Plateau rose extract, preparation method thereof and application thereof in anti-aging products

Abstract

The application discloses a kind of plateau rose extract and its preparation method and application in anti-aging product;First, using macroporous resin has the characteristics of selective adsorption and desorption to different polarity compounds, active ingredients in plateau rose extract are efficiently separated and enriched, and different polarity parts are obtained;After that, using succinate dehydrogenase in living cell mitochondria reduces exogenous MTT into blue-violet formazan crystal, and the survival rate and metabolic activity of cells are indirectly reflected by determining absorbance value, and the protective effect of each separated component on cell viability under oxidative stress and other aging models is systematically evaluated;The application successfully discovers active substances from plateau rose that can significantly improve cell viability, not only fills the gap of plateau rose anti-aging research, but also has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to a plateau rose extract and its preparation method, as well as its application in anti-aging products. Background Technology

[0002] Rose( Rosa rugosa Thunb. The Chinese Pharmacopoeia defines rose as the dried flower bud of the rose plant (Rosa rugosa), a member of the Rosaceae family. It has a long history of cultivation and is highly valued for its ornamental, edible, and medicinal uses. The Compendium of Materia Medica (Bencao Gangmu Shiyi) records its effects of regulating qi and relieving depression, harmonizing blood and relieving pain; it is commonly used to treat liver and stomach pain, irregular menstruation, and other ailments. Roses are rich in over 300 chemical components, including volatile oils, flavonoids, phenolic acids, and polysaccharides. Among these, polysaccharides exhibit significant antioxidant, anti-aging, and anti-inflammatory pharmacological activities. In recent years, there has been considerable research on the Damask rose, which originates from the Middle East and Central Asia.

[0003] Xiaojin County in Sichuan Province boasts a unique semi-mountainous plateau climate, enabling the successful introduction of Damask roses. The survival rate of these roses exceeds 95%, and the flowering period is extended to over three and a half months, setting a superior example for the cultivation of this variety in plateau regions. The planting area has reached 15,000 mu (approximately 1,000 hectares), making it the largest plateau rose base in China. In recent years, research on the chemical composition of Xiaojin plateau roses has yielded results, including component identification and the discovery of high-altitude characteristic components. A component spectrum and quality control system based on modern analytical techniques has been established. Advanced techniques such as gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS / MS) have identified volatile oils (such as geraniol, citronellol, and phenylethyl alcohol), flavonoids, phenolic substances, polysaccharides, and amino acids in the extracts. Simultaneously, strong ultraviolet radiation and diurnal temperature variation significantly enrich bioactive substances such as polyphenols, flavonoids, and anthocyanins, exhibiting remarkable antioxidant and anti-aging activities.

[0004] Aging is a systemic and progressive functional decline that occurs in organisms over time, manifesting as morphological (e.g., loose skin, muscle atrophy), physiological, and molecular-level degeneration. Its root cause lies in the accumulation of cellular damage and the imbalance of molecular homeostasis. Aging is not only a marker of bodily aging but also a core risk factor for chronic diseases such as cardiovascular disease, cancer, and neurodegenerative diseases. During this process, senescent cells release aging-associated secretory phenotypes (SASPs), triggering inflammation and accelerating tissue damage. Therefore, finding safe and effective natural active substances that can intervene in cellular damage and homeostasis imbalance has become a research hotspot for delaying aging and extending healthy lifespan. This invention uses extracts from roses rich in polyphenols, polysaccharides, and other active ingredients to explore their potential effects and mechanisms in delaying aging.

[0005] Currently, there is a lack of systematic research reports on the anti-aging effects of highland roses, and their unique bioactive components and mechanisms of action still need to be explored in depth. To address this research gap, this invention focuses on highland roses grown in extreme environments such as high altitudes and strong ultraviolet radiation, aiming to develop a safe and highly effective natural anti-aging active substance. Summary of the Invention

[0006] This invention utilizes reflux extraction and macroporous resin separation to obtain a plateau rose extract with anti-aging activity. This plateau rose extract can be used to prepare anti-aging drugs or health products.

[0007] The technical solution of the present invention is as follows: An extract of highland rose was prepared by the following method: (1) Using dried petals of plateau rose as raw material, the petals were ground into powder and extracted by reflux with 70%~75% ethanol by volume. The extract was then collected by hot filtration, the solvent was recovered and dried to obtain crude ethanol extract. The preferred ratio of dried petals of highland roses to 70%~75% ethanol is 1:8~10 g / mL. The optimal reflux extraction is performed three times, with each reflux extraction lasting 1.5 to 2 hours. (2) The crude ethanol extract obtained in step (1) was uniformly dispersed in water and added to an AB-8 macroporous resin column. After static adsorption, it was eluted sequentially with 0%~3%, 8%~13%, 28%~33%, 48%~53%, and 95%~100% ethanol by volume fraction, respectively. The elution flow rate was 1~1.5 BV / h, and the elution volume of ethanol for each concentration gradient was 3~5 BV (rinse until the eluent is nearly colorless, and then elute with the next concentration of ethanol). The eluent of each gradient was collected, concentrated under reduced pressure to a paste, and vacuum dried to obtain the eluted fractions (powder) of 0%~3%, 8%~13%, 28%~33%, 48%~53%, and 95%~100% ethanol, namely: plateau rose extract; Before use, AB-8 macroporous resin undergoes the following pretreatment: AB-8 macroporous resin is soaked in 95% ethanol for 12-36 hours and then packed into a glass chromatography column. It is then washed with 95% ethanol until the effluent is clear; then washed with pure water until the effluent has no alcohol odor; next, the resin layer is washed and soaked with 3-5% hydrochloric acid and 3-5% sodium hydroxide solution for 2-4 hours; finally, it is repeatedly washed with pure water for about 2 column volumes until the effluent is neutral, so that the resin in the column is fully compacted and impurities are removed. The preferred crude ethanol extract is uniformly dispersed in water at a material-to-liquid ratio of 1:10~15 g / mL. The preferred static adsorption time is 30 min.

[0008] The plateau rose extract of this invention can be used to prepare anti-aging drugs or health products.

[0009] This invention uses the following method to detect the anti-aging activity of highland rose extract: HepG2 cells in the logarithmic growth phase were selected at a ratio of 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates and allowed to adhere for 24 h. The experiment consisted of a control group, a model group (40 mg / mL D-galactose), and an experimental group (40 mg / mL D-galactose + different concentrations of 0%–100% ethanol eluents), with six replicates in each group. Cell viability was measured using MTT assay, and the effects of extracts eluted with different concentrations of ethanol on D-galactose-induced HepG2 cells were analyzed.

[0010] Based on the analysis, the highland rose extracts eluted with 8%~13% and 28%~33% ethanol were preferred.

[0011] The beneficial effects of this invention are: This invention innovatively employs a strategy combining macroporous resin separation with MTT assay for cell viability: First, the selective adsorption and desorption properties of macroporous resin for compounds of different polarities are utilized to efficiently separate and enrich the active ingredients in plateau rose extract, obtaining fractions of different polarities; then, succinate dehydrogenase in the mitochondria of living cells is used to reduce exogenous MTT to blue-purple formazan crystals, and the absorbance value is measured to indirectly reflect cell viability and metabolic activity, systematically evaluating the protective effect of each separated component on cell viability under aging models such as oxidative stress.

[0012] This invention employs a rigorous scientific approach and has successfully identified active substances in highland roses that can significantly enhance cell vitality. This not only fills a gap in research on the anti-aging properties of highland roses but also has broad application prospects. Attached Figure Description

[0013] Figure 1 : Liquid chromatogram of the ethanol extract of *Rosa simonii*.

[0014] Figure 2 Chromatogram of 10% ethanol extract of *Rosa simonii*.

[0015] Figure 3 Chromatogram of 30% ethanol extract of *Rosa simonii*.

[0016] Figure 4 Effects of different concentrations of D-galactose on the viability of HepG2 cells at different times; A: Induction of different concentrations of D-galactose for 24 hours to establish the model; B: Induction of different concentrations of D-galactose for 48 hours to establish the model.

[0017] Figure 5 The protective effect of different concentrations of ethanol eluents on D-galactose-induced senescence in HepG2 cells; A: 10% ethanol layer; B: 8% ethanol layer; C: 9% ethanol layer; D: 11% ethanol layer; E: 30% ethanol layer; F: 50% ethanol layer; G: 100% ethanol layer; The model group was 40 mg / mL D-galactose. Detailed Implementation

[0018] The present invention is further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0019] In the following embodiments, The plateau roses are from Qingduoxiang Wild Resources Development Co., Ltd., Jiajin Mountain, Xiaojin County, Sichuan Province; the AB-8 macroporous resin is from Beijing Solarbio Technology Co., Ltd.

[0020] Example 1

[0021] 1. Sample preparation

[0022] (1) Take 100.0 g of dried plateau rose powder into a round bottom flask, add 1 L of 75% ethanol, connect a condenser to the top, with water entering at the bottom and exiting at the top, heat under reflux for 1.5 h, filter while hot, retain the filtrate, add 1 L of 75% ethanol to the residue, heat under reflux for 1.5 h, filter, retain the filtrate, add 1 L of 75% ethanol to the residue, heat under reflux for 1.5 h; collect the filtrate (extract) and combine them.

[0023] (2) The solvent was recovered from the extract using a rotary evaporator, and the concentrate was freeze-dried to obtain 36.75 g of crude ethanol extract; the obtained crude extract was analyzed by high performance liquid chromatography (HPLC) at a concentration of 5 mg / mL, such as... Figure 1 ; Chromatographic conditions: High performance liquid chromatography (HPLC) was performed using an H&E SP ODS-A C18 column (250×4.6mm, 5µm) at 30℃. The mobile phase consisted of methanol (A) and 0.1% formic acid aqueous solution, with gradient elution: 0-8 min: 8%-15% A; 8-16 min: 15-20% A; 16-25 min: 20%-22% A; 25-35 min: 22%-25% A; 35-45 min: 25%-30% A; 45-55 min: 30%-37% A; 55-65 min: 37%-45% A; 65-75 min: 45%-50% A; 75-85 min: 50%-60% A; 85-95 min: 60%-75% A; 95-105 min: 75%-95% A. The flow rate was 1.0 mL / min, the detection wavelength was 280 nm, and the injection volume was 20 μL.

[0024] (3) Weigh 3.0 g of the crude extract powder, dissolve it in 30 mL of distilled water, and slowly add it dropwise to an AB-8 macroporous resin column (25 cm high, 4 cm diameter). Let it stand for half an hour to adsorb. Then, perform gradient elution with 0%, 10%, 30%, 50%, and 100% ethanol solutions, respectively, at a flow rate of 1 BV / h. The elution volume for each concentration gradient is 3 BV. Collect the eluent for each gradient, recover the solvent under vacuum, and dry it. The following eluents were obtained: 812.4 mg of 0% ethanol, 256.3 mg of 10% ethanol, 378.4 mg of 30% ethanol, 213.1 mg of 50% ethanol, and 64.6 mg of 100% ethanol. Then, the 10% ethanol eluent was analyzed by HPLC at a concentration of 5 mg / mL (under the same conditions). Figure 1 ),like Figure 2 .

[0025] 2. Cell viability assay

[0026] (1) Cell resuscitation

[0027] HepG2 cells were revived in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a 37 ℃, 5% CO2 cell culture incubator. Cells in the logarithmic growth phase were used for experiments.

[0028] (2) MTT assay for cell viability

[0029] HepG2 cells were fed at a rate of 1×10 5 Cells were seeded at a density of [number] cells / mL in 96-well plates, with 100 μL of cell suspension added to each well and cultured for 24 hours. After complete cell attachment, cells were treated with different concentrations of 0%–100% ethanol effluent (0–200 μg / mL) for 24 hours. The toxicity of different ethanol layers to HepG2 cells was assessed using the MTT assay. Ultimately, ethanol effluent concentrations of 0–150 μg / mL were selected for subsequent experiments.

[0030] (3) Screening of D-galactose concentration

[0031] HepG2 cells were treated with different concentrations of D-galactose for 24 and 48 hours, respectively. Cell viability was assessed by MTT assay. The results are shown below. Figure 4 .Depend on Figure 4It was found that different concentrations of D-galactose were toxic to HepG2 cells, and the cell viability decreased in a concentration-dependent manner; however, the overall survival rate at 48 hours was low (below 50%). Reference studies have found that a cell viability of around 60% is suitable for evaluating the protective effect of cell-active substances. In this invention, the viability of HepG2 cells was 64.95% when the D-galactose concentration was 40 mg / mL; therefore, it was ultimately determined that 40 mg / mL D-galactose was used to induce the cell model for 24 hours.

[0032] 3. Protective effect of 10% ethanol layer compounds on model cells

[0033] (1) Select HepG2 cells in the logarithmic growth phase at a concentration of 1×10⁻⁶. 5 The cells were seeded at a density of 100 μL / mL in 96-well plates and allowed to adhere to the plate for 24 hours.

[0034] (2) The experiment was divided into a control group, a blank group, a model group (40 mg / mL D-galactose), and experimental groups E-25 (40 mg / mL D-galactose + 25 μg / mL 10% ethanol layer compound), E-50 (40 mg / mL D-galactose + 50 μg / mL 10% ethanol layer compound), E-75 (40 mg / mL D-galactose + 75 μg / mL 10% ethanol layer compound), E-100 (40 mg / mL D-galactose + 100 μg / mL 10% ethanol layer compound), and E-150 (40 mg / mL D-galactose + 150 μg / mL 10% ethanol layer compound). Each group had 6 replicates. Cell viability was determined using the MTT assay. 20 μL MTT reagent was added to each well, and after incubation for 4 h, the absorbance was measured at 570 nm using a microplate reader. The protective effects of different concentrations of 10% ethanol layer compounds on D-galactose-induced HepG2 cells were analyzed.

[0035] (3) The cell-protective effect is determined by the following formula.

[0036] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0037] As — Absorbance of the experimental or model group at 570 nm

[0038] Ac – Absorbance of the control group at 570 nm

[0039] Ab – Absorbance of the blank group at 570nm

[0040] The results are as follows Figure 5As shown in Figure A, the 10% ethanol layer compound has a significant protective effect against D-galactose-induced HepG2 cells. Compared with the D-galactose model group, the cell survival rate is significantly improved. For example, at a concentration of 100 μg / mL, the cell survival rate can be restored to 85.6%.

[0041] Example 2

[0042] 1. Sample preparation

[0043] (1) Take 100.0 g of dried plateau rose powder into a round bottom flask, add 0.8 L of 75% ethanol, connect a condenser to the top, with water entering at the bottom and exiting at the top, heat under reflux for 1.5 h, filter, retain the filtrate, add 0.8 L of 75% ethanol to the filter residue, heat under reflux for 1.5 h, filter, retain the filtrate, add 0.8 L of 75% ethanol to the filter residue, heat under reflux for 1.5 h; collect the filtrate (extract) and combine them.

[0044] (2) The solvent was recovered from the extract by rotary evaporator, and the concentrate was freeze-dried to obtain 33.11 g of crude ethanol extract.

[0045] (3) Weigh 3.0 g of the extract powder (crude extract), dissolve it in 40 mL of distilled water, and slowly add it dropwise into an AB-8 macroporous resin column (chromatographic column height 25 cm, diameter 4 cm). Let it stand for half an hour for adsorption. Then, use 0%, 8%, 28%, 48% and 95% ethanol to perform gradient elution in sequence. The elution flow rate is 1 BV / h, and the elution volume of ethanol for each concentration gradient is 3 BV. Collect the eluent of each gradient, recover the solvent under vacuum and dry it. The eluents obtained are 793.81 mg of 0% ethanol, 238.92 mg of 8% ethanol, 362.63 mg of 28% ethanol, 195.46 mg of 48% ethanol, and 65.13 mg of 95% ethanol.

[0046] 2. Cell viability assay

[0047] (1) Cell resuscitation

[0048] HepG2 cells were revived in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a 37 ℃, 5% CO2 cell culture incubator. Cells in the logarithmic growth phase were used for experiments.

[0049] (2) MTT assay for cell viability

[0050] HepG2 cells were loaded at a rate of 1 × 10⁻⁶. 5Cells were seeded at a density of [number] cells / mL in 96-well plates, with 100 μL of cell suspension added to each well and cultured for 24 hours. After complete cell attachment, cells were treated with different concentrations of 0%–100% ethanol effluent (0–200 μg / mL) for 24 hours. The toxicity of different ethanol layers to HepG2 cells was assessed using the MTT assay. Ultimately, ethanol effluent concentrations of 0–150 μg / mL were selected for subsequent experiments.

[0051] (3) Screening of D-galactose concentration

[0052] HepG2 cells were treated with different concentrations of D-galactose for 24 and 48 hours, respectively. Cell viability was assessed by MTT assay. The results are shown below. Figure 4 .Depend on Figure 4 It was found that different concentrations of D-galactose were toxic to HepG2 cells, and the cell viability decreased in a concentration-dependent manner; however, the overall survival rate at 48 hours was low (below 50%). Reference studies have found that a cell viability of around 60% is suitable for evaluating the protective effect of cell-active substances. In this invention, the viability of HepG2 cells was 64.95% when the D-galactose concentration was 40 mg / mL; therefore, it was ultimately determined that 40 mg / mL D-galactose was used to induce the cell model for 24 hours.

[0053] 3. Protective effect of 10% ethanol layer compounds on model cells

[0054] (1) Select HepG2 cells in the logarithmic growth phase at a concentration of 1×10⁻⁶. 5 The cells were seeded at a density of 100 μL / mL in 96-well plates and allowed to adhere to the plate for 24 hours.

[0055] (2) The experiment was divided into a control group, a blank group, a model group (40 mg / mL D-galactose), and experimental groups E-25 (40 mg / mL D-galactose + 25 μg / mL 8% ethanol layer compound), E-50 (40 mg / mL D-galactose + 50 μg / mL 8% ethanol layer compound), E-75 (40 mg / mL D-galactose + 75 μg / mL 8% ethanol layer compound), E-100 (40 mg / mL D-galactose + 100 μg / mL 8% ethanol layer compound), and E-150 (40 mg / mL D-galactose + 150 μg / mL 8% ethanol layer compound). Each group had 6 replicates. Cell viability was measured using MTT assay. 20 μL MTT reagent was added to each well, and after incubation for 4 h, the absorbance was measured at 570 nm using a microplate reader to analyze the protective effect of different concentrations of 8% ethanol layer compound on D-galactose-induced HepG2 cells.

[0056] (3) The cell-protective effect is determined by the following formula.

[0057] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0058] As — Absorbance of the experimental or model group at 570 nm

[0059] Ac – Absorbance of the control group at 570 nm

[0060] Ab – Absorbance of the blank group at 570nm

[0061] The results are as follows Figure 5 As shown in Figure B, the 8% ethanol layer compound has a significant protective effect against D-galactose-induced HepG2 cells. Compared with the D-galactose model group, the cell survival rate is significantly improved. For example, at a concentration of 100 μg / mL, the cell survival rate can be restored to 81.95%.

[0062] Example 3

[0063] 1. Sample preparation

[0064] (1) Take 100.0 g of dried plateau rose powder into a round bottom flask, add 0.9 L of 75% ethanol, connect a condenser to the top, with water entering at the bottom and exiting at the top, heat under reflux for 1.2 h, filter, retain the filtrate, add 0.9 L of 75% ethanol to the residue, heat under reflux for 1.2 h, filter, retain the filtrate, add 0.9 L of 75% ethanol to the residue, heat under reflux for 1.2 h; collect the filtrate (extract) and combine them.

[0065] (2) The solvent was recovered from the extract by rotary evaporator, and the concentrate was freeze-dried to obtain 34.47 g of crude ethanol extract.

[0066] (3) Weigh 3.0 g of the extract powder (crude extract), dissolve it in 35 mL of distilled water, and slowly add it dropwise into an AB-8 macroporous resin column (chromatographic column height 25 cm, diameter 4 cm). Let it stand for half an hour to adsorb, and then perform gradient elution with 0%, 9%, 29%, 49% and 98% ethanol concentrations, respectively. The elution flow rate is 1 BV / h, and the elution volume of ethanol for each concentration gradient is 3 BV. Collect the eluent of each gradient, recover the solvent under vacuum and dry it. The following results were obtained: 807.29 mg of 0% ethanol eluent, 243.28 mg of 9% ethanol eluent, 364.34 mg of 29% ethanol eluent, 198.41 mg of 49% ethanol eluent, and 63.86 mg of 98% ethanol eluent.

[0067] 2. Cell viability assay

[0068] (1) Cell resuscitation

[0069] HepG2 cells were revived in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a 37 ℃, 5% CO2 cell culture incubator. Cells in the logarithmic growth phase were used for experiments.

[0070] (2) MTT assay for cell viability

[0071] HepG2 cells were fed at a rate of 1×10 5 Cells were seeded at a density of [number] cells / mL in 96-well plates, with 100 μL of cell suspension added to each well and cultured for 24 hours. After complete cell attachment, cells were treated with different concentrations of 0%–100% ethanol effluent (0–200 μg / mL) for 24 hours. The toxicity of different ethanol layers to HepG2 cells was assessed using the MTT assay. Finally, ethanol effluents at concentrations of 0–150 μg / mL were used for subsequent experiments.

[0072] (3) Screening of D-galactose concentration

[0073] HepG2 cells were treated with different concentrations of D-galactose for 24 and 48 hours, respectively. Cell viability was assessed by MTT assay. The results are shown below. Figure 4 .Depend on Figure 4 It was found that different concentrations of D-galactose were toxic to HepG2 cells, and the cell viability decreased in a concentration-dependent manner; however, the overall survival rate at 48 hours was low (below 50%). Reference studies have found that a cell viability of around 60% is suitable for evaluating the protective effect of cell-active substances. In this invention, the viability of HepG2 cells was 64.95% when the D-galactose concentration was 40 mg / mL; therefore, it was ultimately determined that 40 mg / mL D-galactose was used to induce the cell model for 24 hours.

[0074] 3. Protective effect of 9% ethanol layer compounds on model cells

[0075] (1) Select HepG2 cells in the logarithmic growth phase at a concentration of 1×10⁻⁶. 5 The cells were seeded at a density of 100 μL / mL in 96-well plates and allowed to adhere to the plate for 24 hours.

[0076] (2) The experiment was divided into a control group, a blank group, a model group (40 mg / mL D-galactose), and experimental groups E-25 (40 mg / mL D-galactose + 25 μg / mL 9% ethanol layer compound), E-50 (40 mg / mL D-galactose + 50 μg / mL 9% ethanol layer compound), E-75 (40 mg / mL D-galactose + 75 μg / mL 9% ethanol layer compound), E-100 (40 mg / mL D-galactose + 100 μg / mL 9% ethanol layer compound), and E-150 (40 mg / mL D-galactose + 150 μg / mL 9% ethanol layer compound). Each group had 6 replicates. Cell viability was measured using MTT assay. 20 μL MTT reagent was added to each well, and after incubation for 4 h, the absorbance was measured at 570 nm using a microplate reader to analyze the protective effect of different concentrations of 9% ethanol layer compound on D-galactose-induced HepG2 cells.

[0077] (3) The cell-protective effect is determined by the following formula.

[0078] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0079] As — Absorbance of the experimental or model group at 570 nm

[0080] Ac – Absorbance of the control group at 570 nm

[0081] Ab – Absorbance of the blank group at 570nm

[0082] The results are as follows Figure 5 As shown in Figure C, the 9% ethanol layer compound has a significant protective effect against D-galactose-induced HepG2 cells. Compared with the D-galactose model group, the cell survival rate is significantly improved, and the cell survival rate can be restored to 81.89% at a concentration of 100 μg / mL.

[0083] Example 4

[0084] 1. Sample preparation

[0085] (1) Take 100.0 g of dried plateau rose powder into a round bottom flask, add 1 L of 70% ethanol, connect a condenser to the top, with water entering at the bottom and exiting at the top, heat under reflux for 1.5 h, filter, retain the filtrate, add 1 L of 70% ethanol to the residue, heat under reflux for 1.5 h, filter, retain the filtrate, add 1 L of 70% ethanol to the residue, heat under reflux for 1.5 h; collect the filtrate (extract) and combine them.

[0086] (2) The solvent was recovered from the extract by rotary evaporator, and the concentrate was freeze-dried to obtain 35.48g of crude ethanol extract.

[0087] (3) Weigh 3.0 g of the extract powder (crude extract), dissolve it in 30 mL of distilled water, and slowly add it dropwise into an AB-8 macroporous resin column (the column height is 25 cm and the diameter is 4 cm). Let it stand for half an hour to adsorb, and then perform gradient elution with 0%, 11%, 31%, 51% and 100% ethanol in sequence. The elution flow rate is 1.2 BV / h, and the elution volume of ethanol for each concentration gradient is 4 BV. Collect the eluent of each gradient, recover the solvent under vacuum and dry it. The following results were obtained: 803.91 mg of 0% ethanol eluent, 239.57 mg of 11% ethanol eluent, 359.37 mg of 31% ethanol eluent, 198.21 mg of 51% ethanol eluent and 62.76 mg of 100% ethanol eluent.

[0088] 2. Cell viability assay

[0089] (1) Cell resuscitation

[0090] HepG2 cells were revived in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a 37 ℃, 5% CO2 cell culture incubator. Cells in the logarithmic growth phase were used for experiments.

[0091] (2) MTT assay for cell viability

[0092] HepG2 cells were fed at a rate of 1×10 5 Cells were seeded at a density of [number] cells / mL in 96-well plates, with 100 μL of cell suspension added to each well and cultured for 24 hours. After complete cell attachment, cells were treated with different concentrations of 0%–100% ethanol effluent (0–200 μg / mL) for 24 hours. The toxicity of different ethanol layers to HepG2 cells was assessed using the MTT assay. Ultimately, ethanol effluent concentrations of 0–150 μg / mL were selected for subsequent experiments.

[0093] (3) Screening of D-galactose concentration

[0094] HepG2 cells were treated with different concentrations of D-galactose for 24 and 48 hours, respectively. Cell viability was assessed by MTT assay. The results are shown below. Figure 4 .Depend on Figure 4It was found that different concentrations of D-galactose were toxic to HepG2 cells, and the cell viability decreased in a concentration-dependent manner; however, the overall survival rate at 48 hours was low (below 50%). Reference studies have found that a cell viability of around 60% is suitable for evaluating the protective effect of cell-active substances. In this invention, the viability of HepG2 cells was 64.95% when the D-galactose concentration was 40 mg / mL; therefore, it was ultimately determined that 40 mg / mL D-galactose was used to induce the cell model for 24 hours.

[0095] 3. Protective effect of 11% ethanol layer compound on model cells

[0096] (1) Select HepG2 cells in the logarithmic growth phase at a concentration of 1×10⁻⁶. 5 The cells were seeded at a density of 100 μL / mL in 96-well plates and allowed to adhere to the plate for 24 hours.

[0097] (2) The experiment was divided into a control group, a blank group, a model group (40 mg / mL D-galactose), and experimental groups E-25 (40 mg / mL D-galactose + 25 μg / mL 11% ethanol layer compound), E-50 (40 mg / mL D-galactose + 50 μg / mL 11% ethanol layer compound), E-75 (40 mg / mL D-galactose + 75 μg / mL 11% ethanol layer compound), E-100 (40 mg / mL D-galactose + 100 μg / mL 11% ethanol layer compound), and E-150 (40 mg / mL D-galactose + 150 μg / mL 11% ethanol layer compound). Each group had 6 replicates. Cell viability was determined using the MTT assay. 20 μL MTT reagent was added to each well, and after incubation for 4 h, the absorbance was measured at 570 nm using a microplate reader. The protective effects of different concentrations of 11% ethanol layer compounds on D-galactose-induced HepG2 cells were analyzed.

[0098] (3) The cell-protective effect is determined by the following formula.

[0099] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0100] As — Absorbance of the experimental or model group at 570 nm

[0101] Ac – Absorbance of the control group at 570 nm

[0102] Ab – Absorbance of the blank group at 570nm

[0103] The results are as follows Figure 5As shown in Figure D, the 11% ethanol layer compound has a significant protective effect against D-galactose-induced HepG2 cells. Compared with the D-galactose model group, the cell survival rate is significantly improved, and the cell survival rate can be restored to 80.83% at a concentration of 50 μg / mL.

[0104] Example 5

[0105] 1. Sample preparation

[0106] (1) Take 100.0 g of dried small golden rose powder into a round bottom flask, add 1 L of 75% ethanol, connect a condenser to the top, with water entering at the bottom and exiting at the top, heat under reflux for 1.5 h, filter, retain the filtrate, add 1 L of 75% ethanol to the residue, heat under reflux for 1.5 h, filter, retain the filtrate, add 1 L of 75% ethanol to the residue, heat under reflux for 1.5 h; collect the filtrate (extract) and combine them.

[0107] (2) The solvent was recovered from the extract using a rotary evaporator, and the concentrate was freeze-dried to obtain 36.751 g of crude ethanol extract; the obtained crude extract was analyzed by HPLC at a concentration of 5 mg / mL, such as... Figure 1 ; (3) Weigh 3.0 g of the crude extract powder, dissolve it in 30 mL of distilled water, and slowly add it dropwise to an AB-8 macroporous resin column (25 cm high and 4 cm in diameter). Let it stand for half an hour to adsorb. Then, perform gradient elution with 0%, 10%, 30%, 50%, and 100% ethanol solutions, respectively, at a flow rate of 1.5 BV / h. The elution volume for each concentration gradient is 5 BV. Collect the eluents for each gradient, recover the solvent under vacuum, and dry them. The eluents obtained are 812.4 mg of 0% ethanol, 256.3 mg of 10% ethanol, 378.4 mg of 30% ethanol, 213.1 mg of 50% ethanol, and 64.6 mg of 100% ethanol. Then, perform HPLC analysis on the obtained 30% ethanol eluent at a concentration of 5 mg / mL (under the same conditions). Figure 1 ),like Figure 3 .

[0108] 2. Cell viability assay

[0109] (1) Cell resuscitation

[0110] HepG2 cells were revived in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a 37 ℃, 5% CO2 cell culture incubator. Cells in the logarithmic growth phase were used for experiments.

[0111] (2) MTT assay for cell viability

[0112] HepG2 cells were fed at a rate of 1×10 5 Cells were seeded at a density of [number] cells / mL in 96-well plates, with 100 μL of cell suspension added to each well and cultured for 24 hours. After complete cell attachment, cells were treated with different concentrations of 0%–100% ethanol effluent (0–200 μg / mL) for 24 hours. The toxicity of different ethanol layers to HepG2 cells was assessed using the MTT assay. Ultimately, ethanol effluent concentrations of 0–150 μg / mL were selected for subsequent experiments.

[0113] (3) Screening of D-galactose concentration

[0114] HepG2 cells were treated with different concentrations of D-galactose for 24 and 48 hours, respectively. Cell viability was assessed by MTT assay. The results are shown below. Figure 4 .Depend on Figure 4 It was found that different concentrations of D-galactose were toxic to HepG2 cells, and the cell viability decreased in a concentration-dependent manner; however, the overall survival rate at 48 hours was low (below 50%). Reference studies have found that a cell viability of around 60% is suitable for evaluating the protective effect of cell-active substances. In this invention, the viability of HepG2 cells was 64.95% when the D-galactose concentration was 40 mg / mL; therefore, it was ultimately determined that 40 mg / mL D-galactose was used to induce the cell model for 24 hours.

[0115] 3. Protective effect of 30% ethanol layer compounds on model cells

[0116] (1) Select HepG2 cells in the logarithmic growth phase at a concentration of 1×10⁻⁶. 5 The cells were seeded at a density of 100 μL / mL in 96-well plates and allowed to adhere to the plate for 24 hours.

[0117] (2) The experiment was divided into a control group, a blank group, a model group (40 mg / mL D-galactose), and experimental groups E-25 (40 mg / mL D-galactose + 25 μg / mL 30% ethanol layer compound), E-50 (40 mg / mL D-galactose + 50 μg / mL 30% ethanol layer compound), E-75 (40 mg / mL D-galactose + 75 μg / mL 30% ethanol layer compound), E-100 (40 mg / mL D-galactose + 100 μg / mL 30% ethanol layer compound), and E-150 (40 mg / mL D-galactose + 150 μg / mL 30% ethanol layer compound). Each group had 6 replicates. Cell viability was determined using the MTT assay. 20 μL MTT reagent was added to each well, and after incubation for 4 h, the absorbance was measured at 570 nm using a microplate reader. The protective effects of different concentrations of 30% ethanol layer compounds on D-galactose-induced HepG2 cells were analyzed.

[0118] (3) The cell-protective effect is determined by the following formula.

[0119] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0120] As — Absorbance of the experimental or model group at 570 nm

[0121] Ac – Absorbance of the control group at 570 nm

[0122] Ab – Absorbance of the blank group at 570nm

[0123] The results are as follows Figure 5 As shown in Figure E, the 30% ethanol layer compound has a significant protective effect against D-galactose-induced HepG2 cells. Compared with the D-galactose model group, the cell survival rate is significantly improved, and the cell survival rate can be restored to 82.26% at a concentration of 50 μg / mL.

[0124] Example 6

[0125] 1. Sample preparation

[0126] (1) Take 100.0 g of dried small golden rose powder into a round bottom flask, add 1 L of 75% ethanol, connect a condenser to the top, with water entering at the bottom and exiting at the top, heat under reflux for 1.5 h, filter, retain the filtrate, add 1 L of 75% ethanol to the residue, heat under reflux for 1.5 h, filter, retain the filtrate, add 1 L of 75% ethanol to the residue, heat under reflux for 1.5 h; collect the filtrate (extract) and combine them.

[0127] (2) The solvent was recovered from the extract using a rotary evaporator, and the concentrate was freeze-dried to obtain 36.751 g of crude ethanol extract; (3) Weigh 3.0 g of the extract powder (crude extract), dissolve it in 30 mL of distilled water, and slowly add it dropwise into an AB-8 macroporous resin column (the column height is 25 cm and the diameter is 4 cm). Let it stand for half an hour to adsorb, and then perform gradient elution with 0%, 10%, 30%, 50% and 100% ethanol in sequence. The elution flow rate is 1 BV / h, and the elution volume of ethanol for each concentration gradient is 4 BV. Collect the eluent of each gradient, recover the solvent under vacuum and dry it. The following results were obtained: 812.4 mg of 0% ethanol eluent, 256.3 mg of 10% ethanol eluent, 378.4 mg of 30% ethanol eluent, 213.1 mg of 50% ethanol eluent and 64.6 mg of 100% ethanol eluent.

[0128] 2. Cell viability assay

[0129] (1) Cell resuscitation

[0130] HepG2 cells were revived in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and cultured in a 37 ℃, 5% CO2 cell culture incubator. Cells in the logarithmic growth phase were used for experiments.

[0131] (2) MTT assay for cell viability

[0132] HepG2 cells were fed at a rate of 1×10 5 Cells were seeded at a density of [number] cells / mL in 96-well plates, with 100 μL of cell suspension added to each well and cultured for 24 hours. After complete cell attachment, cells were treated with different concentrations of 0%–100% ethanol effluent (0–200 μg / mL) for 24 hours. The toxicity of different ethanol layers to HepG2 cells was detected using the MTT assay. According to the results, when the concentrations of 0% and 10% ethanol were 0–200 μg / mL, cell viability did not decrease significantly after 24 hours of treatment. However, when the concentrations of 30%, 50%, and 100% ethanol were 0–100 μg / mL, cell viability decreased slightly, and at concentrations of 100–200 μg / mL, cell viability decreased significantly. For example, in the 30% ethanol layer at 200 μg / mL, the cell survival rate was only 74.26%. Therefore, ethanol effluent concentrations of 0–150 μg / mL were used for subsequent experiments.

[0133] (3) Screening of D-galactose concentration

[0134] HepG2 cells were treated with different concentrations of D-galactose for 24 and 48 hours, respectively. Cell viability was assessed by MTT assay. The results are shown below. Figure 4 .Depend on Figure 4 It was found that different concentrations of D-galactose were toxic to HepG2 cells, and the cell viability decreased in a concentration-dependent manner; however, the overall survival rate at 48 hours was low (below 50%). Reference studies have found that a cell viability of around 60% is suitable for evaluating the protective effect of cell-active substances. In this invention, the viability of HepG2 cells was 64.95% when the D-galactose concentration was 40 mg / mL; therefore, it was ultimately determined that 40 mg / mL D-galactose was used to induce the cell model for 24 hours.

[0135] 3. Protective effect of 100% ethanol layer compounds on model cells

[0136] (1) Select HepG2 cells in the logarithmic growth phase at a concentration of 1×10⁻⁶. 5 The cells were seeded at a density of 100 μL / mL in 96-well plates and allowed to adhere to the plate for 24 hours.

[0137] (2) The experiment was divided into a control group, a blank group, a model group (40 mg / mL D-galactose), and experimental groups E-25 (40 mg / mL D-galactose + 25 μg / mL 50% or 100% ethanol layer compound), E-50 (40 mg / mL D-galactose + 50 μg / mL 50% or 100% ethanol layer compound), E-75 (40 mg / mL D-galactose + 75 μg / mL 50% or 100% ethanol layer compound), E-100 (40 mg / mL D-galactose + 100 μg / mL 50% or 100% ethanol layer compound), and E-150 (40 mg / mL D-galactose + 150 μg / mL 50% or 100% ethanol layer compound). Each group had 6 replicates. Cell viability was determined using the MTT assay. 20 μL MTT reagent was added to each well, and after incubation for 4 h, the absorbance was measured at 570 nm using a microplate reader. The protective effects of different concentrations of 50% or 100% ethanol layer compounds on D-galactose-induced HepG2 cells were analyzed.

[0138] (3) The cell-protective effect is determined by the following formula.

[0139] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%

[0140] As — Absorbance of the experimental or model group at 570 nm

[0141] Ac – Absorbance of the control group at 570 nm

[0142] Ab – Absorbance of the blank group at 570nm

[0143] The results are as follows Figure 5 As shown in Figures F and G, neither the 50% nor 100% ethanol layer compounds had a significant protective effect on D-galactose-induced HepG2 cells, and they did not show a significant improvement compared to the D-galactose model group. Cell viability in both groups was 60%–65%.

Claims

1. A plateau rose extract, characterized in that, It is prepared according to the following method: (1) Using dried petals of plateau rose as raw material, the petals were ground into powder and extracted by reflux with 70%~75% ethanol by volume. The extract was then collected by hot filtration, the solvent was recovered and dried to obtain crude ethanol extract. (2) The crude ethanol extract obtained in step (1) was uniformly dispersed in water and added to an AB-8 macroporous resin column. After static adsorption, it was eluted sequentially with 0%~3%, 8%~13%, 28%~33%, 48%~53%, and 95%~100% ethanol, respectively. The elution flow rate was 1~1.5 BV / h, and the elution volume of ethanol for each concentration gradient was 3~5 BV. The eluent of each gradient was collected, concentrated under reduced pressure to a paste, and dried under vacuum to obtain the eluted fractions of 0%~3%, 8%~13%, 28%~33%, 48%~53%, and 95%~100% ethanol, namely: plateau rose extract.

2. The plateau rose extract as described in claim 1, characterized in that, In step (1), the ratio of dried petals of plateau rose to 70%~75% ethanol is 1:8~10 g / mL.

3. The plateau rose extract as described in claim 1, characterized in that, In step (1), the reflux extraction is performed 3 times, with each reflux extraction lasting 1.5 to 2 hours.

4. The plateau rose extract as described in claim 1, characterized in that, In step (2), the crude ethanol extract is uniformly dispersed in water at a material-to-liquid ratio of 1:10~15 g / mL.

5. The plateau rose extract as described in claim 1, characterized in that, In step (2), the adsorption time is 30 minutes.

6. The use of the plateau rose extract as described in claim 1 in the preparation of anti-aging drugs or health products.

7. The application as described in claim 6, characterized in that, The extract of the plateau rose is the fraction eluted with 8%~13% and 28%~33% ethanol.

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