Preparation method and application of compound pomelo peel extract

Abstract

The present application relates to a kind of preparation method and application of compound shaddock peel extract, belong to the technical field of plant extract preparation.Solve the problem that lack of shaddock peel and bidens pilosa, litchi leaf, pectoria leaf are compounded to prepare extract with anti-inflammatory and antipruritic efficacy in prior art, take shaddock peel, bidens pilosa, litchi leaf and pectoria leaf are mixed according to weight ratio after water decocting extraction, solid-liquid ratio is 1: (15-25) g / mL, after 3-5 hours of soaking, decocting 1-2 hours, filtration is concentrated to obtain water extract;Further add ethanol to water extract to reach 50-65% alcohol concentration, at 2-6 ℃ overnight after centrifugation, take supernatant, after recovery of ethanol, concentrated to obtain refined liquid;Finally, add alcohol to refined liquid to reach 8-18% volume fraction and add sodium citrate to reach 0.01-0.05% mass fraction, mix evenly to obtain.The method can effectively extract active ingredients in compound, and the obtained extract can be used to prepare skin external preparation with bacteriostatic, anti-inflammatory, antipruritic efficacy.

Description

Technical Field

[0001] This invention belongs to the field of plant extract preparation technology, specifically relating to a preparation method and application of a compound grapefruit peel extract. Background Technology

[0002] Grapefruit peel, longan leaves, and lychee leaves are all byproducts of fruit processing, with high yields but low utilization rates. Existing research indicates that grapefruit peel contains various active ingredients such as flavonoids and phenolic acids, possessing certain antibacterial and antioxidant effects. Lychee and longan leaves also contain flavonoids, and traditionally, a decoction of these ingredients is used externally for washing skin itching and inflammation. Bidens pilosa, a plant of the Asteraceae family, has extracts with antibacterial and anti-inflammatory activities. While there are reports of using grapefruit peel, lychee leaves, and longan leaves in skin care, and patents combining grapefruit peel extract with Bidens pilosa extract for food preservation, no technical solutions have been found for compounding grapefruit peel with Bidens pilosa, lychee leaves, and longan leaves to prepare topical skin preparations with anti-inflammatory and antipruritic effects. For example, Chinese patent CN115989855A discloses a food preservative and its preparation method, which prepares the food preservative by mixing grapefruit peel extract, comfrey extract, beggar-ticks extract, and motherwort extract in a certain proportion. The use of this patent is limited to the field of food preservation, which belongs to a different technical field than the topical anti-inflammatory and antipruritic use for skin application involved in this application. Furthermore, its raw material composition differs from this application, omitting lychee leaf and longan leaf. In addition, although there are reports in the prior art of using beggar-ticks extract in topical skin preparations, and patents of combining grapefruit peel with longan, lychee, and other raw materials for treating infantile eczema, their raw material composition and proportions differ from this application, and they do not involve complete extraction, purification, and formulation processes.

[0003] In terms of preparation processes, common methods for preparing plant extracts include water extraction and alcohol precipitation, which are standard techniques in the field. However, for a specific compound raw material combination of grapefruit peel, Bidens pilosa, litchi leaves, and longan leaves, determining suitable extraction conditions (such as material-to-liquid ratio and extraction time) and purification conditions (such as alcohol precipitation concentration and temperature) to ensure the extract exhibits stable anti-inflammatory and antipruritic activities is a technical challenge that needs to be addressed in preparing this type of compound extract. Different medicinal materials have varying types and properties of active ingredients, and simply applying extraction processes for single medicinal materials often fails to ensure the full extraction of active ingredients from each material. Furthermore, the stability of the extract during storage is a crucial factor affecting the actual application effect of the product, necessitating the selection of appropriate formulation excipients to maintain the extract's activity.

[0004] In terms of quality control, for compound extracts containing multiple plant raw materials, establishing reliable quality control methods to ensure the stability of active ingredient content across different batches is a necessary condition for the industrialization of the product. Naringin is one of the characteristic flavonoid components in grapefruit peel and can be used as a quality marker for this compound extract; however, the existing technology lacks a specific method for determining the naringin content in the compound extract described in this application.

[0005] In summary, the existing technology does not have a technical solution for preparing topical skin preparations with anti-inflammatory and antipruritic effects by compounding grapefruit peel with Bidens pilosa, lychee leaves and longan leaves in a specific ratio, nor does it have corresponding preparation processes and quality control methods. Summary of the Invention

[0006] One objective of this invention is to address the lack of a method in the prior art for preparing a topical skin extract with anti-inflammatory and antipruritic effects by combining grapefruit peel with four raw materials: Bidens pilosa, lychee leaves, and longan leaves. The four raw materials have different types and properties of active ingredients, and simply applying extraction processes for a single herb makes it difficult to ensure the full extraction of active ingredients from each herb.

[0007] To achieve the above objectives, the present invention provides a method for preparing a compound grapefruit peel extract, comprising the following steps: Step 1: According to the weight, take 40-60 parts of pomelo peel, 16-24 parts of Bidens pilosa, 12-18 parts of lychee leaves and 12-18 parts of longan leaves and mix them to obtain mixed medicinal materials; Step 2: Add water to the mixed medicinal materials, with a material-to-liquid ratio of 1:(15-25)g / mL. Soak for 3-5 hours, then decoct for 1-2 hours. Filter, collect the filtrate, and concentrate to a volume equivalent to the weight of the mixed medicinal materials to obtain an aqueous extract. Step 3: Add ethanol to the aqueous extract until the alcohol precipitation concentration is 50-65%, let it stand overnight at 2-6℃, centrifuge, take the supernatant, recover the ethanol and concentrate to obtain the purified liquid; Step 4: Add alcohol to the purified solution to a volume fraction of 8-18%, add sodium citrate to a mass fraction of 0.01-0.05%, mix well, and the product is obtained.

[0008] Preferably, in step 1, the weight parts of pomelo peel, beggar-ticks, lychee leaves and longan leaves are 45-55 parts, 18-22 parts, 13-17 parts and 13-17 parts respectively.

[0009] Preferably, in step 1, the ingredients are 50 parts by weight of pomelo peel, 20 parts of Bidens pilosa, 15 parts of lychee leaves, and 15 parts of longan leaves.

[0010] Preferably, in step 2, the decoction is a reflux extraction with a material-to-liquid ratio of 1:20 g / mL, soaking for 4 hours and decocting for 1.5 hours.

[0011] Preferably, in step 3, the alcohol precipitation concentration is 55-60%, and the mixture is left to stand overnight at 4°C, followed by centrifugation at 2500-3500 r / min for 8-15 minutes.

[0012] Preferably, in step 4, alcohol is added to a volume fraction of 10-15%, and sodium citrate is added to a mass fraction of 0.02%.

[0013] Preferably, the decoction extraction in step 2 is a stepwise extraction: add 10-30% ethanol aqueous solution to the mixed medicinal materials, extract at 50-65℃ for 0.5-1.5 hours, raise the temperature to 80-95℃, and continuously add anhydrous ethanol dropwise over 30-60 minutes, so that the volume fraction of ethanol in the extraction solvent is gradually increased from 10-30% to 50-70%, continue extraction for 0.5-1.5 hours, filter, and collect the filtrate.

[0014] Preferably, the ethanol recovery in step 3 is carried out by vacuum distillation. The first distillate, which is 5% to 15% of the volume of the water extract, is collected after the vacuum distillation begins. Water is added to the collected first distillate to reduce the ethanol volume fraction to 15% to 30%. The mixture is then allowed to stand at 2 to 8°C for 2 to 6 hours until the oil layer precipitates and separates from the water layer. In step 3, the separated oil layer is premixed with the ethanol to be added. After the oil layer dissolves, the resulting ethanol-oil mixture and sodium citrate are added to the purified liquid and mixed thoroughly.

[0015] The application of the compound grapefruit peel extract prepared according to any one of the methods provided by this invention in the preparation of preparations with one or more antibacterial, anti-inflammatory, and antipruritic effects.

[0016] Preferably, the preparation is a topical skin wash.

[0017] Preferably, the topical skin wash also contains 10-15% alcohol by volume and 0.02% sodium citrate by mass.

[0018] The present invention has at least the following beneficial effects: 1. Based on the solubility characteristics of the active ingredients in four medicinal herbs, this invention determined the extraction conditions as follows: a material-to-liquid ratio of 1:(15-25) g / mL, soaking for 3-5 hours, and decoction for 1-2 hours, along with an alcohol precipitation concentration of 50-65%. Under these conditions, the combination of water extraction and alcohol precipitation allows for the relatively complete extraction of active ingredients such as flavonoids, phenolic acids, and triterpenoids, while removing some impurities to obtain a purified solution with relatively concentrated active ingredients. HPLC analysis showed that the naringin content in six batches of samples ranged from 1.9987% to 2.3179%, with an average content of approximately 2.0%, indicating good reproducibility of the method across different batches. Adding 8-18% (v / v) alcohol and 0.01-0.05% (w / w) sodium citrate to the purified solution helps maintain the stability of the extract during storage.

[0019] 2. The antibacterial test results showed that the test sample had antibacterial effects against Escherichia coli, Staphylococcus aureus, and Candida albicans at action times of 2 min, 5 min, 10 min, and 20 min. At an action time of 10 min, the antibacterial rates of the four-ingredient formula against the three tested bacteria were 91.4%, 87.6%, and 82.5%, respectively, which were higher than those of single grapefruit peel extract, single Bidens pilosa extract, and the combination of grapefruit peel and Bidens pilosa, demonstrating the synergistic or complementary effect of the active ingredients after the four herbs were combined.

[0020] 3. The anti-inflammatory effects of the extract were evaluated using three mouse inflammation models. In the DNCB-induced mouse dermatitis model, the high-dose group showed a significantly lower ear swelling (16.90±3.26 mg) compared to the model group (34.27±9.74 mg) (P<0.01), and the spleen and thymus indices were also significantly lower. In the xylene-induced mouse ear swelling model, the high-dose group showed a swelling inhibition rate of 51.60%, which was close to that of the positive control calamine lotion group (57.89%). In the carrageenan-induced mouse paw swelling model, the high-dose group showed a significantly lower paw swelling (0.0339±0.00501) compared to the model group (0.0589±0.0179) (P<0.01). These three models simulated allergic contact dermatitis, chemically induced acute inflammation, and non-specific acute inflammation, respectively. The extract showed inhibitory effects in all three models, suggesting that multiple active ingredients may exert their effects through multiple targets and pathways.

[0021] 4. In the 4-AP-induced mouse pruritus model, the number of licks in the high, medium and low dose groups were 8.3±1.33, 7.6±0.84 and 12.3±2.11 times, respectively, all of which were significantly reduced compared with the model group (18.2±1.31 times) (P<0.01), indicating that the extract has an inhibitory effect on 4-AP-induced pruritus behavior in mice.

[0022] 5. This invention also establishes an HPLC method for the determination of naringin content in the compound extract. Method validation results show that naringin exhibits good linearity in the range of 0.0025 to 0.5 mg (R0.0025). 2 =0.9990); the precision test RSD was 0.07%; the test solution was stable within 48 hours (RSD 1.44%); the repeatability test RSD was 0.75%; the recovery rate ranged from 97.05% to 101.43%, with an average recovery rate of 99.98% and an RSD of 1.63%. These data indicate that the precision, stability, repeatability, and accuracy of this assay method are all within acceptable ranges and can be used for the quality control of naringin content in this compound extract.

[0023] 6. Step 2 of this invention employs a stepwise extraction method: First, extraction is performed at 50-65℃ with 10-30% ethanol, preferentially dissolving moderately polar components such as hyperoside from Bidens pilosa. Simultaneously, at this temperature, the dissolution rate and amount of pectin from grapefruit peel are low, reducing pectin's interference with the active ingredients. Then, the temperature is raised to 80-95℃, and ethanol is gradually added to 50-70%. Utilizing the characteristic of pectin's accelerated dissolution at high temperatures followed by flocculation and precipitation at high ethanol concentrations, the encapsulated flavonoid aglycones are released during flocculation, while simultaneously reducing the viscosity of the extract. Compared to water extraction, the extraction rate of naringin increased by 58.7%, and the extraction rate of hyperoside increased by 54.5%; the pectin content of the extract decreased by 82.6%, and the filtration time was shortened by 74.7%. This method achieves simultaneous and efficient extraction of different polar active ingredients in the same extraction process, with mild and controllable extraction conditions, suitable for industrial production.

[0024] 7. In the process of recovering ethanol by vacuum distillation, this invention collects the initial distillate, dilutes it to reduce the ethanol volume fraction to 15%-30%, allows it to stand at low temperature, separates the oil layer, and reuses it in the product. This method achieves effective capture and recovery of volatile oil distilled with ethanol, reducing the loss of active ingredients in the raw materials. After reusing the captured volatile oil in the product, the inhibition rates against Escherichia coli, Staphylococcus aureus, and Candida albicans are increased by 3.8, 4.4, and 4.4 percentage points, respectively, compared to the method without reuse, and the effect is better than adding an equal amount of commercial grapefruit peel essential oil. By pre-dissolving the oil layer in alcohol before adding it, uniform dispersion of the volatile oil in the formulation is ensured. This method utilizes the existing vacuum distillation process simultaneously, without adding additional equipment, and has a high degree of process integration.

[0025] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0026] Figure 1The chromatogram of naringin reference standard; Figure 2 Chromatogram of the test sample for the washing solution; Figure 3 This is the standard curve for naringin. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description. It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials described are commercially available unless otherwise specified. It should be understood that terms such as "having," "comprising," and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0028] Example 1: Preparation of Compound Grapefruit Peel Extract Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, 15g of dried lychee leaves, and 15g of dried longan leaves, coarsely grind them separately, and then mix them to obtain 100g of mixed medicinal materials. Add 2000mL of water (material-to-liquid ratio of 1:20g / mL) to the mixed medicinal materials, soak for 4 hours, then boil and reflux for 1.5 hours, filter, and collect the filtrate. Repeat the extraction once with the residue under the same conditions, combine the two filtrates, evaporate and concentrate to 100mL to obtain the compound pomelo peel aqueous extract.

[0029] Take 100 mL of the above aqueous extract and slowly add 122-150 mL of anhydrous ethanol while continuously stirring, gradually increasing the ethanol concentration to 55-60%. Refrigerate at 4°C overnight, centrifuge at 3000 r / min for 10 minutes, and filter. Recover the ethanol from the resulting alcohol precipitate by vacuum distillation, filter off any small amount of precipitate, and then concentrate to 20 mL to obtain the compound grapefruit peel refined solution.

[0030] Add 3-4.5 mL of alcohol to the above purified solution to achieve an alcohol volume fraction of 10-15% (v / v). Separately weigh 0.06 g of sodium citrate, dissolve it in an appropriate amount of sterile water, add it to the purified solution, and add sterile water to bring the total volume to 30 mL. Mix thoroughly to obtain the compound grapefruit peel extract.

[0031] Example 2: HPLC determination of naringin content in compound grapefruit peel extract 2.1 Chromatographic conditions Column: YMC-Triart C18 (4.6mm×250mm, 5μm); Mobile phase: Acetonitrile was used as mobile phase A, and 0.025 mol / L potassium dihydrogen phosphate aqueous solution containing 0.1% (V / V) phosphoric acid was used as mobile phase B. The gradient elution program was as follows: 0-30 minutes, the volume percentage of mobile phase A linearly increased from 20% to 50%; detection wavelength: 280 nm; column temperature: 30 ℃; injection volume: 10 μL; flow rate: 1.0 mL / min.

[0032] 2.2 Preparation of the test solution Take 100 μL of the antibacterial solution prepared in Example 1, transfer it to a stoppered conical flask, dilute to 10 mL with 30% methanol, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate.

[0033] 2.3 Preparation of reference solution Accurately weigh 5 mg of naringin reference standard, add 30% methanol to a final volume of 10 mL, shake well, filter through a 0.45 μm microporous membrane, and collect the filtrate (concentration 0.5 mg / mL).

[0034] 2.4 Determination Method Accurately pipette 10 μL each of the test solution and reference solution and inject them into the high-performance liquid chromatograph (HPLC). Record the chromatograms. The chromatographic peaks of naringin were all separated from other adjacent peaks, with a resolution ≥1.5. The tailing was controlled within the range of 0.95-1.05, indicating good specificity of the method. See the chromatogram below. Figure 1 and Figure 2 ,in, Figure 1 The chromatogram of naringin reference standard is shown below. Figure 2 The chromatogram of the test sample for the washing solution.

[0035] Pipette 0.05 ml, 0.10 ml, 0.50 ml, 1.0 ml, 2.5 ml, and 5.0 ml of the reference solution into 5 ml volumetric flasks, respectively, and dilute to the mark with 30% methanol. Mix well to obtain six series of reference solutions with different concentrations. Measure the peak area under chromatographic conditions. Plot the reference concentration on the x-axis and the peak area on the y-axis, and perform linear regression to establish the standard curve equation for the determination of naringin content: y = 12302x - 11.364, R0. 2 =0.9990, within the range of 0.0025 ~ 0.5 mg, such as Figure 3 As shown, the naringin content has a good linear relationship with the peak area value.

[0036] Take a 0.1 mg / mL reference solution and inject it 6 times consecutively under the above chromatographic conditions. Record the peak area. As shown in Table 1, the RSD of the precision test is 0.07%, which is less than 2.0%, indicating that the instrument has high precision and can meet the testing requirements.

[0037] Table 1 Instrument Precision Test Take the same washing solution of the test sample (batch number: 20251031), prepare the sample in the same way, and inject it sequentially at 0, 4, 8, 16, 24 and 48 h to determine the peak area of ​​naringin. As shown in Table 2, the RSD of the stability test is 0.92%, indicating that the test sample is stable within 48 h.

[0038] Table 2 Stability Experiment Take the test sample (batch number: 20251031) and extract 6 100 μL portions. Prepare the samples using the same method and determine the peak area of ​​naringin. As shown in Table 3, the RSD of the repeatability test is 1.49%, indicating that this method for determining naringin has good repeatability.

[0039] Table 3 Repeatability Experiments Six 100 μL aliquots of the same sample (batch number: 20251031) were precisely pipetted, and naringin reference standard equivalent to one times the concentration in the sample was precisely added to each. The samples were prepared using the same method, and the naringin content was determined. The recovery rate was calculated. Table 4 shows that the naringin recovery rate ranged from 97.05% to 101.43%, with an RSD of 1.63%, less than 2%. This meets the requirements, indicating that the method for determining the content of naringin in the antibacterial solution of this invention has good accuracy and can accurately reflect the content of the sample.

[0040] Table 4 Recovery rate experiment Different batches of test sample solutions were prepared and precisely pipetted, and the content of naringin in each sample was determined. The results are shown in Table 5. The content of naringin in the collected 6 batches of samples ranged from 1.9987% to 2.3179%, with an average of 2.1%.

[0041] Table 5. Results of the determination of naringin content in 6 batches of washing solution samples The naringin content in the six batches of antibacterial solutions prepared in Example 1 ranged from 1.9987% to 2.3179%, with an average of 2.1%.

[0042] Example 3: Evaluation of the in vitro antibacterial effect of compound grapefruit peel extract The tests were conducted according to Appendix E.6.1 of GB 15979-2024 "Hygienic Requirements for Disposable Sanitary Products". The compound grapefruit peel extract prepared in Example 1 was used as the test sample, and *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 6538, and *Candida albicans* ATCC 10231 were used as test strains. The stock solution of the test sample was mixed with the suspensions of each test bacteria, and the contact times were 2 min, 5 min, 10 min, and 20 min, respectively. Three parallel samples were set at each time point, and the test was repeated three times to determine the antibacterial rate.

[0043] The results showed that the compound grapefruit peel extract prepared in Example 1 had antibacterial effects against Escherichia coli, Staphylococcus aureus and Candida albicans.

[0044] Table 7. Antibacterial effect against Escherichia coli Table 8. Antibacterial effect against Staphylococcus aureus Table 9. Antibacterial effect against Candida albicans Example 4: Anti-inflammatory effect of the prepared compound grapefruit peel extract on a mouse dermatitis model. Methods: Sixty CL-grade Kunming mice (half male and half female) were used. One day before the experiment, the hair on the backs of the mice was removed, and 4% sodium sulfide nonahydrate was applied to the backs to remove hair, covering an area of ​​approximately 2 cm × 2 cm. 100 μL of 5% DNCB acetone-olive oil solution was applied to the backs of the mice for sensitization induction. On the 4th day, the mice were divided into 6 groups (n=10 per group) according to the degree of scab formation and redness on their backs: blank control group (Control), model group (Model), positive control group (LGS, given calamine lotion), high-dose group of compound grapefruit peel extract (FYXY-G), medium-dose group (FYXY-Z), and low-dose group (FYXY-D). The 10 mice in the blank group were not used for modeling. All mice were labeled and numbered. On the 5th day, 20 μL of 3% DNCB acetone-olive oil solution was applied to the inner side of the right ear of the mice for challenge, while acetone-olive oil (4:1) was applied to the left ear as a solvent control. The challenge was repeated every 2 days for 3 times. The presence of varying degrees of redness, papules, vesicles, erosion, exudation, crusting, and desquamation on the right ear and back skin of mice indicated successful model establishment. Twelve hours after the initial challenge, the control and model groups were only washed with purified water, while the other four groups were repeatedly treated with the corresponding concentration of the drug solution at 0.05 ml twice daily for seven consecutive days. Thirty minutes after the last administration, the mice were euthanized by cervical dislocation. Both ears were removed, and circular ear pieces were harvested symmetrically from the left and right ears using an 8 mm diameter skin punch. The ear pieces were weighed, and the degree of swelling and swelling inhibition rate were calculated. The thymus and spleen of each group were harvested and weighed, and the thymus index and spleen index were calculated. The lesion tissue from the right ear piece was fixed and preserved in 10% formaldehyde. Swelling degree (mg) = Right ear piece weight (mg) - Left ear piece weight (mg); Thymus or spleen index (mg / g) = Thymus or spleen mass (mg) / Body weight (g).

[0045] Table 10 Effects of compound grapefruit peel extract on ear swelling, spleen index, and thymus index in mice with dermatitis (n=10, X±S) Note: Compared with the blank group, ## P < 0.01, compared with the model group, ＊ P<0.05 ＊＊ P < 0.01.

[0046] Results: As shown in the table above, the ear swelling and spleen index in the model group were significantly higher than those in the blank group (P < 0.01), indicating that the immune function of mice was abnormal after induction with 2,4-dinitrochlorobenzene, and the model was successfully established. The ear swelling, spleen, and thymus index in the drug group were significantly lower than those in the model group (P < 0.05), indicating that the three concentrations of the compound antibacterial solution had a certain alleviating effect on skin inflammation in mice and improved the immune dysfunction caused by external skin stimulation.

[0047] Example 5 Sixty CL-grade Kunming mice (half male, half female, weighing 18-22g) were used and acclimatized for 3 days. They were then randomly divided into 6 groups of 10 mice each: a blank control group (Control), a model group (Model), a positive control group (LGS, treated with calamine lotion), a high-dose group (FYXY-G), a medium-dose group (FYXY-Z), and a low-dose group (FYXY-D) of compound grapefruit peel extract. The corresponding drug was applied bilaterally to the left hind paw of each mouse at a dose of 0.05 mL. The blank control and model control groups were treated with the same volume of distilled water twice daily for 7 consecutive days. Thirty minutes after the last administration, 0.05 mL of 0.5 mg / mL 4-aminopyridine (4-AP) was injected subcutaneously into the paw of each mouse. The blank control group received an equal volume of physiological saline. The time was 10 minutes, and the number of times the mice licked their paws within 10 minutes was observed. The licking response was defined as the repeated head-turning and licking of the left hind paw. A single lick was counted as a short pause following continuous licking. All data are expressed as mean ± standard deviation (X±S). One-way ANOVA was performed using SPSS software. LSD-t test was used for comparisons between groups. P < 0.05 was considered statistically significant.

[0048] Table 11 Effects of compound grapefruit peel extract on 4-AP-induced pruritus in mice (n=10, X±S) Note: Compared with the blank group, ## P < 0.01, compared with the model group, ＊ P<0.05 ＊＊ P<0.01 As shown in the table above, compared with the model group, the number of times mice licked their bodies was significantly reduced in the high, medium and low dose groups of compound grapefruit peel extract (P < 0.01), indicating that compound grapefruit peel extract can improve the degree of 4-AP-induced itching in mice.

[0049] Example 6: Anti-inflammatory effect of compound grapefruit peel extract on xylene-induced mouse ear swelling and inflammation model Sixty CL-grade Kunming mice (half male and half female, weighing 18-22g) were used. After acclimatization for 3 days, they were grouped according to the method in Example 4. 0.05mL of the corresponding drug was applied bilaterally to the inner and outer sides of the right ear of each mouse. The blank control group and the model control group were given the same volume of distilled water twice daily for 7 consecutive days. Thirty minutes after the last administration, 30μL of xylene was applied to both sides of the right auricle of each mouse using a pipette; the left ear served as a control. After 30 minutes, the mice were euthanized by cervical dislocation. Both ears were cut off along the auricle baseline, and circular ear pieces were punched at the same location on both ears using an 8mm diameter punch. The pieces were weighed, and the degree of swelling and swelling inhibition rate were calculated.

[0050] Table 12 Effects of compound grapefruit peel antibacterial agent on xylene-induced ear swelling in mice (n=10, `X±S) Results: The ear swelling in the model group was 19.07±2.75 mg, while that in the control group was 0.98±0.21 mg (P<0.01), indicating successful model establishment. Compared with the model group, the ear swelling in mice in the FYXY-G group (9.23±2.27 mg) and the positive control group (8.03±1.90 mg) was significantly reduced (P<0.01), with swelling inhibition rates of 51.60% and 57.89%, respectively, indicating that the compound grapefruit peel extract has an inhibitory effect on xylene-induced ear swelling in mice.

[0051] Example 7: Anti-inflammatory effect of compound grapefruit peel extract on carrageenan-induced acute inflammatory model of mouse paw edema Sixty SPF-grade Kunming mice (half male, half female, weighing 18-22g) were used. After acclimatization for 3 days, they were grouped according to the method in Example 4. Each group of mice received 0.05mL of the corresponding drug applied to the dorsal side of their left hind paw. The blank control group and the model control group received the same volume of distilled water twice daily for 7 consecutive days. On day 7, before drug application, the height and circumference of the left hind paw were measured three times, and the average value was used to calculate the paw volume before carrageenan injection. After drug application, 0.05mL of 3% carrageenan was injected subcutaneously into the left hind paw. Two hours after injection, the height and circumference of the left hind paw were measured again three times, and the average value was used. The difference in paw volume before and after carrageenan injection was used as the degree of paw swelling.

[0052] Table 13 Effects of compound grapefruit peel antibacterial agent on carrageenan-induced paw edema in mice (n=10, X±S) Note: Compared with the blank group, ## P < 0.01, compared with the model group, ＊ P<0.05 ＊＊ P<0.01 Results: The paw swelling degree in the model group was 0.0589±0.0179, while that in the blank group was 0.00896±0.00543 (P<0.01), indicating successful modeling. Compared with the model group, the paw swelling degree in mice in all FYXY groups was reduced (P<0.05). Among them, the FYXY-G group (0.0339±0.00501) and the positive group (0.0291±0.00849) showed comparable effects, indicating that the compound grapefruit peel extract can effectively alleviate carrageenan-induced paw swelling in mice and has anti-inflammatory effects.

[0053] Comparative experiment To verify the effectiveness of the present invention, the following comparative examples were set up: Comparative Example 1: Preparation of a single grapefruit peel extract Take 100g of dried pomelo peel and prepare it using the same water extraction, alcohol precipitation and compounding process as in Example 1 to obtain a single pomelo peel antibacterial solution.

[0054] Comparative Example 2: Preparation of a single Bidens pilosa extract Take 100g of dried Bidens pilosa and prepare it using the same water extraction, alcohol precipitation and compounding process as in Example 1 to obtain a single Bidens pilosa antibacterial solution.

[0055] Comparative Example 3: Preparation of a compound extract of grapefruit peel and Bidens pilosa Take 50g of dried pomelo peel and 50g of dried Bidens pilosa, totaling 100g, and prepare the compound antibacterial solution using the same water extraction, alcohol precipitation, and compounding process as in Example 1.

[0056] Comparative Example 3 is based on CN115989855A, which discloses a combination of grapefruit peel extract and Bidens pilosa extract for food preservation. However, this document does not cover its use for topical anti-inflammatory and antipruritic purposes and does not include litchi leaves and longan leaves. This comparative example simulates the core combination (grapefruit peel + Bidens pilosa) of this document, using the same preparation process and evaluation methods as this application, to verify whether the addition of litchi leaves and longan leaves produces a synergistic effect.

[0057] Comparative Example 4: Preparation of a compound extract of grapefruit peel, Bidens pilosa, and lychee leaf Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, and 30g of dried lychee leaves, totaling 100g, and prepare the three-ingredient compound antibacterial solution using the same water extraction, alcohol precipitation, and compounding process as in Example 1.

[0058] Comparative Example 5: Preparation of a compound extract of grapefruit peel, Bidens pilosa, and longan leaf Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, and 30g of dried longan leaves, totaling 100g, and prepare the three-ingredient compound antibacterial solution using the same water extraction, alcohol precipitation, and compounding process as in Example 1.

[0059] Comparative Example 6: Preparation of a compound extract of grapefruit peel, lychee leaf, and longan leaf (excluding Bidens pilosa). Take 50g of dried pomelo peel, 25g of dried lychee leaves, and 25g of dried longan leaves, totaling 100g, and prepare the solution using the same water extraction, alcohol precipitation, and compounding process as in Example 1 to obtain a three-ingredient compound antibacterial liquid that does not contain Bidens pilosa.

[0060] Experiment 1: Comparison of anti-inflammatory effects of different raw material combinations on a DNCB-induced mouse dermatitis model A mouse model of DNCB-induced dermatitis was established according to the method in Example 4. The mice were administered the compound grapefruit peel extract (four-ingredient formula) prepared in Example 1 and the antibacterial solutions prepared in Comparative Examples 1-6, respectively, at the same dosage (high-dose group, 0.05 mL / time) for 7 consecutive days. The ear swelling, spleen index, and thymus index of each group of mice were measured, and the results are shown in Table 14.

[0061] Table 14 Comparison of anti-inflammatory effects of different raw material combinations on DNCB-induced mouse dermatitis model (n=10, X ±S) Note: Compared with the blank group, ## P < 0.01; compared with the model group, ＊ P < 0.05 ＊＊ P < 0.01.

[0062] As shown in the table above, the auricular swelling in the four-ingredient formula group of Example 1 was significantly lower than that in all comparative examples, and comparable to the positive control group. Compared with Comparative Example 3, the auricular swelling of the four-ingredient formula was reduced by 43.3%; compared with Comparative Examples 4 and 5, it was reduced by 30.6% and 29.5%, respectively; and compared with Comparative Example 6 (excluding Bidens pilosa, 21.54 mg), it was reduced by 47.7%. Regarding the spleen index, the four-ingredient formula group of Example 1 was comparable to the positive control group, but significantly lower than that of Comparative Examples 3 and 6. These results indicate that the four herbs—pomelo peel, Bidens pilosa, litchi leaf, and longan leaf—produced a synergistic effect in a ratio of 50:20:15:15. The anti-inflammatory effect of the four-ingredient formula could not be achieved by omitting any one of these herbs or using only two herbs.

[0063] Experiment 2: Comparison of the antipruritic effects of different combinations of raw materials on a 4-AP-induced mouse pruritus model A 4-AP-induced pruritus model was established in mice according to the method in Example 5. The compound grapefruit peel extract (four-ingredient formula) prepared in Example 1 and the antibacterial solutions prepared in Comparative Examples 1-6 were administered topically at the same dose (high-dose group, 0.05 mL / time) for 7 consecutive days. The number of times mice licked their bodies within 10 minutes was measured in each group, and the results are shown in Table 15.

[0064] Table 15 Comparison of antipruritic effects of different raw material combinations on 4-AP-induced mouse pruritus model (n=10, X±S) Note: Compared with the blank group, ## P < 0.01; compared with the model group, ＊ P < 0.05 ＊＊ P < 0.01.

[0065] As shown in the table above, the number of licks in the four-ingredient formula group of Example 1 was significantly lower than that in all comparative examples. Compared with Comparative Example 3, the number of licks decreased by 27.8%; compared with Comparative Examples 4 and 5, it decreased by 15.3% and 11.7%, respectively; and compared with Comparative Example 6, it decreased by 34.6%. The antipruritic effect of the four-ingredient formula group was better than that of the positive control group, indicating that the compound has a significant synergistic effect in relieving itching. The above results further confirm that the complete combination of the four medicinal materials—grapefruit peel, Bidens pilosa, litchi leaf, and longan leaf—is a necessary condition for achieving the best antipruritic effect; the absence or reduction of any of the ingredients significantly reduced the antipruritic effect.

[0066] Experiment 3: Comparison of the anti-inflammatory effects of different raw material combinations on a xylene-induced mouse ear swelling and inflammation model. A xylene-induced mouse ear swelling and inflammation model was established according to the method in Example 6. The compound grapefruit peel extract (four-ingredient formula) prepared in Example 1 and the antibacterial solutions prepared in Comparative Examples 1-6 were administered topically at the same dose (high-dose group, 0.05 mL / time) for 7 consecutive days. The degree of ear swelling and swelling inhibition rate of each group of mice were measured, and the results are shown in Table 16.

[0067] Table 16 Comparison of anti-inflammatory effects of different raw material combinations on xylene-induced ear swelling in mice (n=10, X±S) Note: Compared with the blank group, ## P < 0.01; compared with the model group, ＊ P < 0.05 ＊＊ P < 0.01.

[0068] As can be seen, the swelling inhibition rate of the Four-Ingredient Formula group (Example 1) reached 51.60%, which was close to that of the positive control group (57.89%) and significantly higher than that of Comparative Example 3 (pomelo peel + Bidens pilosa, 37.39%), Comparative Example 4 (44.62%), Comparative Example 5 (45.94%), and Comparative Example 6 (30.36%). From Comparative Example 3 to Comparative Example 4 (with the addition of litchi leaves), the swelling inhibition rate increased from 37.39% to 44.62%, an increase of 7.23 percentage points; from Comparative Example 4 to the Four-Ingredient Formula (with the addition of longan leaves), the swelling inhibition rate increased from 44.62% to 51.60%, a further increase of 6.98 percentage points. This progressively increasing synergistic effect proves that the addition of litchi leaves and longan leaves is not a simple additive of raw materials, but rather produces a synergistic effect. The swelling inhibition rate of Comparative Example 6 (without Bidens pilosa) was only 30.36%, which was significantly lower than that of the various regimens containing Bidens pilosa, indicating that Bidens pilosa is also indispensable in this compound.

[0069] Experiment 4: Comparison of the anti-inflammatory effects of different raw material combinations on a carrageenan-induced acute paw edema model in mice. A carrageenan-induced acute inflammatory model of paw edema in mice was established according to the method in Example 7. The mice were administered the compound grapefruit peel extract (four-ingredient formula) prepared in Example 1 and the antibacterial solutions prepared in Comparative Examples 1-6, respectively, at the same dosage (high-dose group, 0.05 mL / time) for 7 consecutive days. The degree of paw edema in each group was measured, and the results are shown in Table 17.

[0070] Table 17 Comparison of anti-inflammatory effects of different raw material combinations on carrageenan-induced paw edema in mice (n=10, X±S) Note: Compared with the blank group, ## P < 0.01; compared with the model group, ＊ P < 0.05 ＊＊ P < 0.01.

[0071] The results showed that the foot swelling degree in the four-ingredient formula group (Example 1) (0.0339) was closest to that in the positive control group (0.0291), and significantly lower than that in all control groups. The four-ingredient formula group showed a reduction of 18.3% compared to control group 3 (pomelo peel + Bidens pilosa, 0.0415) and a reduction of 23.5% compared to control group 6 (without Bidens pilosa, 0.0443), further confirming the necessity and synergistic effect of the complete combination of the four herbs.

[0072] Experiment 5: Comparison of in vitro antibacterial effects of different raw material combinations The experiment was conducted in accordance with Appendix E.6.1 of GB 15979-2024. The compound grapefruit peel extract (four-ingredient formula) prepared in Example 1 and the antibacterial solutions prepared in Comparative Examples 1-6 were used as test samples. Escherichia coli, Staphylococcus aureus and Candida albicans were used as test strains. The action time was 10 min. The antibacterial rate of each test sample was determined. The results are shown in Table 18.

[0073] Table 18 Comparison of the inhibition rates of different raw material combinations on three tested bacteria (action time 10 min) The results showed that the four-herb formula (Example 1) exhibited significantly higher inhibition rates against all three tested bacteria than all comparative examples. The inhibition rate against *Escherichia coli* reached 91.4%, an increase of 7.8 percentage points compared to Comparative Example 3 (grapefruit peel + Bidens pilosa, 83.6%); the inhibition rate against *Staphylococcus aureus* reached 87.6%, an increase of 8.2 percentage points compared to Comparative Example 3 (79.4%); and the inhibition rate against *Candida albicans* reached 82.5%, an increase of 10.4 percentage points compared to Comparative Example 3 (72.1%). From Comparative Example 3 (two herbs) to Comparative Examples 4 / 5 (three herbs) and then to Example 1 (four herbs), the inhibition rate showed a progressively increasing trend, indicating that the addition of each herb contributed to the antibacterial effect. Furthermore, the combined antibacterial effect of the four herbs exceeded the simple sum of the effects of each herb alone, demonstrating a synergistic effect. The antibacterial rates of Comparative Example 6 (without Bidens pilosa) (78.5%, 74.6%, 68.2%) were significantly lower than those of the various regimens containing Bidens pilosa, further confirming the key role of Bidens pilosa in this compound.

[0074] Example 8: Stepwise extraction of compound grapefruit peel extract To further optimize the extraction method, a stepwise variable temperature extraction method for compound grapefruit peel extract was designed.

[0075] Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, 15g of dried lychee leaves, and 15g of dried longan leaves, coarsely grind them separately, and then mix them to obtain 100g of mixed medicinal materials. Add 2000mL of 20% ethanol aqueous solution (material-to-liquid ratio of 1:20g / mL) to the mixed medicinal materials, and extract at 60℃ for 1 hour. Then, raise the extraction temperature to 90℃ and continuously add anhydrous ethanol dropwise over 45 minutes, gradually increasing the volume fraction of ethanol in the extraction solvent from 20% to 60%, and continue extraction for 1 hour. After extraction, filter, collect the filtrate, and concentrate it to 100mL to obtain the compound pomelo peel aqueous extract.

[0076] Take 100 mL of the above aqueous extract and slowly add 122-150 mL of anhydrous ethanol while continuously stirring, gradually increasing the ethanol concentration to 55-60%. Refrigerate at 4°C overnight, centrifuge at 3000 r / min for 10 minutes, and filter. Recover the ethanol from the resulting alcohol precipitate by vacuum distillation, filter off any small amount of precipitate, and then concentrate to 20 mL to obtain the compound grapefruit peel refined solution.

[0077] Add 3-4.5 mL of alcohol to the above purified solution to achieve an alcohol volume fraction of 10-15% (v / v). Separately weigh 0.06 g of sodium citrate, dissolve it in an appropriate amount of sterile water, add it to the purified solution, and add sterile water to bring the total volume to 30 mL. Mix thoroughly to obtain the compound grapefruit peel extract.

[0078] Comparative Example 7: Water Extraction (Single Temperature, Pure Water Extraction) Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, 15g of dried lychee leaves, and 15g of dried longan leaves, mix them, add 2000mL of pure water, reflux at 100℃ for 2 hours, filter, collect the filtrate, and concentrate to 100mL. The subsequent alcohol precipitation and compounding steps are the same as in Example 8.

[0079] This comparative model simulates the traditional extraction method of external washing with decoction and the optimal conditions for water extraction of Bidens pilosa, representing the most basic extraction scheme.

[0080] Comparative Example 8: Constant High Temperature Alcohol Extraction Method (Single Temperature, Constant Ethanol Concentration) Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, 15g of dried lychee leaves, and 15g of dried longan leaves, mix them, add 2000mL of 60% ethanol aqueous solution, reflux extract at 90℃ for 2 hours, filter, collect the filtrate, and concentrate to 100mL. The subsequent alcohol precipitation and compounding steps are the same as in Example 8.

[0081] This comparative example simulated the alcohol extraction process conditions of total flavonoids from grapefruit peel and total flavonoids from Bidens pilosa. The ethanol concentration (60%) and extraction temperature (90℃) were both within the range of conventional optimal conditions.

[0082] Comparative Example 9: Flavonoid Extraction Followed by Pectin Precipitation Method (Stepwise Extraction-Separation Mode) Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, 15g of dried lychee leaves, and 15g of dried longan leaves. Mix them and add 2000mL of 85% ethanol aqueous solution. Reflux at 80℃ for 2 hours, filter, collect the filtrate (flavonoid extract), and concentrate to 100mL. Repeat the extraction once with the residue under the same conditions, and combine the filtrates. The subsequent alcohol precipitation and compounding steps are the same as in Example 8.

[0083] This comparative example simulates the technical route of extracting flavonoids first in the co-production process of flavonoids and pectin in grapefruit peel, that is, using high-concentration ethanol to preferentially extract flavonoid components, while pectin remains in the filter residue.

[0084] Comparative Example 10: Pectin Precipitation followed by Flavonoid Extraction Method (Stepwise Extraction-Separation Mode) Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, 15g of dried lychee leaves, and 15g of dried longan leaves, mix them, add 2000mL of pure water, and extract at 90℃ for 1.5 hours. Filter and collect the filtrate. Add anhydrous ethanol to the filtrate to make the ethanol volume fraction reach 60%, let it stand to precipitate pectin, filter to remove the pectin precipitate, recover the ethanol from the filtrate, and concentrate to 100mL. The subsequent alcohol precipitation and compounding steps are the same as in Example 8.

[0085] This comparative example simulates the technical approach of recovering flavonoids from waste liquid after pectin extraction, namely, removing pectin precipitate first and then extracting flavonoid components.

[0086] Effect Experiment Experiment 6: Comparison of the extraction rate of active ingredients and pectin content in compound extracts using different extraction methods Compound grapefruit peel aqueous extracts (samples before alcohol precipitation) were prepared according to the methods of Example 8 and Comparative Examples 7-10, respectively. The extraction rates of naringin and hyperoside, and the pectin content in the extracts were used as evaluation indicators to compare the extraction effects of different extraction methods. The naringin content was determined by HPLC according to the method in Example 2, the hyperoside content was determined according to the literature method, and the pectin content was determined by carbazole-sulfuric acid colorimetric method.

[0087] Table 19 Effects of different extraction methods on the extraction rate of active ingredients and pectin content Results analysis: (1) Regarding the extraction rate of naringin, the extraction rate of Example 8 (13.68 mg / g) was significantly higher than that of all comparative examples. It was 20.5% higher than that of Comparative Example 8 (constant high temperature alcohol extraction, 11.35 mg / g); 25.8% higher than that of Comparative Example 9 (flavonoids extracted first, then pectin precipitated, 10.87 mg / g); 58.7% higher than that of Comparative Example 7 (water extraction, 8.62 mg / g); and 97.4% higher than that of Comparative Example 10 (pectin precipitated first, then flavonoids extracted, 6.93 mg / g).

[0088] (2) Regarding the extraction rate of hyperoside, the extraction rate of Example 8 (2.89 mg / g) was also significantly higher than that of all comparative examples. It was 19.9% ​​higher than that of Comparative Example 8 (2.41 mg / g); 47.4% higher than that of Comparative Example 9 (1.96 mg / g); 54.5% higher than that of Comparative Example 7 (1.87 mg / g); and 90.1% higher than that of Comparative Example 10 (1.52 mg / g).

[0089] (3) Regarding the pectin content of the extract, the pectin content of Example 8 (2.15 mg / mL) was significantly lower than that of Comparative Example 7 (12.34 mg / mL) and Comparative Example 8 (7.86 mg / mL), but higher than that of Comparative Example 9 (3.52 mg / mL) and Comparative Example 10 (1.28 mg / mL). This indicates that the stepwise temperature-switching extraction in Example 8 effectively controlled the amount of pectin dissolved during the extraction process—the initial low temperature and low alcohol stage inhibited the early large-scale dissolution of pectin, and the pectin flocculated and precipitated at a high alcohol concentration during the temperature-raising and alcohol-supplementing stage, and finally the pectin content in the extract remained at a low level.

[0090] Comparative Example 7 (water extraction) had the highest pectin content (12.34 mg / mL). This was because high-temperature pure water extraction caused a large amount of pectin in the grapefruit peel to dissolve, forming a viscous colloidal system. This colloidal system had an encapsulation effect on components such as hyperoside, resulting in the lowest extraction rate of hyperoside (1.87 mg / g). At the same time, the high viscosity of the extract also affected the mass transfer efficiency of naringin. Although the addition of ethanol reduced the dissolution of pectin in Comparative Example 8 (constant high-temperature ethanol extraction) (7.86 mg / mL), pectin still dissolved to a considerable extent at a 60% ethanol concentration, and the extraction rate of hyperoside (2.41 mg / g) was still lower than that in Example 8, indicating that a single temperature condition cannot achieve the optimal balance between pectin dissolution and active ingredient extraction. Comparative Example 9 (flavonoids extracted first, then pectin precipitated) used 85% high-concentration ethanol for extraction. Pectin dissolution was minimal (3.52 mg / mL), but the high-concentration ethanol had low extraction efficiency for moderately polar components such as hyperoside in Bidens pilosa (1.96 mg / g), resulting in a low hyperoside extraction rate. This comparative example illustrates that prioritizing flavonoid extraction sacrifices the extraction efficiency of moderately polar components. Comparative Example 10 (pectin precipitated first, then flavonoids extracted) had the lowest pectin content in the extract (1.28 mg / mL), but the pectin precipitation process also adsorbed and co-precipitated some flavonoid components, resulting in the lowest extraction rates for naringin and hyperoside (6.93 mg / g and 1.52 mg / g, respectively). This indicates that while prioritizing pectin precipitation removed pectin, it caused a significant loss of active ingredients.

[0091] Example 8 employed a stepwise temperature-switching extraction strategy. Initially, extraction was performed at 50-65℃ with 10-30% ethanol, preferentially dissolving moderately polar components such as hyperoside from Bidens pilosa while simultaneously inhibiting the early, large-scale dissolution of pectin from grapefruit peel. Subsequently, the temperature was increased to 80-95℃ with a gradual addition of ethanol to 50-70%. Utilizing the characteristic of pectin's accelerated dissolution at high temperatures followed by immediate flocculation and precipitation at high ethanol concentrations, the pectin released its bound flavonoid aglycones during precipitation. Ultimately, Example 8 achieved the highest extraction rates of naringin (13.68 mg / g) and hyperoside (2.89 mg / g) while maintaining a low pectin content (2.15 mg / mL). These results demonstrate that stepwise temperature-switching extraction, through the synergistic control of temperature and ethanol concentration, effectively solved the problem of pectin from grapefruit peel interfering with the dissolution of other active ingredients in the compound, achieving simultaneous and efficient extraction of active components with different polarities.

[0092] Experiment 7: Comparison of the appearance and filtration performance of extracts obtained by different extraction methods Compound grapefruit peel aqueous extract (samples before alcohol precipitation) was prepared according to the methods of Example 8 and Comparative Examples 7-10, respectively. The appearance of the extract was observed, and the filtration time of the same volume (100 mL) of extract under the same conditions (Buchner funnel, qualitative filter paper) was recorded.

[0093] Table 20 Comparison of the appearance and filtration performance of extracts obtained by different extraction methods Results analysis: The extract of Comparative Example 7 was the most viscous and had the longest filtration time (186 s). This was because high-temperature water extraction caused a large amount of pectin in the grapefruit peel to dissolve, forming a high-viscosity colloidal system. In Comparative Example 8, the addition of ethanol reduced pectin dissolution, resulting in a lower viscosity. The extracts of Comparative Examples 9 and 10 were relatively clear and had shorter filtration times, but this came at the cost of sacrificing the extraction rate of active ingredients (see Experiment 6). The extract of Example 8 had good fluidity, and its filtration time (47 s) was close to that of Comparative Example 9 (58 s), but much shorter than that of Comparative Examples 7 (186 s) and 8 (112 s). This indicates that stepwise temperature-controlled extraction effectively controlled pectin dissolution without affecting the extraction efficiency of active ingredients, and the extract had good filterability, which is beneficial for subsequent industrial production.

[0094] Based on the combined results of Experiments 6 and 7, the stepwise variable temperature extraction method of Example 8 has the following advantages compared with Comparative Examples 7-10: (1) Compared with water extraction (Comparative Example 7), the stepwise variable temperature extraction inhibited the early dissolution of pectin by using initial low temperature and low alcohol conditions, avoiding the encapsulation effect of high viscosity colloidal system on active ingredients. The extraction rates of naringin and hyperoside were increased by 58.7% and 54.5%, respectively, and the filtration time of the extract was shortened by 74.7%. (2) Compared with constant high temperature alcohol extraction (Comparative Example 8), the stepwise variable temperature extraction, through the gradient synergistic change of temperature and ethanol concentration, enabled different polar components to obtain the optimal dissolution conditions at different stages. The extraction rates of naringin and hyperoside were increased by 20.5% and 19.9%, respectively, and the pectin content of the extract was reduced by 72.6%. (3) Compared with extracting flavonoids first and then precipitating pectin (Comparative Example 9), the initial low-alcohol conditions of stepwise temperature-varying extraction are more conducive to the dissolution of moderately polar components such as hyperoside in Bidens pilosa, and the extraction rate of hyperoside is increased by 47.4%. (4) Compared with precipitating pectin first and then extracting flavonoids (Comparative Example 10), stepwise temperature-varying extraction avoids the adsorption and co-precipitation loss of flavonoid components during pectin precipitation, and the extraction rates of naringin and hyperoside are increased by 97.4% and 90.1%, respectively.

[0095] The above results show that the stepwise temperature-varying extraction method of Example 8 effectively solves the problem of pectin in grapefruit peel interfering with the dissolution of other active ingredients in the compound medicine by synergistic gradient control of temperature and ethanol concentration. It achieves synchronous and efficient extraction of active ingredients with different polarities in the same extraction process, and overcomes the defects of component loss or insufficient extraction caused by the separation extraction mode in the prior art.

[0096] Example 9: Preparation of Compound Grapefruit Peel Extract Grapefruit peel contains a certain amount of volatile oil components. During vacuum distillation, some of the volatile oil evaporates along with ethanol-water vapor and enters the condensate. If this volatile oil is not recovered and is directly discarded, it will result in the loss of active ingredients in the raw material. Furthermore, these volatile oils themselves have certain antibacterial activity, and their loss may weaken the antibacterial effect of the final product. Therefore, an improved method for preparing a compound grapefruit peel extract containing volatile oil for capture and reuse is provided.

[0097] Take 50g of dried pomelo peel, 20g of dried Bidens pilosa, 15g of dried lychee leaves, and 15g of dried longan leaves, coarsely grind them separately, and then mix them to obtain 100g of mixed medicinal materials. Add 2000mL of pure water (material-to-liquid ratio of 1:20g / mL) to the mixed medicinal materials, soak for 4 hours, then boil and reflux for 1.5 hours, filter, and collect the filtrate. Repeat the extraction once with the residue under the same conditions, combine the two filtrates, evaporate and concentrate to 100mL to obtain the compound pomelo peel aqueous extract.

[0098] Take 100 mL of the above aqueous extract and slowly add 122-150 mL of anhydrous ethanol while continuously stirring, gradually increasing the ethanol concentration to 55-60%. Refrigerate at 4°C overnight, centrifuge at 3000 rpm for 10 minutes, and filter. Perform vacuum distillation on the resulting ethanol precipitate to recover the ethanol. Collect the first distillate, which is 10% (10 mL) of the aqueous extract volume, after the start of vacuum distillation. Separate this first distillate from the subsequent distillates and collect them separately. Add water to the collected first distillate to reduce the ethanol volume fraction to approximately 20%, and let it stand at 4°C for 4 hours. After the oil layer precipitates and separates from the water layer, approximately 0.12 mL of oil is separated.

[0099] The remaining supernatant was further distilled under reduced pressure to recover ethanol, and concentrated to 20 mL to obtain a compound grapefruit peel extract. 3-4.5 mL of ethanol was added to this extract to achieve an ethanol volume fraction of 10-15% (v / v). Separately, 0.06 g of sodium citrate was weighed and dissolved in an appropriate amount of sterile water. Approximately 0.12 mL of the separated oil layer was premixed with the ethanol to be added. After the oil layer dissolved, the resulting ethanol-oil mixture and the sodium citrate solution were added to the extract, and sterile water was added to bring the total volume to 30 mL. The mixture was thoroughly mixed to obtain the compound grapefruit peel extract.

[0100] Comparative Example 11: No volatile oil capture (vacuum distillation) Take the same batch of alcohol precipitation supernatant as in Example 9, and directly perform vacuum distillation to recover ethanol. Do not separate the initial distillate for collection; discard all distillates after mixing. The remaining operations (including raw materials, extraction, alcohol precipitation, and compounding) are exactly the same as in Example 9. This comparative example simulates conventional ethanol recovery methods, i.e., without capturing and reusing the volatile oils distilled off with the ethanol during distillation.

[0101] Comparative Example 12: Capturing volatile oils but not re-adding them Take the same batch of alcohol precipitation supernatant as in Example 9, collect the initial distillate and separate the oil layer according to the method in Example 9, but discard the separated oil layer and do not add it to the purified liquid. The remaining operations (including raw materials, extraction, alcohol precipitation, compounding, etc.) are exactly the same as in Example 9.

[0102] This comparative example was used to verify the difference in effect between trapping without re-addition and trapping with re-addition, to demonstrate the contribution of volatile oil reuse itself to the antibacterial effect.

[0103] Comparative Example 13: Adding commercial grapefruit peel essential oil instead of natural volatile oil Take the same batch of alcohol precipitation supernatant as in Example 9, and perform vacuum distillation according to the method of Comparative Example 11. Do not collect volatile oil. After concentrating to obtain a refined liquid, add an equal amount (about 0.12 mL) of commercial grapefruit peel essential oil (main component is limonene) to the oil layer collected in Example 9 during the compounding stage, instead of the self-collected volatile oil. The remaining operations are the same as in Example 9.

[0104] This comparative example is used to verify whether there is a difference in efficacy between the self-collected volatile oil and the added commercial grapefruit peel essential oil, in order to prove the identity specificity of the self-collected volatile oil. The volatile oil derived from the same compound extract has a higher matching degree and better compatibility or synergistic effect compared with the added essential oil.

[0105] Effect Experiment Experiment 8: Effects of different volatile oil treatment methods on the antibacterial effect of compound grapefruit peel extract Compound grapefruit peel extracts were prepared according to the methods in Example 9 and Comparative Examples 11-13, respectively. Tests were conducted according to Appendix E of GB 15979-2024 "Hygienic Requirements for Disposable Sanitary Products," using *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 6538, and *Candida albicans* ATCC 10231 as test strains. The stock solutions of each test sample were mixed with the suspensions of the respective test bacteria, and the reaction time was 10 min. Three replicates were prepared for each sample, and the antibacterial rate was determined.

[0106] Table 21 Effect of different volatile oil treatment methods on the antibacterial rate of compound grapefruit peel extract (action time 10 min) Results analysis: (1) Comparative Example 11 (no capture and no re-addition) had the lowest antibacterial rate, with antibacterial rates of 89.7%, 85.4%, and 80.3% for the three tested bacteria, respectively. This is the baseline level of the process. (2) The antibacterial rates of Comparative Example 12 (capture but no re-addition) (89.9%, 85.7%, 80.6%) were not significantly different from those of Comparative Example 11 (89.7%, 85.4%, 80.3%) (P>0.05), indicating that the simple capture operation itself does not change the antibacterial effect; the key lies in re-addition. (3) The antibacterial rate of Comparative Example 13 (with added commercial grapefruit peel essential oil) (91.2%, 87.1%, 82.0%) was higher than that of Comparative Example 11, indicating that grapefruit peel essential oil itself has certain antibacterial activity, and the addition of essential oil can enhance the antibacterial effect to a certain extent. However, this enhancement effect is limited—after adding an equal amount of commercial essential oil, the antibacterial rate only increased by about 1.5-1.7 percentage points compared to Comparative Example 11. (4) The antibacterial rate of Example 9 (capturing and re-adding self-sourced volatile oil) (93.5%, 89.8%, 84.7%) was significantly higher than that of all comparative examples (P < 0.05). Compared with Comparative Example 11, the antibacterial rate increased by 3.8-4.4 percentage points; compared with Comparative Example 13 (adding commercial essential oil), the antibacterial rate increased by 2.3-2.7 percentage points.

[0107] The antibacterial effect of Example 9 was significantly better than that of Comparative Example 13, suggesting that although the self-sourced volatile oil and the added commercial grapefruit peel essential oil are from similar sources, both being grapefruit peel, their effects differ. The possible reason is that the self-sourced volatile oil is collected from the alcohol precipitation supernatant of the compound extract itself, and its composition is completely consistent with the inherent volatile components of the compound extract, resulting in better compatibility with the formulation matrix after re-addition. In contrast, the extraction process of commercial grapefruit peel essential oil is usually steam distillation or pressing, and the source variety may differ from that of this product, leading to a less complete match in composition. The main component of grapefruit peel volatile oil is d-limonene, with a content ranging from 42% to 89%, and it also contains various terpenoid compounds such as myrcene and linalool. Grapefruit peel essential oils obtained from different varieties and extraction methods differ in composition and relative content. Although the commercial grapefruit peel essential oil in this experiment also exhibited a certain antibacterial enhancement effect, its effect was lower than that of the self-sourced volatile oil, possibly due to the incomplete match between its composition and that of the product's extract. Furthermore, there was no significant difference between Comparative Example 12 (capture but not re-addition) and Comparative Example 11 (no capture and no re-addition), indicating that the simple capture operation itself does not affect the antibacterial effect.

[0108] The above results indicate that the self-sourced volatile oil capture and reuse scheme of the present invention (Example 9) significantly improves the antibacterial rate of compound grapefruit peel extract against Escherichia coli, Staphylococcus aureus, and Candida albicans compared to the non-capture scheme (Comparative Example 11), with an increase of approximately 3.8-4.4 percentage points. Compared to the scheme using commercial grapefruit peel essential oil (Comparative Example 13), the self-sourced volatile oil reuse scheme has a better effect (an increase of approximately 2.3-2.7 percentage points), which may be related to the component compatibility between the self-sourced volatile oil and the formulation matrix.

[0109] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for preparing a compound grapefruit peel extract, characterized in that, Includes the following steps: Step 1: According to the weight, take 40-60 parts of pomelo peel, 16-24 parts of Bidens pilosa, 12-18 parts of lychee leaves and 12-18 parts of longan leaves and mix them to obtain mixed medicinal materials; Step 2: Add water to the mixed medicinal materials, with a material-to-liquid ratio of 1:(15-25)g / mL. Soak for 3-5 hours, then decoct for 1-2 hours. Filter, collect the filtrate, and concentrate to a volume equivalent to the weight of the mixed medicinal materials to obtain an aqueous extract. Step 3: Add ethanol to the aqueous extract until the alcohol precipitation concentration is 50-65%, let it stand overnight at 2-6℃, centrifuge, take the supernatant, recover the ethanol and concentrate to obtain the purified liquid; Step 4: Add alcohol to the purified liquid to a volume fraction of 8-18%, add sodium citrate to a mass fraction of 0.01-0.05%, mix well, and the product is ready.

2. The preparation method according to claim 1, characterized in that, In step 1, by weight, there are 50 parts of pomelo peel, 20 parts of Bidens pilosa, 15 parts of lychee leaves, and 15 parts of longan leaves.

3. The preparation method according to claim 1, characterized in that, In step 2, the decoction is a reflux extraction with a material-to-liquid ratio of 1:20 g / mL. The mixture is soaked for 4 hours and then decocted for 1.5 hours.

4. The preparation method according to claim 1, characterized in that, In step 3, the alcohol concentration is 55-60%, and the mixture is left to stand overnight at 4°C. The centrifugation conditions are 2500-3500 r / min for 8-15 minutes.

5. The preparation method according to claim 1, characterized in that, In step 4, add alcohol to a volume fraction of 10-15% and sodium citrate to a mass fraction of 0.02%.

6. The preparation method according to claim 1, characterized in that, In step 2, the decoction extraction is a stepwise extraction: add 10-30% ethanol aqueous solution to the mixed medicinal materials, extract at 50-65℃ for 0.5-1.5 hours, raise the temperature to 80-95℃, and continuously add anhydrous ethanol dropwise over 30-60 minutes, so that the volume fraction of ethanol in the extraction solvent is gradually increased from 10-30% to 50-70%, continue extraction for 0.5-1.5 hours, filter, and collect the filtrate.

7. The preparation method according to claim 1, characterized in that, Ethanol recovery is achieved through vacuum distillation. The initial distillate, which is 5% to 15% of the volume of the water extract, is collected after the start of vacuum distillation. Water is added to the collected initial distillate to reduce the ethanol volume fraction to 15% to 30%. The mixture is then allowed to stand at 2 to 8°C for 2 to 6 hours until the oil layer separates from the water layer. The separated oil layer is premixed with the ethanol to be added. After the oil layer dissolves, the resulting ethanol-oil mixture is added to the purified solution along with sodium citrate and mixed thoroughly.

8. The application of the compound grapefruit peel extract prepared by any one of the preparation methods according to any one of claims 1-7 in the preparation of preparations with one or more antibacterial, anti-inflammatory, and antipruritic effects.

9. The application according to claim 8, characterized in that, The preparation is a topical skin wash.

10. The application according to claim 9, characterized in that, The topical skin wash also contains 10-15% alcohol by volume and 0.02% sodium citrate by mass.

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