A method for mechanical chemistry-assisted extraction of total flavonoids from hibiscus flowers

Abstract

This invention discloses a mechanochemical-assisted extraction method for total flavonoids from Hibiscus mutabilis flowers. Hibiscus mutabilis flowers and an auxiliary agent are added to a ball mill jar, zirconium beads are added as the grinding medium, and the jar is placed in a ball mill for co-grinding. After the reaction, the product is separated from the zirconium beads, and the ball-milled product is ultrasonically extracted with water to obtain a crude extract of total flavonoids from Hibiscus mutabilis flowers. This invention utilizes a mechanical ball milling reaction between the auxiliary agent and Hibiscus mutabilis flower powder. Under mechanical force, the particle size of the dried coarse Hibiscus mutabilis flower powder decreases, and the cell structure is destroyed, promoting the release of its active ingredients. Furthermore, by selectively adding solid reagents during co-grinding, utilizing the physicochemical properties of flavonoids, the flavonoids and auxiliary agent form salts under mechanical force, thereby increasing the extraction rate of flavonoids in water. Compared with existing extraction methods, this method has advantages such as environmental friendliness, good water solubility, high extraction rate, and simple operation, and has broad market application prospects.

Description

Technical Field

[0001] This invention belongs to the field of total flavonoid extraction technology, specifically relating to a method for mechanochemical-assisted extraction of total flavonoids from okra flowers. Background Technology

[0002] The dried corolla of *Abelmoschus manihot* (L.) Medic., a plant in the Malvaceae family, is also known as okra, cotton flower, and water cotton. *Abelmoschus manihot* flowers contain thousands of compounds, mainly including flavonoids, reducing sugars, tannins, sterols, polysaccharides, and other components. They possess various effects such as antioxidant, antitumor, lipid-lowering, liver protection, immune regulation, and promotion of burn wound healing. Flavonoids are the main active components of *Abelmoschus manihot* flowers. These mainly include hyperoside, quercetin, isoquercetin, quercetin-3-O-glucoside, myricetin, and quercetin-3-O-aphoroside. Currently, common methods for extracting total flavonoids include reflux extraction, warm maceration, Soxhlet extraction, and ultrasonic extraction. These methods either process small sample volumes, have low yields, require large amounts of organic solvents, or have high equipment requirements and are cumbersome to operate.

[0003] In recent years, mechanochemical technology has brought significant progress to various fields, including organic synthesis, materials science, pharmaceutical formulation, and plant extraction. Mechanochemical reactions involve the application of mechanical forces (such as shear, friction, and impact) to solid objects to achieve supramolecular chemical reactions between small molecule drugs and excipients. Under the influence of mechanical forces, the particle size of dried plant powder decreases and its cell structure is disrupted, promoting the release of its effective active ingredients. Furthermore, by selectively adding solid reagents based on the physicochemical properties of the target compound, bioactive substances can react with the reagents to form salts, glycosides, or undergo other chemical reactions under the influence of mechanochemical forces, thereby increasing the extraction rate of the target compound in the solvent. Mechanochemical-assisted extraction not only improves the extraction rate of target components but also shortens the extraction time and reduces the requirements for extraction conditions, making it a highly efficient and environmentally friendly extraction method. The use of mechanochemical technology to assist in the extraction of total flavonoids from Hibiscus syriacus flowers is currently unreported, representing an innovative process for extracting total flavonoids from Hibiscus syriacus flowers. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a method for mechanochemical-assisted extraction of total flavonoids from okra flowers.

[0005] The specific technical solution is as follows:

[0006] A method for mechanochemical-assisted extraction of total flavonoids from Hibiscus syriacus flowers involves adding Hibiscus syriacus flowers and an auxiliary agent into a ball mill jar, adding zirconium beads as the grinding medium, placing the ball mill jar in a ball mill, and performing a co-grinding reaction. After the reaction is completed, the product is separated from the zirconium beads, and the ball-milled product is ultrasonically extracted with water to obtain a crude extract of total flavonoids from Hibiscus syriacus flowers.

[0007] Furthermore, the additive is one of tartaric acid, sodium carbonate, calcium carbonate, sodium bicarbonate, quartz sand, and magnesium carbonate, preferably magnesium carbonate.

[0008] Furthermore, the adjuvant accounts for 1%-30% of the mass of the okra flower, preferably 10%.

[0009] Furthermore, the ratio of zirconium beads to the total mass of raw materials is 1:15-50, preferably 1:40.

[0010] Furthermore, the ball milling speed is 100-400 r / min, preferably 300 r / min; the ball milling time is 10-60 min, preferably 20 min; and the diameter of the zirconium beads is 10 mm.

[0011] Further, after extraction, the pH of the solution is adjusted to neutral with 10% citric acid solution, and the filtrate is taken as crude extract of total flavonoids from okra flowers.

[0012] Furthermore, the extraction time is 10-60 minutes, preferably 30 minutes.

[0013] Furthermore, the mass ratio of the ball-milled product to the volume ratio of water is 1g:100-400mL, preferably 1g:200mL.

[0014] The beneficial effects of this invention are as follows:

[0015] 1) In the presence of additives, the present invention extracts total flavonoids by mechanical grinding. No organic waste liquid is added or solvent residue is left during the extraction process, thus avoiding the environmental pollution problems that are easily caused by solvent removal.

[0016] 2) This invention utilizes mechanochemical-assisted extraction. Under the action of high-energy mechanical force, the material is crushed and salts are formed with the auxiliaries. This method not only improves the extraction rate of the target components, but also shortens the extraction time and simplifies the extraction steps. It is an efficient and environmentally friendly extraction method.

[0017] 3) The total flavonoid extract of Hibiscus syriacus flowers of the present invention can be applied to the clinical treatment of various oral inflammations and chronic bronchitis, with significant analgesic effects and a wide range of applications. Attached Figure Description

[0018] Figure 1 Figure showing the yield of total flavonoids extracted from Hibiscus syriacus flowers using different adjuvants;

[0019] Figure 2 Figure showing the yield of total flavonoids extracted from Hibiscus syriacus flowers by magnesium carbonate at different mass ratios;

[0020] Figure 3 Figure showing the yield of total flavonoids extracted from Hibiscus syriacus flowers by ball milling with different ball-to-material ratios;

[0021] Figure 4 Figure showing the yield of total flavonoids extracted from Hibiscus syriacus flowers under different ball milling times;

[0022] Figure 5 Figure showing the yield of total flavonoids extracted from Hibiscus syriacus flowers at different ball milling speeds;

[0023] Figure 6 Figure showing the yield of total flavonoids extracted from Hibiscus syriacus flowers at different extraction times;

[0024] Figure 7 The graph shows the total flavonoid yield from okra flowers extracted with different material-to-liquid ratios. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] A. Construction of the standard curve for total flavonoids: Accurately weigh 10 mg of rutin, dissolve it in 5 ml of ethanol, and then dilute to 50 mL with water to prepare the standard solution. Measure 2, 3, 4, 5, 6, and 7 mL of the standard solution into 25 mL volumetric flasks, respectively. Add 8, 7, 6, 5, 4, and 3 mL of water to each flask to make a total volume of 10 mL. Then add 1 ml of 5% sodium nitrite solution, shake well, and let stand for 6 min. Next, add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min. Finally, add 10 ml of 5% sodium hydroxide solution, dilute to 25 mL with water, shake well, and let stand for 30 min. Measure the absorbance at 510 nm. Plot the standard curve for total flavonoids from Hibiscus syriacus flower with rutin standard concentration (mg / mL) on the x-axis and absorbance (A) on the y-axis: y = 10.449x + 0.0049 (R² = 0.999).

[0027] B. Determination of total flavonoid yield from Hibiscus syriacus flowers: The product was extracted with water for 30 min, and the pH was adjusted to neutral by adding 10% citric acid solution. After filtration, the filtrate was brought to a final volume of 100 ml. 10 ml of the filtrate was transferred to a 25 ml volumetric flask, 1 ml of 5% sodium nitrite solution was added, and the mixture was shaken and allowed to stand for 6 min. Then, 1 mL of 10% aluminum nitrate solution was added, and the mixture was shaken and allowed to stand for 6 min. Finally, 10 ml of 5% sodium hydroxide solution was added, and the volume was brought to a final volume of 25 mL. The mixture was shaken, allowed to stand for 30 min, and the absorbance was measured at 510 nm.

[0028] Total flavonoid yield from Hibiscus mutabilis flowers (mg / g)

[0029] In the formula, C1 is the total flavonoid concentration obtained from the standard line, in mg / mL;

[0030] V1 is the dissolution volume, mL;

[0031] N is the dilution factor;

[0032] M1 represents the mass of okra flowers, in grams.

[0033] Example 1

[0034] Total flavonoids were extracted using sodium carbonate as an auxiliary agent.

[0035] Sodium carbonate (0.32g), okra flowers (2.93g), and 20 zirconium beads (65.0g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from okra flowers was obtained.

[0036] The total flavonoid yield of okra flowers extracted with sodium carbonate as an adjuvant was determined to be 30.98 mg / g.

[0037] Example 2

[0038] Total flavonoids were extracted using calcium carbonate as an auxiliary agent.

[0039] Add calcium carbonate (0.32g), okra flowers (2.93g), and 20 zirconium beads (65.0g) with a diameter of 10mm to a 50mL polytetrafluoroethylene ball milling jar. Grind at 300r / min for 30min. After the reaction, separate the zirconium beads from the ball milling product. Sonicate the ball milling product with water at a material-to-liquid ratio of 1:200 for 30min. After extraction, adjust the pH of the solution to neutral with 10% citric acid solution. After filtration, the filtrate is the crude extract of total flavonoids from okra flowers.

[0040] The total flavonoid yield of okra flowers extracted with calcium carbonate as an adjuvant was determined to be 29.33 mg / g.

[0041] Example 3

[0042] Total flavonoids were extracted using tartaric acid as an auxiliary agent.

[0043] Add tartaric acid (0.32g), okra flower (2.93g), and 20 zirconium beads (65.0g) with a diameter of 10mm to a 50mL polytetrafluoroethylene ball milling jar. Grind at 300r / min for 30min. After the reaction, separate the zirconium beads from the ball milling product. Sonicate the ball milling product with water at a material-to-liquid ratio of 1:200 for 30min. After extraction, adjust the pH of the solution to neutral with 10% citric acid solution. After filtration, the filtrate is the crude extract of total flavonoids from okra flower.

[0044] The yield of total flavonoids extracted from Hibiscus syriacus flowers using tartaric acid as an adjuvant was determined to be 21.91 mg / g.

[0045] Example 4

[0046] Total flavonoids were extracted using sodium bicarbonate as an auxiliary agent.

[0047] Sodium bicarbonate (0.32 g), okra flowers (2.93 g), and 20 zirconium beads (65.0 g) with a diameter of 10 mm were added to a 50 mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300 r / min for 30 min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30 min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from okra flowers was obtained.

[0048] The total flavonoid yield of okra flowers extracted with sodium bicarbonate as an adjuvant was determined to be 33.30 mg / g.

[0049] Example 5

[0050] Extraction of total flavonoids using quartz sand as an auxiliary agent

[0051] Add 0.32 g of quartz sand, 2.93 g of okra flowers, and 20 zirconium beads (65.0 g) with a diameter of 10 mm to a 50 mL polytetrafluoroethylene ball milling jar. Grind at 300 r / min for 30 min. After the reaction is complete, separate the zirconium beads from the ball milling product. Sonicate the ball milling product with water at a material-to-liquid ratio of 1:200 for 30 min. After extraction, adjust the pH of the solution to neutral with 10% citric acid solution. After filtration, take the filtrate as the crude extract of total flavonoids from okra flowers.

[0052] The yield of total flavonoids extracted from Hibiscus syriacus flowers using quartz sand as an auxiliary agent was determined to be 27.52 mg / g.

[0053] Example 6

[0054] Extraction of total flavonoids using magnesium carbonate as an auxiliary agent

[0055] Magnesium carbonate (0.32g), okra flowers (2.93g), and 20 zirconium beads (65.0g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from okra flowers was obtained.

[0056] The yield of total flavonoids extracted from Hibiscus syriacus flowers using magnesium carbonate as an adjuvant was determined to be 34.38 mg / g.

[0057] Examples 1 to 6 selected different adjuvants, from Figure 1 It was observed that when magnesium carbonate was used as the adjuvant, the total flavonoid yield was relatively high. The increased alkalinity of the adjuvant enhanced the acid-base neutralization reaction between the adjuvant and the aglycone, thereby improving the extraction rate of flavonoids. However, adjuvants with excessive alkalinity, such as sodium carbonate, may cause glycosidic bond breakage or even damage to the parent nucleus, leading to deglycosidization and deglycosidization reactions, which in turn reduced the extraction yield of various flavonoids. Therefore, magnesium carbonate was preferred as the adjuvant, and the total flavonoid yield extracted from Hibiscus syriacus flowers was 34.38 mg / g.

[0058] Example 7

[0059] Total flavonoids (1%) were extracted using magnesium carbonate as an auxiliary agent.

[0060] Magnesium carbonate (0.03g), Hibiscus rosa-sinensis flowers (3.22g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0061] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 25.12 mg / g.

[0062] Example 8

[0063] Total flavonoids (5%) were extracted using magnesium carbonate as an auxiliary agent.

[0064] Magnesium carbonate (0.16g), Hibiscus rosa-sinensis flowers (3.09g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0065] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 34.13 mg / g.

[0066] Example 9

[0067] Total flavonoids (10%) were extracted using magnesium carbonate as an auxiliary agent.

[0068] Magnesium carbonate (0.32g), Hibiscus rosa-sinensis flowers (2.93g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0069] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 34.37 mg / g.

[0070] Example 10

[0071] Total flavonoids (20%) were extracted using magnesium carbonate as an auxiliary agent.

[0072] Magnesium carbonate (0.65g), Hibiscus rosa-sinensis flowers (2.60g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0073] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 31.19 mg / g.

[0074] Example 11

[0075] Total flavonoids (30%) were extracted using magnesium carbonate as an auxiliary agent.

[0076] Magnesium carbonate (0.97g), Hibiscus rosa-sinensis flowers (2.28g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0077] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 26.12 mg / g.

[0078] In Examples 7 to 11, the proportion of magnesium carbonate to the total mass of the raw materials was 1%, 5%, 10%, 20%, and 30%, respectively, and the results were as follows: Figure 2 Increasing the amount of magnesium carbonate can increase the yield of total flavonoids from okra flowers. However, when the amount increases to a certain level, the effect tends to level off, and the extraction yield no longer increases or even decreases slightly. Therefore, it is preferable that the proportion of magnesium carbonate to the total mass of raw materials is 10%. At this time, the yield of total flavonoids extracted from okra flowers is 34.37 mg / g.

[0079] Example 12

[0080] Total flavonoids were extracted using magnesium carbonate as an auxiliary agent (1:15).

[0081] Magnesium carbonate (0.43g), Hibiscus rosa-sinensis flowers (3.91g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0082] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 33.19 mg / g.

[0083] Example 13

[0084] Total flavonoids were extracted using magnesium carbonate as an auxiliary agent (1:20).

[0085] Magnesium carbonate (0.32g), Hibiscus rosa-sinensis flowers (2.93g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0086] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 34.37 mg / g.

[0087] Example 14

[0088] Total flavonoids were extracted using magnesium carbonate as an auxiliary agent (1:30).

[0089] Magnesium carbonate (0.22g), Hibiscus rosa-sinensis flowers (1.95g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0090] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 34.53 mg / g.

[0091] Example 15

[0092] Total flavonoids were extracted using magnesium carbonate as an auxiliary agent (1:40).

[0093] Magnesium carbonate (0.16g), Hibiscus rosa-sinensis flowers (1.46g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0094] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 35.13 mg / g.

[0095] Example 16

[0096] Total flavonoids were extracted using magnesium carbonate as an auxiliary agent (1:50).

[0097] Magnesium carbonate (0.13g), Hibiscus rosa-sinensis flowers (1.17g), and 20 zirconium beads (65.00g) with a diameter of 10mm were added to a 50mL polytetrafluoroethylene ball milling jar. The mixture was ground at 300r / min for 30min. After the reaction was completed, the zirconium beads were separated from the ball milling product. The ball milling product with a material-to-liquid ratio of 1:200 was ultrasonically extracted with water for 30min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then filtered and the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers was obtained.

[0098] The yield of total flavonoids extracted from Hibiscus syriacus flowers was determined to be 31.93 mg / g.

[0099] The ratio of total raw material mass to zirconium bead mass was 1:15, 1:20, 1:30, 1:40, and 1:50, respectively, and the results are as follows: Figure 3 Increasing the ratio of raw materials to zirconium beads can increase the yield of total flavonoids from Hibiscus syriacus flowers. However, when the amount of raw materials is increased to a certain extent, the extraction yield no longer increases and may even decrease slightly. Therefore, the optimal ratio of total raw material mass to zirconium bead mass is 1:40. At this time, the yield of total flavonoids extracted from Hibiscus syriacus flowers is 35.13 mg / g.

[0100] Example 17

[0101] Optimal ball milling time

[0102] Magnesium carbonate (0.16 g), Hibiscus syriacus flowers (1.46 g), and 20 zirconium beads (65.00 g) with a diameter of 10 mm were added to a 50 mL polytetrafluoroethylene ball mill jar. The mixture was ground at 300 r / min for 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. After the reaction, the zirconium beads were separated from the ball milling product. The ball milling product (material-to-liquid ratio 1:200) was ultrasonically extracted with water for 30 min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then collected as the crude extract of total flavonoids from Hibiscus syriacus flowers. The extraction yield of total flavonoids from Hibiscus syriacus flowers is as follows: Figure 4 As shown in the figure, the yield of total flavonoids increases with increasing ball milling time. The increase in yield is most significant after 20 minutes of ball milling, but further increases in milling time cease and may even show a slight decreasing trend. This is likely because the reaction is essentially complete at around 20 minutes of milling, and further increases in milling time do not improve the yield of the target product. Furthermore, excessively long reaction times can lead to excessive heat accumulation in the milling jar, potentially damaging the effective components of *Hippophae rhamnoides* flowers. Therefore, a milling time of 20 minutes is preferred. At this time, the yield of total flavonoids extracted from *Hippophae rhamnoides* flowers is 39.70 mg / g.

[0103] Example 18

[0104] Optimal ball mill speed

[0105] Magnesium carbonate (0.16 g), Hibiscus syriacus flowers (1.46 g), and 20 zirconium beads (65.00 g) with a diameter of 10 mm were added to a 50 mL polytetrafluoroethylene ball mill jar. The mixture was milled for 20 min at speeds of 100 r / min, 200 r / min, 300 r / min, and 400 r / min, respectively. After the reaction, the zirconium beads were separated from the milling product. The milled product (material to liquid ratio 1:200) was ultrasonically extracted with water for 30 min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then collected as the crude extract of total flavonoids from Hibiscus syriacus flowers. The extraction yield of total flavonoids from Hibiscus syriacus flowers is as follows: Figure 5 As shown in the figure, with the increase of rotational speed, the zirconium beads grind the material more thoroughly, thereby increasing the cell wall breakage rate and releasing the active ingredients. However, when the rotational speed reaches 400 r / min, the total flavonoid yield no longer increases but instead shows a decreasing trend. This may be because excessively high rotational speed generates too much energy, raising the temperature of the reaction system inside the ball mill jar, causing side reactions, leading to the destruction of active ingredients and a slight decrease in yield. Therefore, the preferred ball milling speed is 300 r / min, at which point the yield of total flavonoids extracted from Hibiscus syriacus is 39.70 mg / g.

[0106] Example 19

[0107] Optimal extraction time

[0108] Magnesium carbonate (0.16 g), Hibiscus syriacus flowers (1.46 g), and 20 zirconium beads (65.00 g) with a diameter of 10 mm were added to a 50 mL polytetrafluoroethylene ball mill jar. The mixture was ground at 300 r / min for 20 min. After the reaction, the zirconium beads were separated from the ball milling product. Water was added at a mass ratio of 1:200 to the product, and the mixture was extracted for 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then collected as the crude extract of total flavonoids from Hibiscus syriacus flowers. The extraction yield of total flavonoids from Hibiscus syriacus flowers is as follows: Figure 6 As shown in the figure, increasing the extraction time improves the extraction efficiency. However, after a certain extraction time, the diffusion of active ingredients in the solvent reaches equilibrium, and the extraction time is no longer directly proportional to the extraction rate. Furthermore, prolonged extraction can lead to the precipitation of other impurities, increasing the difficulty of separating and purifying the active ingredients. Therefore, the optimal extraction time is 30 minutes, at which point the yield of total flavonoids from *Hippophae rhamnoides* flowers is 39.70 mg / g.

[0109] Example 20

[0110] Optimal material-liquid ratio

[0111] Magnesium carbonate (0.16 g), Hibiscus rosa-sinensis flowers (1.46 g), and 20 zirconium beads (65.00 g) with a diameter of 10 mm were added to a 50 mL polytetrafluoroethylene ball mill jar. The mixture was ground at 300 r / min for 20 min. After the reaction, the zirconium beads were separated from the ball milling product. The mass ratio of product to water was 1:100, 1:200, 1:300, and 1:400, respectively. The mixture was extracted for 30 min. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. The filtrate was then collected as the crude extract of total flavonoids from Hibiscus rosa-sinensis flowers. The determination of the total flavonoid yield was the same as in Example 1. The extraction yield of total flavonoids from Hibiscus rosa-sinensis flowers was as follows: Figure 7 When the material-to-liquid ratio is too low, the extracted substances have a low concentration difference due to their high relative concentration, resulting in a weak diffusion driving force and a slow leaching rate, thus failing to completely extract the active ingredients. As shown in the figure, the yield of total flavonoids from *Hippophae rhamnoides* flowers reaches a high level when the material-to-liquid ratio is 1:200 g / mL. As the material-to-liquid ratio continues to increase, the yield no longer increases. Therefore, a material-to-liquid ratio of 1:200 g / mL is preferred, at which point the yield of total flavonoids extracted from *Hippophae rhamnoides* flowers is 39.70 mg / g.

Claims

1. A method for mechanochemical-assisted extraction of total flavonoids from Hibiscus mutabilis flowers, characterized in that, The flowers and additives of Hibiscus syriacus were added to a ball mill jar, and zirconium beads were added as the grinding medium. The jar was then placed in a ball mill for co-grinding. After the reaction, the product was separated from the zirconium beads, and the ball-milled product was ultrasonically extracted with water. After extraction, the pH of the solution was adjusted to neutral with 10% citric acid solution. After filtration, the filtrate was taken as the crude extract of total flavonoids from Hibiscus syriacus. The additive was magnesium carbonate. The mass ratio of the ball-milled product to the volume of water was 1 g: 200 mL, the total mass ratio of raw materials to zirconium beads was 1:40, the ball milling time was 20 min, the ball milling speed was 300 r / min, the extraction time was 30 min, the zirconium bead diameter was 10 mm, and the additive accounted for 10% of the mass ratio of Hibiscus syriacus.

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