Preparation method and use of almond oligosaccharide

Abstract

The present invention relates to a method for preparing almond oligosaccharides and its use. The method comprises taking Xinjiang almond seeds, crushing them, extracting and defatting them with supercritical carbon dioxide, extracting and concentrating the defatted powder, and then precipitating the supernatant with ethanol to obtain crude almond oligosaccharides. The crude oligosaccharides are then purified, decolorized, and small molecule components are removed, and then separated by gel column chromatography to obtain neutral oligosaccharides. The obtained neutral oligosaccharides have an average molecular weight of 1070 Da and are composed of glucose and galactose monosaccharides. The almond oligosaccharides obtained by the method of the present invention are highly pure and have strong freckle-removing and whitening activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation technology of almond oligosaccharide extract, and relates to a preparation method of almond oligosaccharide and application thereof. Background Art

[0002] Melanin is a biological pigment synthesized by melanocytes. Within the melanosomes of melanocytes, melanin typically starts with L-tyrosine. Tyrosinase sequentially hydroxylates and oxidizes melanin to produce L-dopa and dopaquinone. Dopaquinone is unstable and rapidly converts to other intermediates, which then undergo multiple reactions to ultimately synthesize melanin. Research has shown that melanin production is linked to the actions of tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). Tyrosinase is the primary rate-limiting enzyme, catalyzing two consecutive rate-limiting steps in melanin biosynthesis. Normally, this pigment protects skin cells from the harmful effects of ultraviolet radiation, oxidative stress, and other environmental pollutants. However, excessive melanin synthesis can cause a variety of skin diseases, such as freckles, age spots, and chloasma, and can also lead to cancerous transformation, including melanoma. Consequently, the development of drugs and cosmetics that can prevent and treat conditions such as hyperpigmentation and melanoma has attracted significant attention. Currently, most commercially available cosmetics and skin-whitening agents are based on small-molecule compounds, such as arbutin, hydroquinone, kojic acid, and gallic acid. However, while these traditional melanin inhibitors offer promising whitening and antioxidant benefits, they also exhibit significant irritation, allergic reactions, and other adverse skin reactions. Consequently, the search for safe and effective melanin inhibitors derived from natural products has become a hot topic in cosmetic research and is gaining increasing popularity among consumers.

[0003] Oligosaccharides, also known as oligosaccharides, are linear or branched polymers composed of 2-10 monosaccharide molecules linked by glycosidic bonds. Research has shown that oligosaccharides possess a variety of pharmacological activities, including anti-tumor, antiviral, antibacterial, antidepressant, immune-modulating, and hematopoietic enhancement. In recent years, bio-derived oligosaccharides have garnered widespread attention in the development and application of cosmetic ingredients. These natural plant-derived oligosaccharides have low molecular weight, are relatively safe for the human body, have high absorption and utilization rates, and exhibit unique physical and chemical properties, such as moisturizing, antioxidant, and antibacterial properties.

[0004] Almond (Prunus dulcis (Mill.) DA Webb or Amygdalus communis L.), also known as almond, is a world-renowned nut tree in the genus Amygdalus, subfamily Prunaceae, Rosaceae. Native to the southern mountainous regions of West and Central Asia, it was introduced to North and South America, South Africa, and Australia, and is now found in over 60 countries. In my country, large-scale cultivation is only found in areas like Kashgar in Xinjiang, with Shache County, China's largest almond production base, boasting abundant resources. Almonds are listed in the "Ministry of Health Drug Standards - Uyghur Medicine Volume" and are believed to strengthen the body and brain, improve eyesight and complexion, and relieve coughs. They are used for symptoms of weakness, cough with phlegm, chest tightness, constipation, and blurred vision and a dark complexion.

[0005] Systematic studies of almonds have revealed that oligosaccharides are one of their main active ingredients. However, there are currently no reports on the extraction, separation, purification, and whitening efficacy of uniform oligosaccharides from Xinjiang almonds. Therefore, the present invention prepares a uniform oligosaccharide from almonds in Shache County, Xinjiang, with relatively stable quality and properties and more pronounced activity, providing a theoretical basis and scientific foundation for the development and utilization of Xinjiang almond resources as a cosmetic ingredient. Summary of the Invention

[0006] The object of the present invention is to provide a preparation method and use of almond oligosaccharides. The method comprises the following steps: taking Xinjiang almond seeds, crushing them, extracting and defatting them with supercritical carbon dioxide, extracting and concentrating the defatted powder, and taking the supernatant through ethanol precipitation to obtain crude almond oligosaccharides. The crude oligosaccharides are then purified, decolorized, and small molecule components are removed, and then separated through gel column chromatography to obtain neutral oligosaccharides. The obtained neutral oligosaccharides have an average molecular weight of 1070 Da and are composed of glucose and galactose monosaccharides. The almond oligosaccharides obtained by the method of the present invention are of high purity and have strong freckle-removing and whitening activity.

[0007] The preparation method of almond oligosaccharides described in the present invention is carried out according to the following steps:

[0008] a. Grind the selected Xinjiang almond kernels and extract them with supercritical carbon dioxide to obtain defatted almond powder, add water with a solid-liquid ratio of 1:10-1:30, extract twice in water at a temperature of 20-90°C for 1-2 hours, filter, and combine the supernatants to obtain an almond total extract;

[0009] b. Concentrate the total extract obtained in step a at 55° C. to obtain a concentrate concentrated to 1 / 5-1 / 6 of the original extract;

[0010] c. The concentrate obtained in step b was precipitated with 95% ethanol at a volume ratio of 1:4 overnight. The supernatant was taken and filtered the next day to obtain a clear supernatant;

[0011] d. freeze-drying or spray-drying the supernatant obtained in step c to obtain a crude extract;

[0012] e. The crude extract obtained in step d was preliminarily separated and purified by AB-8 macroporous adsorption resin, and eluted with deionized water to obtain crude oligosaccharides;

[0013] f. The crude oligosaccharide obtained in e) was dialyzed through a 1000 Da dialysis bag; the portion in the dialysis bag was further purified using a Sephadex G-25 gel column as a separation column and distilled water as the mobile phase. The fractions were collected and freeze-dried to obtain relatively pure oligosaccharides containing a small amount of peptide or protein components;

[0014] g. The components obtained by Sephadex G-25 gel column in f are further separated again by Sephadex G-50 gel column, and distilled water is used as the mobile phase for elution and purification again to remove the mixed peptide or protein components. The components are collected and freeze-dried to obtain purified almond oligosaccharides.

[0015] The almond oligosaccharides obtained by the method are used in the preparation of whitening cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of almond oligosaccharides of the present invention on the viability of B16F10 mouse melanoma cells;

[0017] Figure 2 The present invention shows the effect of almond oligosaccharides on the melanin content of B16F10 mouse melanoma cells induced by Fosrskolin (FSK), and the effect of almond oligosaccharides on the tyrosinase activity of B16F10 mouse melanoma cells induced by Fosrskolin (FSK) (**P<0.01, ***P<0.001, ****<0.0001 compared with the FSK model control group). DETAILED DESCRIPTION

[0018] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the embodiments, and the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0019] Example 1

[0020] a. Select 100 g of defatted almond powder after supercritical carbon dioxide extraction, add 3000 mL of purified water (30 times the weight of the raw material), extract twice in water at 90°C for 2 hours, filter, and combine the two supernatants to obtain the total extract;

[0021] b. Concentrate the total extract obtained in step a by using a rotary evaporator to reduce the volume to 1 / 6 at 55° C. to obtain a concentrated solution;

[0022] c. The concentrate obtained in step b was precipitated with 95% ethanol at a volume ratio of 1:4 overnight. The supernatant was taken and filtered the next day to obtain a clear supernatant;

[0023] d. Freeze-drying the supernatant obtained in step c to obtain a crude extract; namely, 15.59 g of crude almond oligosaccharide, with an oligosaccharide extraction rate of 15.59%. Analysis results showed that the oligosaccharide content of the extract was 27.01%, and the protein content was 17.46%;

[0024] e. The crude extract obtained in step d was preliminarily separated and purified by AB-8 macroporous adsorption resin, and eluted with deionized water. The deionized water eluate was mainly composed of carbohydrates to obtain crude oligosaccharides;

[0025] f. The crude oligosaccharide obtained in e) was dialyzed through a 1000 Da dialysis bag for 48 h; the portion in the dialysis bag was further purified using a Sephadex G-25 gel column as a separation column and distilled water as the mobile phase. The fractions were collected and freeze-dried to obtain relatively pure oligosaccharides containing a small amount of peptide or protein components;

[0026] g. The components obtained by Sephadex G-25 gel column in f are further separated again by Sephadex G-50 gel column, and distilled water is used as the mobile phase for elution and purification again to remove the mixed peptide or protein components. The components are collected and freeze-dried to obtain purified almond oligosaccharides.

[0027] Example 2

[0028] a. Select 100 g of defatted almond powder after supercritical carbon dioxide extraction, add 1000 ml of purified water (10 times the weight of the raw material), extract twice in water at 85°C for 1.5 hours, filter, and combine the two supernatants to obtain the total extract;

[0029] b. Concentrate the total extract obtained in step a by using a rotary evaporator to reduce the volume to 1 / 6 at 55° C. to obtain a concentrated solution;

[0030] c. The concentrate obtained in step b was precipitated with 95% ethanol at a volume ratio of 1:4 overnight. The next day, the supernatant was taken and filtered to obtain a clear supernatant;

[0031] d. Freeze-drying the supernatant obtained in step c to obtain a crude extract; namely, 18.57 g of crude almond oligosaccharide, with an oligosaccharide extraction rate of 18.57%. Analysis results showed that the oligosaccharide content of the extract was 30.34%, and the protein content was 20.46%.

[0032] e. The crude extract obtained in step d was preliminarily separated and purified by AB-8 macroporous adsorption resin, and eluted with deionized water. The deionized water eluate was mainly composed of carbohydrates to obtain crude oligosaccharides;

[0033] f. The crude oligosaccharide obtained in e) was dialyzed through a 1000 Da dialysis bag; the portion in the dialysis bag was further purified using a Sephadex G-25 gel column as a separation column and distilled water as the mobile phase. The fractions were collected and freeze-dried to obtain relatively pure oligosaccharides containing a small amount of peptide or protein components;

[0034] g. The components obtained by Sephadex G-25 gel column in f are further separated again by Sephadex G-50 gel column, and distilled water is used as the mobile phase for elution and purification again to remove the mixed peptide or protein components. The components are collected and freeze-dried to obtain purified almond oligosaccharides.

[0035] Example 3

[0036] a. Select 100 g of defatted almond powder after supercritical carbon dioxide extraction, add 2000 ml of purified water (20 times the weight of the raw material), and extract twice in water at 30°C, each extraction for 1 hour; filter and combine the two supernatants to obtain the total extract;

[0037] b. Concentrate the total extract obtained in step a by using a rotary evaporator to reduce the volume to 1 / 6 at 55° C. to obtain a concentrated solution;

[0038] c. The concentrate obtained in step b was precipitated with 95% ethanol at a volume ratio of 1:4 overnight. The supernatant was taken and filtered the next day to obtain a clear supernatant;

[0039] d. Freeze-drying the supernatant obtained in step c to obtain a crude extract; namely, 20.52 g of crude almond oligosaccharide, with an oligosaccharide extraction rate of 18.57%. Analysis results showed that the oligosaccharide content of the extract was 32.75%, and the protein content was 19.46%;

[0040] e. The crude extract obtained in step d was preliminarily separated and purified by AB-8 macroporous adsorption resin, and eluted with deionized water. The deionized water eluate was mainly composed of carbohydrates to obtain crude oligosaccharides;

[0041] f. The crude oligosaccharide obtained in e) was dialyzed through a 1000 Da dialysis bag; the portion in the dialysis bag was further purified using a Sephadex G-25 gel column as a separation column and distilled water as the mobile phase. The fractions were collected and freeze-dried to obtain relatively pure oligosaccharides containing a small amount of peptide or protein components;

[0042] g. The components obtained by Sephadex G-25 gel column in f are further separated again by Sephadex G-50 gel column, and distilled water is used as the mobile phase for elution and purification again to remove the mixed peptide or protein components. The components are collected and freeze-dried to obtain purified almond oligosaccharides.

[0043] Example 4

[0044] CCK-8 assay was used to evaluate the cytotoxicity of almond oligosaccharides on B16F10 mouse melanoma cells:

[0045] Mouse skin melanoma B16F10 cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1×10 4 B16F10 cells / well were seeded in 96-well plates and cultured for 24 h. Different concentrations of almond oligosaccharides were added to the 96-well plates and treated for 48 h. The cell viability was determined using the CCK-8 method. The results are shown in Table 1. Figure 1 As shown:

[0046] Table 1. Effects of almond oligosaccharides on the viability of B16F10 mouse melanoma cells

[0047]

[0048]

[0049] Note: Oligosaccharide samples were sterilized by filtration using a 0.22 μM filter membrane before addition to cells.

[0050] Table 1 shows the cell viability of B16F10 cells tested using the CCK-8 assay. At concentrations of 0.1, 0.5, and 1.0 mg / mL, almond oligosaccharides achieved cell viability rates of 100.4%, 99.67%, and 96.18%, respectively, demonstrating virtually no cytotoxicity. This result suggests that almond oligosaccharides at concentrations of 0.1-1 mg / mL are within a safe concentration range for B16F10 cells.

[0051] Example 5

[0052] Determination of intracellular melanin content in B16F10 mouse melanoma cells:

[0053] Forskolin (FSK) was used to induce B16F10 melanoma cells to establish a melanin pigmentation model. Almond oligosaccharides and the positive control kojic acid (KA) were then treated. The melanin content of the cells was detected by the NaOH lysis method. The effect of almond oligosaccharides on melanin production was evaluated by the ratio of the melanin content to the corresponding protein content. The specific results were as follows: B16F10 cells were passaged and cultured in DMEM (high glucose cell culture medium) containing 10% fetal bovine serum (FBS). 0.5×10 5 B16F10 cells were seeded in 6-well plates and cultured for 24 h. After 0.5 h of treatment with kojic acid and almond oligosaccharides, 4 μM rucic acid was added and the cells were cultured for 72 h. Samples were collected and lysed with 100 μL of RIPA buffer. After centrifugation, the supernatant was removed and 200 μL of 1 M NaOH containing 10% DMSO was added. The cells were placed in a water bath at 80°C for 1 h. The absorbance was measured at 405 nm. Each group was repeated three times. The relative content of melanin and the inhibition rate of melanin production were calculated (Table 2, Figure 2 ).

[0054] Table 2. Effects of almond oligosaccharides on melanin content in FSK-induced B16F10 mouse melanoma cells

[0055]

[0056] Note: Oligosaccharide samples were sterilized by filtration using a 0.22 μM filter membrane before addition to cells.

[0057] As can be seen in Table 2, compared with the blank control group, the melanin content in the FSK pigmentation model group increased significantly, indicating that the melanin model was established. Melanin content decreased in the groups treated with different concentrations of almond oligosaccharides. Almond oligosaccharides at a concentration of 0.1-1 mg / mL significantly inhibited melanin production activity, with a melanin content inhibition rate of 26.86% at a concentration of 1 mg / mL, demonstrating significant melanin production inhibition activity.

[0058] Example 6

[0059] Intracellular tyrosinase activity assay in B16F10 mouse melanoma cells:

[0060] Tyrosinase is the key rate-limiting enzyme in the biosynthesis of melanin. The determination of tyrosinase inhibition activity is an important evaluation index for screening whitening agents and is also the most commonly used index in the evaluation of whitening efficacy. The FSK-induced B16F10 mouse melanoma cell pigmentation model was selected and treated with almond oligosaccharides. The cell tyrosinase activity was detected by the L-DOPA method. The ratio of tyrosinase activity to the corresponding protein content was used to evaluate the effect of almond oligosaccharides on tyrosinase activity. The details are as follows: After passage, B16F10 cells were cultured in a high-glucose cell culture medium DMEM containing 10% fetal bovine serum (FBS). 1×10 5 B16F10 cells were seeded into 6-well plates and cultured for 24 hours. After 0.5 hours of treatment with kojic acid and almond oligosaccharides, 4 μM rucic acid was added and cultured for another 48 hours. Samples were collected and lysed with PBS (pH 6.8) containing 1% Triton X-100 and 1% sodium deoxycholate for 30 minutes. 70 μL of the lysate was added to 10 μL of L-DOPA (10 mM) and incubated at 37°C in the dark for 30 minutes. The relative activity of tyrosinase and the inhibition rate of tyrosinase activity were calculated. The results are shown in Table 3. Figure 2 :

[0061] Table 3. Effects of almond oligosaccharides on tyrosinase activity in FSK-induced B16F10 mouse melanoma cells

[0062]

[0063] Note: Oligosaccharide samples were sterilized by filtration using a 0.22 μM filter membrane before addition to cells.

[0064] As shown in Table 3, compared with the blank control group, the melanin content in the FSK pigmentation model group increased significantly, indicating that the melanin pigmentation model was established. Compared with the FSK pigmentation model group, tyrosinase activity decreased in the almond oligosaccharide groups at different concentrations. Almond oligosaccharides at concentrations ranging from 0.1 to 1 mg / mL significantly inhibited tyrosinase activity in the cells. At a concentration of 0.1 mg / mL, almond oligosaccharides exhibited an inhibition rate of 16.94% on FSK-induced tyrosinase activity in B16F10 cells, exceeding the 13.34% inhibition rate of the positive control drug. At a concentration of 0.5 mg / mL, almond oligosaccharides exhibited an inhibition rate of 25.79% on FSK-induced tyrosinase activity in B16F10 cells. At a concentration of 1 mg / mL, the inhibition rate reached 31.39%, demonstrating excellent tyrosinase inhibition.

Claims

1. A method for preparing almond oligosaccharides, characterized in that: Follow these steps: a. Grind the selected Xinjiang almond kernels and extract them with supercritical carbon dioxide to obtain defatted almond powder, add water with a solid-liquid ratio of 1:10-1:30, extract twice in water at a temperature of 20-90°C for 1-2 hours, filter, and combine the supernatants to obtain an almond total extract; b. Concentrate the total extract obtained in step a at 55° C. to obtain a concentrate concentrated to 1 / 5-1 / 6 of the original extract; c. The concentrate obtained in step b was precipitated with 95% ethanol at a volume ratio of 1:4 overnight. The supernatant was taken and filtered the next day to obtain a clear supernatant; d. freeze-drying or spray-drying the supernatant obtained in step c to obtain a crude extract; e. Preliminary separation and purification of the crude extract obtained in step d by passing it through AB-8 macroporous adsorption resin, and eluting it with deionized water to obtain crude oligosaccharides; f. The crude oligosaccharide obtained in step e was dialyzed through a 1000 Da dialysis bag; the portion in the dialysis bag was further separated and purified using a Sephadex G-25 gel column as a separation column and distilled water as a mobile phase, and the components were collected and freeze-dried to obtain relatively pure oligosaccharides containing a small amount of peptide or protein components; g. The components obtained by Sephadex G-25 gel column in step f are further separated again by Sephadex G-50 gel column, and distilled water is used as the mobile phase for elution and purification again to remove the mixed peptide or protein components. The components are collected and freeze-dried to obtain purified almond oligosaccharides.

2. Use of almond oligosaccharides obtained according to the method of claim 1 in the preparation of whitening cosmetics.

Images