Preparation method and application of chaenomeles speciosa leaf multi-effect refined extract

Abstract

The invention provides a preparation method and application of a chaenomeles speciosa leaf multi-effect refined extract. The method comprises the following steps: extracting with an ethanol solution, filtering, concentrating and drying to obtain the multiple-effect chaenomeles speciosa leaf fine extract. The refined extract has relatively strong antioxidant activity, antibacterial activity and hypoglycemic activity, and the antioxidant activity is three times that of vitamin C. The compound can be used for preparing antioxidative, antibacterial and hypoglycemic drugs. Or can be used for preparing wash supplies; or is used for preparing an external preparation for external antibiosis and anti-inflammation; or can be used for preparing food or medicine additives. The preparation technology of the refined extract is simple, safe and reliable, the prepared refined extract is high in yield, active ingredients are not prone to inactivation, and large-scale production is easy to expand.

Description

Technical Field

[0001] This invention relates to a method for preparing and using a multi-effect extract of papaya leaves. Background Technology

[0002] Smooth-skinned papaya is the dried, ripe fruit of Chaemmeles sinemis (Thodlin) Koemne., a plant in the Rosaceae family. It was once included in the quality standards of traditional Chinese medicine in Shandong, Hubei, and other provinces. Because its surface is smooth and wrinkle-free, it is given the alternative name "smooth-skinned papaya" to distinguish it from the papaya listed in the 2025 edition of the Chinese Pharmacopoeia.

[0003] The medicinal value of papaya leaves has been recorded in my country for thousands of years. The Compendium of Materia Medica clearly states its effects of calming the liver and stomach, relaxing muscles and tendons, dispelling dampness and reducing swelling, and clearing heat and detoxifying. Research from Shandong Agricultural University has found that papaya leaves contain polyphenols, flavonoids, saponins, polysaccharides, and other active substances. These active substances are all natural substances with broad pharmacological activities, exhibiting strong antioxidant, anti-inflammatory, antibacterial, and antiviral properties, as well as effects such as lowering blood lipids, regulating blood sugar, anti-tumor activity, and improving cardiovascular health.

[0004] In literature reports, the extraction processes of active ingredients in papaya leaves mostly focus on the extraction of flavonoids from wrinkled papaya leaves and carnation leaves, while there is very little research on the extraction of active ingredients from smooth papaya leaves, and most of it is concentrated in basic laboratory research. In terms of application, it is mainly used to make papaya leaf tea and to make cold dishes after blanching; or to be used as feed or fermented into organic fertilizer.

[0005] Research and development on papaya leaves has largely focused on the extraction, content analysis, and bioactivity of polyphenols or flavonoids, neglecting the yield of the extracts. The yield of extracts is a crucial factor in maximizing raw material utilization and reducing subsequent processing costs. It is a core requirement for cost reduction, efficiency improvement, quality control, and stable efficacy in traditional Chinese medicine production, and a key link between raw materials and finished products, permeating the entire process of process development, production monitoring, and quality assessment. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing and using a multi-effect extract of papaya leaves. The method involves extraction with an ethanol solution, filtration, concentration, and drying to obtain the multi-effect extract of papaya leaves. This extract not only has a high yield, exceeding 33.41%, but also exhibits strong antioxidant, antibacterial, and hypoglycemic activities, with its antioxidant activity being three times that of vitamin C.

[0007] Analysis of the obtained extracts using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-MS / MS) revealed that the extracts of *Papaya scabra* leaves contain a significant amount of polyphenols (total relative content exceeding 40%) and flavonoids (total relative content exceeding 20%), such as catechins, proanthocyanidins B1, apigenin-7-O-glucuronide, luteolin-7-glucuronide, quercetin, hyperoside, and cryptochlorogenic acid. These polyphenols and flavonoids generally possess strong antioxidant, antibacterial, anti-inflammatory, antiviral, cardiovascular protective, hepatoprotective, and hypoglycemic activities. These polyphenols and flavonoids form the material basis for the liver-soothing, stomach-regulating, muscle-relaxing, heat-clearing, and detoxifying effects of *Papaya scabra* leaves. In addition, the refined extract also contains a certain amount of mannitol (relative content of 15.784%) and citric acid (relative content of 11.388%). Mannitol can be used to prevent acute renal failure (such as after major surgery or trauma) and maintain renal perfusion through diuresis; it can also be used as an adjunct treatment for edema caused by nephrotic syndrome. Citric acid can indirectly regulate the activity of antioxidant enzymes and also regulate the acid-base balance of blood and urine. This may be the main material basis for the dampness-removing and swelling-reducing effects of papaya leaves.

[0008] The present invention discloses a method for preparing a multi-effect extract of papaya leaves, which comprises the following steps:

[0009] a. Soak the leaves of the smooth-skinned papaya in 10-30 times their volume of 30-95% ethanol solution for 0-3 hours, then extract using ultrasound or reflux. Ultrasonic extraction temperature is 35-65℃, extraction time is 0.5-1 hour; reflux extraction temperature is 50-100℃, extraction time is 0.5-2 hours; extract 1-3 times to obtain the extract.

[0010] b. Filter the extract obtained in step a through a 260-mesh filter cloth, concentrate the filtrate under reduced pressure at 50°C, dry it at 40°C, and pulverize it to obtain a multi-effect refined extract of papaya leaves with smooth skin.

[0011] The use of the multi-effect extract of papaya leaves obtained by the method in the preparation of antioxidant drugs.

[0012] The use of the multi-effect extract of papaya leaves obtained by the method in the preparation of antibacterial drugs.

[0013] The use of the multi-effect extract of papaya leaves obtained by the method in the preparation of hypoglycemic drugs.

[0014] The use of the multi-effect extract of papaya leaves obtained by the method in the preparation of topical formulations for anti-inflammatory and anti-inflammatory drugs.

[0015] The use of the multi-effect extract of papaya leaves obtained by the method in the preparation of personal care products.

[0016] The use of the multi-effect extract of papaya leaves obtained by the method in the preparation of food or pharmaceutical additives.

[0017] This invention discloses a method for preparing a multi-effect extract from the leaves of *Papaya sclerotium* (smooth-skinned papaya). This method fully utilizes and develops the resources of *Papaya sclerotium* from Heze, providing a method for preparing a multi-effect extract from the leaves and its applications. The preparation process of this extract is simple, safe, reliable, has a high yield, and low cost. The *Papaya sclerotium* leaf extract obtained by this method can be used in the preparation of drugs with antioxidant, antibacterial, and hypoglycemic effects; it can also be used in the preparation of personal care products; or in the preparation of topical anti-inflammatory and anti-inflammatory preparations; or as a food or pharmaceutical additive.

[0018] This invention discloses a method for preparing a multi-effect refined extract from the leaves of papaya. The extract is concentrated under reduced pressure, dried, pulverized, and sieved using conventional methods to obtain a solid refined extract powder. The yield of the refined extract from the papaya leaves is calculated using conventional methods. The antioxidant activity of the extract is determined using conventional methods, specifically its DPPH free radical scavenging effect. The antibacterial activity of the extract is determined using conventional methods, specifically its inhibition of Staphylococcus aureus. The hypoglycemic activity of the extract is determined using conventional methods, specifically its inhibition of protein tyrosine phosphatase 1B (PTP1B). The active components of the extract are analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry. Attached Figure Description

[0019] Figure 1 This is the TIC diagram of the multi-effect extract of papaya leaves in positive ion mode according to the present invention;

[0020] Figure 2 This is the TIC diagram of the multi-effect extract of papaya leaves under negative ion mode of the present invention;

[0021] Figure 3 This is a TIC image of a blank sample in positive ion mode according to the present invention;

[0022] Figure 4 This is a TIC image of a blank sample in the negative ion mode of this invention. Detailed Implementation

[0023] Example 1

[0024] Take 1.0 kg of dried, smooth-skinned papaya leaves, add 10 times the volume of 30% ethanol solution, and directly reflux extract three times at 80℃ for 1.5 hours each time. Combine the extracts.

[0025] The extract was filtered through a 260-mesh filter cloth, concentrated under reduced pressure at 50°C, dried at 40°C, and pulverized to obtain a multi-effect refined extract of papaya leaves with smooth skin.

[0026] Example 2

[0027] Take 1.0 kg of dried, smooth-skinned papaya leaves, add 16 times the volume of 50% ethanol solution, soak for 2 hours, reflux extract once, extract temperature 50℃, time 0.5 hours, to obtain the extract;

[0028] The extract was filtered through a 260-mesh filter cloth, concentrated under reduced pressure at 50°C, dried at 40°C, and pulverized to obtain a multi-effect refined extract of papaya leaves with smooth skin.

[0029] Example 3

[0030] Take 1.0 kg of dried, smooth-skinned papaya leaves, add 20 times the volume of 70% ethanol solution, soak for 3 hours, reflux extract once at 60℃ for 2 hours to obtain the extract;

[0031] The extract was filtered through a 260-mesh filter cloth, concentrated under reduced pressure at 50°C, dried at 40°C, and pulverized to obtain a multi-effect refined extract of papaya leaves with smooth skin.

[0032] Example 4

[0033] Take 1.0 kg of dried, smooth-skinned papaya leaves, add 30 times their volume of 60% ethanol solution, soak for 1 hour, and then extract twice with ultrasound at 65℃, 0.5 hours each time. Combine the extracts.

[0034] The extract was filtered through a 260-mesh filter cloth, concentrated under reduced pressure at 50°C, dried at 40°C, and pulverized to obtain a multi-effect refined extract of papaya leaves with smooth skin.

[0035] Example 5

[0036] Take 1.0 kg of dried, smooth-skinned papaya leaves, add 24 times the volume of 80% ethanol solution, and directly reflux extract twice at 100℃ for 2 hours each time. Combine the extracts.

[0037] The extract was filtered through a 260-mesh filter cloth, concentrated under reduced pressure at 50°C, dried at 40°C, and pulverized to obtain a multi-effect refined extract of papaya leaves with smooth skin.

[0038] Example 6

[0039] Take 1.0 kg of dried, smooth-skinned papaya leaves, add 30 times the volume of 95% ethanol solution, soak for 3 hours, and then extract by ultrasonication 3 times at 30℃ for 1 hour each time. Combine the extracts.

[0040] The extract was filtered through a 260-mesh filter cloth, concentrated under reduced pressure at 50°C, dried at 40°C, and pulverized to obtain a multi-effect refined extract of papaya leaves with smooth skin.

[0041] Example 7

[0042] After weighing the multi-effect extracts of papaya leaves obtained in Examples 1-6, the yield of the extracts was calculated as shown in Table 1. The formula for calculating the yield is as follows:

[0043]

[0044] Table 1. Yield of multi-effect extract from Papaya leaves (smooth-skinned papaya)

[0045]

[0046] Example 8

[0047] The antioxidant activity of the multi-effect extracts of papaya leaves obtained in Examples 1-6 was determined, specifically the scavenging rate against 2,2-biphenyl-1-picrylhydrazyl (DPPH) free radicals, to assess the antioxidant capacity of the samples. The specific determination method is as follows:

[0048] Dissolve an appropriate amount of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in anhydrous ethanol to prepare a stock solution; dilute the stock solution with anhydrous ethanol to a concentration of 2 × 10⁻⁶. -4 mol / L; Take 100 μL of samples at different dilutions and DPPH dilution (concentration 2 × 10⁻⁶). -4 Mix the 1 mol / L (mol / L) solution thoroughly and react at room temperature in the dark for 30 min. Measure the absorbance (A) at 515 nm. Simultaneously, measure the absorbance (A) of the blank sample without DPPH. c The absorbance (A0) of DPPH with or without sample (100 μL of 50% ethanol was used instead of sample) was measured in parallel three times, with vitamin C as a positive control.

[0049] The formula for calculating the clearance rate is:

[0050]

[0051] The IC50 of the multi-effect extract of papaya leaves against DPPH free radical scavenging was calculated using Origin software. 50 The values ​​are shown in Table 2:

[0052] Table 2. Scavenging effect of multi-effect extracts from Papaya leaves on DPPH free radicals.

[0053]

[0054]

[0055] Example 9

[0056] The hypoglycemic activity of the multi-effect extracts of papaya leaves obtained in Examples 1-6 was screened by determining their inhibitory effect on protein tyrosine phosphatase 1B (PTP1B). The specific method is as follows:

[0057] The experiment used disodium p-nitrophenyl phosphate (PNPP) as a substrate. The activity of protein tyrosine phosphatase 1B (PTP1B) was determined by detecting changes in the product disodium p-nitrophenyl phosphate. The total reaction volume was 200 μL: 179 μL of phosphate buffer solution containing protein tyrosine phosphatase 1B (PTP1B) protein solution, 1 μL of the test sample, and 20 μL of 35 mmol / L disodium p-nitrophenyl phosphate (PNPP). After shaking and mixing, the reaction was carried out at room temperature for 30 min, and the absorbance was measured at 405 nm. The enzyme-free solution system was used as a blank, and 3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethyl-benzofuran-6-sulfonic acid-(4-(thiazol-2-sulfonamide)-aniline was used as a positive control.

[0058] Test sample: Take an appropriate amount of the multi-effect extract of papaya leaves, weigh it accurately, dissolve it in DMSO, and shake well;

[0059] Formula for calculating inhibition rate:

[0060]

[0061] The IC50 of the multi-effect extract of papaya leaves on protein tyrosine phosphatase 1B (PTP1B) was calculated using Origin software. 50 The values ​​are shown in Table 3:

[0062] Table 3. Inhibitory effect of multi-effect extracts from Papaya leaves on PTP1B.

[0063]

[0064] Example 10

[0065] The size of the inhibition zone of the multi-effect extract of papaya leaves obtained in Examples 1-6 against Staphylococcus aureus was determined to assess the antibacterial ability of the multi-effect extract. The specific method is as follows:

[0066] The antibacterial activity of the samples was detected using the paper disc agar diffusion method. Gram-positive Staphylococcus aureus was cultured overnight at 37°C and 150 rpm. The overnight culture was diluted 1000 times and then evenly spread onto a solid medium. A certain amount of the multi-effect extract of papaya leaves was dissolved in DMSO and spotted onto sterile paper discs (5.0 mg / disc). The discs were dried under a fan in a clean bench. The dried discs were then placed on the inoculated culture medium and incubated at 37°C for 24 hours. The diameter of the inhibition zone was then measured. Physiological saline was used as a negative control. The antibacterial effects are shown in Table 4.

[0067] Table 4. Antibacterial effects of multi-effect extracts from Papaya leaves (smooth-skinned papaya)

[0068]

[0069] Example 11

[0070] The multi-effect extracts of papaya leaves obtained in Examples 1-6 were analyzed using Thermo QE plus liquid chromatography-tandem high-resolution mass spectrometry and Compound Discover data processing software to determine the chemical components in the extracts and identify their pharmacodynamic material basis. The specific determination methods are as follows:

[0071] Preparation of test solution:

[0072] Take 200 mg of the purified extract, add 10 mL of 50% methanol aqueous solution (v:v, methanol:water = 50:50), sonicate for 30 min, filter through a 0.22 μm microporous membrane, and treat the blank sample under the same conditions.

[0073] Experimental conditions:

[0074] Liquid phase conditions:

[0075] Chromatographic column: ACQUITY UPLC HSS T3 column (2.1×100mm, 1.8μm);

[0076] Column temperature: 35℃; Injection volume: 10μL; Flow rate: 0.3mL / min;

[0077] Mobile phase: A is deionized water containing 0.1% formic acid; B is acetonitrile containing 0.1% formic acid. Gradient elution is used, and the specific gradient elution conditions are as follows:

[0078] Table 5 Elution conditions of the liquid mobile phase

[0079]

[0080] Mass spectrometry conditions:

[0081] Mass spectrometry data acquisition was performed using Q Exactive Orbitrap high-resolution mass spectrometry, with Full MS-ddMS detection mode. 2 Positive and negative ion modes were scanned separately, with a scan range of m / z 100-1200, MS. 1 Resolution set to 70000, MS 2Resolution set to 17500, ion source voltage to 3.2kV, capillary ion transfer tube temperature to 320℃, auxiliary gas heater temperature to 350℃, sheath gas flow rate to 40L / min, auxiliary gas flow rate to 15L / min, AGC Target to 1e6, TopN to 5, and trigger MS. 2 The collision energy for scanning was determined using a stepped fragmentation voltage NCE, set to 30, 40, and 50.

[0082] result:

[0083] TIC scan images: TIC scan images of the extract of papaya leaf in positive ion mode and negative ion mode are shown below. Figure 1 and Figure 2 As shown;

[0084] Component analysis: Characteristic peaks were extracted from raw RAW mass spectrometry data using Compound Discover 3.2 software. The mass deviation for characteristic peak elemental matching, molecular formula prediction, and isotope distribution matching was set to within 5 ppm. Characteristic peak identification was performed using the mzcloud online database and the locally built mzVault database of traditional Chinese medicine natural products. Positive results were selected based on a mass deviation < 5 ppm, conformity to isotope distribution, and a matching score > 70 points in the mzVault best match database. After manual confirmation and duplicate result removal, a total of 106 compounds were identified, of which 60 had a relative content of 0.08% or higher, with a total relative content of 97.948%. Specific results are shown in Table 6.

[0085] Table 6. Information on compounds identified in the multi-effect extract of Papaya leaves.

[0086]

[0087]

[0088]

[0089]

[0090] The multi-effect extract of papaya leaves obtained by the method described in this invention can be used in the preparation of drugs with antioxidant, antibacterial, and hypoglycemic effects; or in the preparation of personal care products with antioxidant and antibacterial effects; or in the preparation of topical antibacterial and anti-inflammatory preparations; or in the preparation of food or pharmaceutical additives.

Claims

1. A method for preparing a multi-effect extract from the leaves of papaya with smooth peel, characterized in that... The method is performed according to the following steps: a. Soak the leaves of the smooth-skinned papaya in 10-30 times their volume of 30-95% ethanol solution for 0-3 hours, and then extract them by ultrasound or reflux. The ultrasound extraction temperature is 35-65℃ and the extraction time is 0.5-1 hour; the reflux extraction temperature is 50-100℃ and the extraction time is 0.5-2 hours. Extract 1-3 times to obtain the extract; b. Filter the extract obtained in step a through a 260-mesh filter cloth, concentrate the filtrate under reduced pressure at 50°C, dry it at 40°C, and pulverize it to obtain a multi-effect refined extract of papaya leaves with smooth skin.

2. The use of the multi-effect extract of papaya leaves obtained by the method according to claim 1 in the preparation of antioxidant drugs.

3. The use of the multi-effect extract of papaya leaves obtained by the method according to claim 1 in the preparation of antibacterial drugs.

4. The use of the multi-effect extract of papaya leaves obtained by the method according to claim 1 in the preparation of hypoglycemic drugs.

5. The use of the multi-effect extract of papaya leaves obtained by the method according to claim 1 in the preparation of topical formulations for anti-inflammatory and de-inflammatory drugs.

6. The use of the multi-effect extract of papaya leaves obtained by the method according to claim 1 in the preparation of personal care products.

7. The use of the multi-effect extract of papaya leaves obtained by the method according to claim 1 in the preparation of food or pharmaceutical additives.

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