A method for increasing the content and molecular weight of aloe polysaccharides in aloe

Abstract

The present invention relates to a method for increasing the content and molecular weight of aloe polysaccharides in aloe vera. The method comprises the following steps: firstly removing the blank part of the tip and tail of the aloe vera, retaining the aloe vera leaves, and cultivating the leaves with pure water; peeling and beating the aloe vera leaves to obtain aloe vera gel; placing the obtained aloe vera gel in a light irradiation reaction tank; setting an agitator at the tank mouth of the light irradiation reaction tank; placing a color-adjustable LED lamp above the light irradiation reaction tank, and subjecting the aloe vera gel to alternating light treatments of blue light, red light, and no light. The present invention successfully increases the content and molecular weight of aloe polysaccharides in aloe vera gel by using an external field such as a red and blue alternating light field. In addition, the application of appropriate light regulation can simulate the natural system and produce biological macromolecules (such as polysaccharides) by controlling the polymerization reaction through the external field. Since the high molecular weight and content of aloe polysaccharides are crucial to the biological activity of aloe polysaccharides, the present invention is of great significance to the development of the medicinal value of aloe polysaccharide products.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and particularly relates to a method for increasing the content and molecular weight of aloe polysaccharides in aloe. Background Art

[0002] Aloe polysaccharide is a polymer derived from aloe vera. The 2019 issue of the journal Molecules showed that aloe polysaccharide exhibits a special bidirectional function: inhibiting the activity of diseased cells while promoting the activity of normal cells. (Liu et al., 2019), and a large number of studies have shown that polysaccharides have biological activities such as regulating blood sugar, anti-microbial, anti-tumor, regulating immunity, anti-virus, and anti-oxidation.

[0003] The Dutch journal Immunopharmacology evaluated 32 aloe vera products on the US market provided by the International Aloe Society (IASC). The article concluded: "The manufacturing process of aloe vera cannot destroy high molecular weight polysaccharides, which are crucial to the biological activity of aloe polysaccharides" (Turner, Williamson, Stroud, & Talley, 2004). Therefore, maximizing the molecular weight of aloe polysaccharides is important to ensure their biological activity.

[0004] Over the past few decades, there has been a growing interest in developing advanced polymerization technologies using external fields, such as temperature, light, electricity, magnetic fields, ultrasound, etc. Chemically regulated polymerization reactions may contain foreign substances, which may lead to the accumulation of impurities and even adverse side reactions. External field regulated polymerization technology is a non-invasive technology. It has excellent and unique capabilities in process intensification and spatial and temporal regulation. The parameters of the external field can be adjusted to adjust the onset and rate of the polymerization reaction.

[0005] So far, there has been no report on the technology of using an external field such as a light field to regulate the polymerization reaction of aloe polysaccharides to increase the molecular weight and content of aloe polysaccharides. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a method for increasing the content and molecular weight of aloe polysaccharides in aloe vera, which method utilizes light irradiation to regulate the polymerization reaction of aloe polysaccharides to increase the content and molecular weight of aloe polysaccharides. Specifically, it is achieved by alternating irradiation with blue light, red light and no light.

[0007] A method for increasing the content of macromolecular aloe polysaccharides in aloe, comprising the following steps:

[0008] Remove the white part at the tip and tail of the aloe vera, keep the aloe vera leaves, and cultivate them with pure water after processing;

[0009] The cultivated aloe leaves are peeled and pulped to obtain aloe vera gel; the amount of gelling in the pulping barrel at a time does not exceed 40L, and the gelling time is not less than 20 minutes and not more than 30 minutes;

[0010] The obtained aloe vera gel is placed in a light irradiation reaction tank, which is made of opaque material, has an open top, preferably has a volume of 300 L, a diameter of 85 cm, and a depth of 75 cm. The light irradiation reaction tank is equipped with a circulating water jacket and is externally connected to a cold water circulation unit, and the circulating water temperature is 9-12°C;

[0011] A stirrer is installed at the mouth of the light reaction tank, and the stirring rate is adjusted to a suitable rate when the ripples caused by the stirring just disappear at the edge of the tank;

[0012] A color-adjustable LED lamp is arranged above the illumination reaction tank to perform alternating treatments of blue light, red light and no light. The alternating time is 60 minutes, and the distance between the light source and the liquid surface is 60-70 cm.

[0013] Furthermore, the aloe vera is preferably Aloe vera, and the growth period of the aloe vera is more than 10 months. Furthermore, the aloe vera of the present invention comes from a dry and hot river valley.

[0014] The length of the aloe vera tip removed is 15-20 cm.

[0015] The present invention successfully increases the content and molecular weight of aloe polysaccharides in aloe vera gel by using an external field such as a red and blue alternating light field. In addition, the application of appropriate light regulation can simulate the natural system and produce biological macromolecules (such as polysaccharides) by controlling the polymerization reaction through the external field. Since the high molecular weight and content of aloe polysaccharides are crucial to the biological activity of aloe polysaccharides, the present invention is of great significance to the development of the medicinal value of aloe polysaccharide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the chromatogram of the control sample;

[0017] Figure 2 It is the chromatogram of the experimental group samples. DETAILED DESCRIPTION

[0018] In order to make the technical personnel in the field understand the present invention more clearly and intuitively, the present invention will be further described below in conjunction with the accompanying drawings. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0019] A method for increasing the content and molecular weight of aloe polysaccharides in aloe, comprising the following steps:

[0020] Remove the white part at the tip and tail of the aloe vera, keep the aloe vera leaves, and cultivate them with pure water after processing;

[0021] The cultivated aloe leaves are peeled and pulped to obtain aloe vera gel; the amount of gelling in the pulping barrel at a time does not exceed 40L, and the gelling time is not less than 20 minutes and not more than 30 minutes;

[0022] The obtained aloe vera gel is placed in a light irradiation reaction tank, the volume of the light irradiation reaction tank is preferably 300L, the diameter is 85cm, the depth is 75cm, the light irradiation reaction tank is equipped with a circulating water jacket, an external cold water circulation unit, and the circulating water temperature is 9-12°C;

[0023] A stirrer is installed at the mouth of the light reaction tank, and the stirring rate is adjusted to a suitable rate when the ripples caused by the stirring just disappear at the edge of the tank;

[0024] Place a color-adjustable LED light on the light reaction tank for alternating treatment with blue light, red light, and no light. The alternation time is 60 minutes, and the distance between the light source and the liquid surface is 60-70 cm. When the blue light is treated, the LED light is blue, and when the red light is treated, the LED light is red. When the no light is treated, the LED light is off.

[0025] Furthermore, the aloe vera is preferably Aloe vera, and the growth period of the aloe vera is more than 10 months. Furthermore, the aloe vera of the present invention comes from a dry and hot river valley.

[0026] Preferred Embodiments

[0027] A method for increasing the content and molecular weight of aloe polysaccharides comprises the following steps:

[0028] (1) Select Aloe vera grown in dry and hot valleys, and the growth period of the Aloe vera is more than 10 months.

[0029] (2) Cut off 15-20 cm of the front end of the aloe vera obtained in step (1), remove the blank part of the tail, and cultivate it in pure water for 24 hours after the treatment is completed.

[0030] (3) Peeling and beating the aloe vera leaves cultivated in step (2). The single glue-applying amount in the beating barrel is 40L, and the glue-applying time is preferably 25 minutes. During the experiment, a group of experimental groups with a glue-applying time of more than 30 minutes was set in this step. It was found that a glue-applying time of more than 30 minutes would reduce the viscosity of the aloe vera gel. Finally, the molecular weight of the polysaccharide in the aloe vera gel was reduced. Therefore, the best glue-applying time obtained by the present invention is no more than 30 minutes;

[0031] (4) 300 L of aloe vera gel obtained in step (3) is placed in light irradiation reaction tanks No. 1, 2, and 3 respectively. A cold water circulation unit is installed outside the light irradiation reaction tank. The temperature of the circulating water is 9° C.

[0032] (5) Set a stirrer at the mouth of the light reaction tank. The stirring rate is such that the ripples just disappear at the edge of the pot. This is the appropriate rate.

[0033] (6) Place a color-adjustable LED light above the No. 1 light reaction tank. The light source is white light. The distance between the light source and the liquid surface is 65 cm. The reaction time is 48 hours. Set as the control group (1).

[0034] (7) Place a color-adjustable LED light above the No. 2 light reaction tank. The light source is blue light → red light → no light in a cyclic alternating pattern. The alternating time is 60 minutes. The distance between the light source and the liquid surface is 65 cm. The reaction time is 48 hours. This is set as experimental group (1).

[0035] (8) Place a color-adjustable LED light above the No. 3 light reaction tank. The light source is red light → blue light → no light in a cyclic alternating pattern. The alternating time is 60 minutes. The distance between the light source and the liquid surface is 65 cm. The reaction time is 48 hours. This is set as experimental group (2).

[0036] (9) 100 ml of aloe vera gel from the No. 1, No. 2, and No. 3 light irradiation reaction tanks were taken respectively to detect the content and molecular weight of aloe polysaccharide in the aloe vera gel.

[0037] Determination of aloe polysaccharide content:

[0038] The detection method for the aloe polysaccharide content in aloe vera gel is carried out in accordance with the polysaccharide detection method in the light industry standard QB / T2489-2018 of the People's Republic of China.

[0039] The test results are as follows: the content of aloe polysaccharide in the aloe vera gel of the control group (1) is 6759.51 mg / kg, the content of aloe polysaccharide in the aloe vera gel of the experimental group (1) is 7660.13 mg / kg, and the content of aloe polysaccharide in the aloe vera gel of the experimental group (2) is 7223.08 mg / kg. Result analysis: The content of polysaccharide in the experimental group (1) increased by The polysaccharide content in the experimental group (2) increased compared with that in the control group. It can be seen that the use of alternating red and blue light process can effectively increase the content of aloe polysaccharides in the product, and the lighting sequence of blue light-red light-no light is better than the effect of red light-blue light-no light on the content of aloe polysaccharides.

[0040] Take another batch of aloe vera leaves and repeat steps (1)-(3);

[0041] (4) Then 300L of aloe vera gel is placed in the No. 4, 5, and 6 light reaction tanks. A cold water circulation unit is installed outside the light reaction tank. The temperature of the circulating water is 9°C.

[0042] (5) Set a stirrer at the mouth of the light reaction tank. The stirring rate is such that the ripples just disappear at the edge of the pot. This is the appropriate rate.

[0043] (6) Place a color-adjustable LED light above the No. 4 light reaction tank. The light source is white light. The distance between the light source and the liquid surface is 65 cm. The reaction time is 48 hours. Set as the control group (2).

[0044] (7) Place a color-adjustable LED light above the No. 5 light irradiation tank. The light source is blue light → red light → no light, alternating for 60 minutes. The distance between the light source and the liquid surface is 65 cm, and the reaction time is 48 hours. This is set as experimental group (3).

[0045] (8) Place a color-adjustable LED light above the No. 6 light reaction tank. The light source is red light → blue light → no light, alternating for 60 minutes. The distance between the light source and the liquid surface is 65 cm, and the reaction time is 48 hours. This is set as experimental group (4).

[0046] (9) 100 ml of aloe vera gel from the No. 4, No. 5, and No. 6 light irradiation reaction tanks were taken respectively to detect the content and molecular weight of aloe polysaccharide in the aloe vera gel.

[0047] Determination of aloe polysaccharide content:

[0048] The detection method for the aloe polysaccharide content in aloe vera gel is carried out in accordance with the polysaccharide detection method in the light industry standard QB / T2489-2018 of the People's Republic of China.

[0049] The test results are as follows: the content of aloe polysaccharide in the aloe vera gel of the control group (2) is 2017.51 ​​mg / kg, the content of aloe polysaccharide in the aloe vera gel of the experimental group (3) is 2188.71 mg / kg, and the content of aloe polysaccharide in the aloe vera gel of the experimental group (4) is 2096.24 mg / kg. Result analysis: The content of polysaccharide in the experimental group (3) is increased by 1.3% compared with that in the control group. The polysaccharide content in the experimental group (4) increased compared with that in the control group. It can be seen that the use of red and blue light alternating process can effectively increase the content of aloe polysaccharides in the product. And the effect of the blue light-red light-no light illumination sequence on the aloe polysaccharide content is better than the effect of the red light-blue light-no light illumination sequence on the aloe polysaccharide content.

[0050] Relative molecular weight determination:

[0051] (1) 100 ml of aloe vera gel from the first batch of control group (1) and experimental group (1) were respectively taken and precipitated with 95% alcohol, and then the obtained polymer was freeze-dried.

[0052] (2) Dextran with different molecular weights was used as standard, and high performance gel permeation chromatography (HPGPC) was used for detection using a high performance gel permeation chromatography column and a differential detector. The results were analyzed using GPC software. The logarithm of the relative molecular mass of the standard was used as the ordinate and the retention time of the corresponding chromatographic peak was used as the abscissa to develop a calibration curve equation for the determination of the molecular weight and molecular weight distribution of the polysaccharide.

[0053] (3) Experimental samples

[0054] Table 1 Experimental sample information

[0055] Serial number Aloe Vera Gel Sample Name 1 Control group (1) 2 Experimental group (1)

[0056] (4) Standard substances

[0057] Table 2 Standard substance information

[0058]

[0059] (5) Experimental instruments

[0060] Table 3 Experimental instrument information

[0061] Instrument Name brand model One hundred thousandth electronic balance Shimadzu AUW 220D 1% electronic balance Double Hero JJ-1000Y Ultrasound Kunshan KQ-400DE High performance liquid chromatography Shimadzu LC-2050C3D Differential detector Shimadzu RID-20A

[0062] (6) Experimental methods

[0063] a. Establishment of molecular weight calibration curve

[0064] Take 5 dextran reference substances of known molecular weight, dissolve them in water and dilute them to prepare dextran solutions containing 2 mg of dextran per 1 ml.

[0065] b. Preparation of test solution

[0066] Take appropriate amount of aloe vera gel from the control group (1) and the experimental group (1), weigh accurately, dissolve and dilute with water to make a solution containing about 1 mg to 2 mg of aloe vera gel per 1 ml, shake well, filter, and take the filtrate to obtain.

[0067] Note: When the sample is poorly water-soluble, dissolve it by ultrasound or heating.

[0068] Table 4 Test solution preparation information

[0069] Serial number Sample name Sampling volume mg Dilution multiple Dissolution method 1 Control group (1) 21.71 15 heating 2 Experimental group (1) 20.21 15 heating

[0070] c. Chromatographic conditions

[0071] Instrument: High performance liquid chromatography

[0072] Detector: Differential detector RID-20A

[0073] Chromatographic column: TSK GPWXL chromatographic column (7.8mm*300mm, particle size 13μm)

[0074] Mobile phase: water

[0075] Flow rate: 0.6ml / min

[0076] Column temperature: 30°C

[0077] Detector temperature: 4

[0078] Injection volume: 20μ

[0079] (7) Experimental results

[0080] a. The results of the calibration curve are shown in Table 5.

[0081] Table 5 Calibration curve results

[0082]

[0083] b. Sample test results

[0084] The test results of the chromatogram of the test sample control group (1) and the chromatogram of the test sample experimental group (1) are as follows: Figure 1 , 2 The corresponding monitor data are shown in Table 6 and Table 7.

[0085] Table 6

[0086] <Peak table>

[0087] Detector B Ch1

[0088] Peak Retention time Compound Name area area% high Theoretical Plate Number (USP) Resolution (USP) Tailing Factor 1 12.088 670000 74429 32.675 715 -- -- -- 2 13.816 D6 150342 66.002 1774 423 -- -- 3 14.738 D4 3012 1.322 129 -- -- -- total 227784 100.000 2618 -- -- --

[0089] Table 7

[0090] <Peak table>

[0091] Detector B Ch1

[0092] Peak Retention time Compound Name area area% high Theoretical Plate Number (USP) Resolution (USP) Tailing Factor 1 11.709 670000 127230 35.594 933 39 -- -- 2 14.099 D6 222866 62.349 2652 624 0.488 -- 3 14.738 D4 7352 2.057 320 -- -- -- total 357448 100.000 3905 -- -- --

[0093] (8) Result analysis:

[0094] a) By Figure 1 and Figure 2 It can be seen that the area of ​​the part with a molecular weight greater than that of the standard product 5 (dextran 67000) in the experimental group (1) is 127230, and the area of ​​the control group (1) is 74429. The fact that the area of ​​the experimental group (1) is greater than that of the control group (1) indicates that the part with a molecular weight greater than that of the standard product 5 (dextran 67000) in the experimental group (1) is greater than that in the control group (1).

[0095] b) By Figure 1, Figure 2 As shown in Tables 6 and 7, the area of ​​the experimental group (1) is 222866 for the molecular weight greater than standard 2 (dextran D6) and less than standard 5 (dextran 67000), and the area of ​​the control group (1) is 150342. The fact that the area of ​​the experimental group (1) is larger than that of the control group (1) indicates that the area of ​​the experimental group (1) with a molecular weight greater than that of the standard 2 (dextran D6) and less than that of the standard 5 (dextran 67000) is greater than that of the control group (1).

[0096] In summary, the experimental group (1) has a significantly higher molecular weight of aloe polysaccharide than the control group (1), indicating that the method of alternately irradiating with blue light, red light and no light of the present invention can effectively increase the molecular weight of aloe polysaccharide in aloe gel.

[0097] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the embodiments herein, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for increasing the content and molecular weight of aloe polysaccharides in aloe, characterized in that: The following steps are involved: Remove the white part at the tip and tail of the aloe vera, keep the aloe vera leaves, and cultivate them with pure water after processing; The cultivated aloe leaves are peeled and pulped to obtain aloe gel; The obtained aloe vera gel is placed in a light irradiation reaction tank, and the temperature of the heavy circulating water in the circulating water jacket of the light irradiation reaction tank is 9-12°C; A color-adjustable LED lamp is placed above the light reaction tank to alternately irradiate the aloe vera gel with red light → blue light → no light or blue light → red light → no light; The alternating time of the cyclic alternating irradiation was 60 minutes.

2. The method for increasing the content and molecular weight of aloe polysaccharides in aloe according to claim 1, characterized in that: The aloe vera is Aloe vera, and the growth period of the aloe vera is more than 10 months.

3. The method for increasing the content and molecular weight of aloe polysaccharides in aloe according to claim 1, characterized in that: The time for applying aloe vera glue in the beating barrel is 20 to 30 minutes.

4. The method for increasing the content and molecular weight of aloe polysaccharides in aloe according to claim 1, characterized in that: The order of alternating light treatment is blue light → red light → no light in a cyclic alternating manner.

5. The method for increasing the content and molecular weight of aloe polysaccharides in aloe according to claim 1, characterized in that: The distance between the light source and the liquid surface is 60-70cm.

6. The method for increasing the content and molecular weight of aloe polysaccharides in aloe according to claim 1, characterized in that: The time for aloe vera to be cultivated in pure water is 24 hours.

7. The method for increasing the content and molecular weight of aloe polysaccharides in aloe according to claim 1, characterized in that: A stirrer is provided at the mouth of the light irradiation reaction tank, and the stirring rate is adjusted so that the ripples caused by the stirring just disappear at the edge of the tank.

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