range: 0.1–2.0 mg / mL) using ultrapure water. Experiments were conducted according to the following reaction systems: Experimental group (A1) consisted of 200 μL ABTS working solution plus 50 μL sample solution; Zeroing group (A2) consisted of 50 μL sample solution plus 200 μL ultrapure water; Blank group (A0) consisted of 200 μL ABTS working solution plus 50 μL ultrapure water. After reacting at 37℃ in the dark for 30 min, the absorbance of each well was measured at 734 nm. Vitamin C (Vc) at the same concentration gradient was used as a positive control. The ABTS free radical scavenging rate was calculated using the following formula:
[0059] Clearance rate (%) = (1 - (A1 - A2) / A0) × 100%
[0060] Calculate the half-maximal inhibitory concentration (IC50) of each polysaccharide extract. 50 Value. For example... Figure 2 The results showed that the polysaccharide from the Yinlong variety of *Hylocereus undatus* exhibited the strongest scavenging activity against ABTS free radicals, with an IC50 value of [missing value]. 50 The value was 0.4780 mg / mL; the IC50 values for Saigon Red, Danxia, Ruyi, and Yuanshuai were... 50 The values were 0.6372 mg / mL, 1.167 mg / mL, 0.6409 mg / mL, and 0.6434 mg / mL, respectively; the IC50 of the positive control vitamin C was... 50 The value was 0.02848 mg / mL. The scavenging ability of polysaccharides from all varieties of *Heliotropium indicum* against ABTS free radicals increased with increasing concentration.
[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides, characterized in that, Includes the following steps: S1. Take the raw material of Blood Leaf Orchid, dry it, pulverize it, and sieve it to obtain Blood Leaf Orchid powder; S2. Add the *Hypericum erythrorhizon* powder to a solvent and extract using ultrasound-assisted extraction to obtain an extract; S3. The extract is centrifuged, the supernatant is collected, and the volume is adjusted to obtain the *Heliotropium indicum* polysaccharide extract; S4. The polysaccharide content in the *Heliotropium indicum* polysaccharide extract was determined using the phenol-sulfuric acid method; S5. The antioxidant activity of the *Heliotropium indicum* polysaccharide extract was evaluated using the DPPH radical scavenging method and / or the ABTS radical scavenging method.
2. The method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides according to claim 1, characterized in that: In step S1, the drying temperature is 55~70℃; after pulverization, the powder is passed through a No. 5 sieve.
3. The method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides according to claim 1, characterized in that: In step S2, the extraction solvent is ultrapure water, the material-to-liquid ratio is 1:30~1:50 (g:mL), the temperature of ultrasonic-assisted extraction is 50~70℃, the ultrasonic power is 200~400 W, the extraction time is 40~100 min, and the number of extractions is 3~5.
4. The method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides according to claim 1, characterized in that: In step S2, the material-to-liquid ratio is 1:40 g:mL, the ultrasonic power is 300 W, the extraction time is 80 min, and the extraction is performed 3 times.
5. The method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides according to claim 1, characterized in that: In step S3, the centrifugation speed is 4000~4500 r / min and the centrifugation time is 8~15 min.
6. The method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides according to claim 1, characterized in that: In step S4, the specific operation of the phenol-sulfuric acid method is as follows: take 1 mL of the supernatant after volume adjustment, add 1 mL of phenol solution with a mass fraction of 5%, shake well, slowly add 5 mL of concentrated sulfuric acid, let stand for 5 min, heat in a boiling water bath for 15 min, and after cooling, measure the absorbance at a wavelength of 490 nm.
7. The method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides according to claim 1, characterized in that: In step S5, the DPPH free radical scavenging system is as follows: 150 μL of DPPH solution and 50 μL of *Heliotropium indicum* polysaccharide extract are reacted at 37℃ for 30 min, and the absorbance is measured at a wavelength of 517 nm. The scavenging rate is calculated according to the formula scavenging rate (%) = (1-(A1-A2) / A0)×100%, where A0 is the absorbance of the blank group, A1 is the absorbance of the experimental group, and A2 is the absorbance of the zeroing group.
8. The method for extracting and evaluating the antioxidant activity of *Heliotropium indicum* polysaccharides according to claim 1, characterized in that: In step S5, the ABTS free radical scavenging system is as follows: 200 μL of ABTS working solution and 50 μL of *Hemiberlesia javanica* polysaccharide extract are reacted at 37℃ for 30 min, and the absorbance is measured at a wavelength of 734 nm. The scavenging rate is calculated according to the formula scavenging rate (%) = (1-(A1-A2) / A0)×100%, where A0 is the absorbance of the blank group, A1 is the absorbance of the experimental group, and A2 is the absorbance of the zeroing group.