CN1969682A - Application of bamboo shoot polysaccharide in preparation of health food - Google Patents

Abstract

The invention relates to a new application of bamboo shoot crude polysaccharide in preparing health food with immune regulation function. The present invention uses bamboo shoots or their dried bamboo shoots as raw materials, extracts crude polysaccharides therein, and develops them as active ingredients and other acceptable raw materials and auxiliary materials in food technology into suitable health food preparations with immune regulation functions, such as capsules Agents (soft capsules, hard capsules), dropping pills, tablets, granules, powders, oral liquids, etc. Experiments have proved that the bamboo shoot crude polysaccharide has a good effect in promoting the function of cellular immunity in vitro, and has little toxic and side effects, and has a good development and application prospect.

Description

Application of bamboo shoot crude polysaccharide in the preparation of health food

Technical field:

The present invention relates to the use of bamboo shoot crude polysaccharide, especially the use in health food.

technical background:

Bamboo shoot polysaccharides are soluble polysaccharides extracted from discarded bamboo shoots and bamboo shoots. Polysaccharide is a polymer composed of more than 10 monosaccharides. As a soluble dietary fiber, it has the general physiological functions of dietary fiber. Modern science has proved that dietary fiber can prevent and treat hypertension, coronary heart disease, etc. and remove exogenous harmful substances. And other functions,. Many diseases of modern civilization, such as hypertension, hyperlipidemia, obesity, etc., are related to the lack of dietary fiber. At the same time, polysaccharides often have more important physiological activities, such as the regulation of polysaccharides and immune function, and the recognition of cells. The transport of substances between cells is closely related to the diagnosis and treatment of cancer. Polysaccharide is also a good adjuvant in medicine. In addition, it can control cell division and differentiation, regulate cell growth and aging and various biological activities. At present, the chemical research and biological function of polysaccharides are in the ascendant, and the potential functions of polysaccharides from different sources need to be further studied.

The physiological functions of functional polysaccharides discovered so far are mainly in the following two aspects:

1. Anti-tumor

People have obtained a variety of polysaccharides with anti-tumor activity from different biological materials. For example, the lentinan obtained from Lentinus edodes has strong anti-tumor activity. Its anti-cancer and anti-tumor effects do not directly act on transplanted cancer cells, but It works through host regulation.

2. Enhance immune function

It has been confirmed that different polysaccharides have different immune-promoting effects. Lentinan is an ideal immune enhancer, which acts as an adjuvant for T cell positioning and helps T cell stimulation to participate in the body's immune response. It has been found that it has a highly specific immune enhancing effect. Tremella polysaccharide can promote the transformation of lymphocytes, enhance the phagocytosis of mouse peritoneal macrophages, significantly promote the synthesis of liver protein and nucleic acid, promote bone marrow hematopoietic function, and improve humoral immunity. Black fungus polysaccharide can effectively increase the phagocytic index and percentage of mouse macrophages. Pharmacological experiments have proved that tea polysaccharide can enhance immune function and promote phagocytosis of monocyte-macrophage system. Studies have shown that: tea polysaccharide is an immune enhancer, which can significantly promote the phagocytosis of macrophages and increase the weight of the immune organ spleen. The cells have different enhancement effects, which can enhance the cellular immune function of the body.

Although there are many research documents on polysaccharides, the research on polysaccharides from bamboo shoots is relatively rare. After literature search, only scattered functional reports are found, and the reported existing functions are anti-tumor (animal experiments).

Contents of the invention

The purpose of the present invention is to provide a new application of the crude polysaccharide from bamboo shoots, that is, the application in the functional positioning of health food.

The present invention is characterized in that the bamboo shoot crude polysaccharide is used in health food.

The invention specifically relates to the new application of bamboo shoot crude polysaccharide in the preparation of health food with immune regulation function.

The present invention uses bamboo shoots or their dried bamboo shoots as raw materials, extracts crude polysaccharides therein, and develops them as active ingredients and other acceptable raw materials and auxiliary materials in food technology into suitable health food preparations with immune regulation functions, such as capsules Agents (soft capsules, hard capsules), dropping pills, tablets, granules, powders, oral liquids, etc.

In order to better understand the essence of the present invention, the results of immunomodulatory function research and acute toxicity experiment of the crude polysaccharide from bamboo shoots are used to illustrate its use in the preparation of health food with immunomodulatory function.

1: Immunomodulatory test of crude polysaccharides from bamboo shoots: ConA-induced mouse spleen lymphocyte transformation test (MTT method)

I: Test principle: 1. Principle: When T lymphocytes are stimulated by the ConA mitogen to undergo mother cell transformation, living cells, especially proliferating cells, decompose MTT (a light yellow azole salt) into blue-purple through mitochondrial hydrolase Formazan crystallizes and develops color, and its optical density value can reflect the proliferation of cells.

II: Test equipment and reagents:

Sterile room, ultra-clean workbench N H ₄ CI Brogeramine Positive pressure device RPMI1640 calf serum Filter (0.22μm, 0.45μm) Glutamine penicillin, streptomycin Thick glass bottle (500ml, 250ml, 100ml, 50ml) penicillin bottle and cap _Na HCO ₃ Hanks solution Various droppers, droppers, straws, suction heads, glass tubes, pipettes ConA Trypan Blue Staining Solution 200 mesh screen _NaCl Bamboo shoot crude polysaccharide (freeze-dried, vacuum-dried) Tweezers, scissors, straight and curved ophthalmic scissors, pairs of ophthalmic forceps KCl Petri dishes MTT Microsampler (1ml, 20ul, 200ul) Isopropanol 96-well cell plate ethanol 24-well cell plate NaCl HCl The above equipment and reagents need to be sterilized during the experiment, which can be divided into ultraviolet sterilization, moist heat sterilization, filter sterilization, etc. according to the situation.

III: Test steps:

1. Reagent preparation:

1.1 Prepare RPMI1640 complete culture solution: prepare RPMI1640 culture solution according to the instructions of RPMI1640, filter and sterilize, add 10% calf serum, 1% glutamine (200mmol/L), penicillin (100U/ml), streptomycin before use Sodium (100ug/L) and 5×10 ^-5 mol/L 2-mercaptoethanol, and use sterile 1mol/L HCl or 1mol/L NaOH to adjust the pH to 7.0-7.2, namely the complete culture solution.

1.2 ConA solution was prepared into a 100ug/mL solution with double distilled water, sterilized by filtration, and stored in a low-temperature refrigerator (-20°C). Prepare to 5μl/ml before use

1.3PBS solution: 0.02M pH7.2 1L

1.4 0.155M NH ₄ Cl 0.83g to 100ml

1.5 Sterile Hanks solution was adjusted to pH 7.2-7.4 with 3.5% sterile NaHCO3 before use.

1.6 MTT solution 5mgMTT is dissolved in 1mL PBS of pH7.2, and it is used now.

1.7 Acidic isopropanol solution Add 4ml of 1mol/L HCL to 96mL of isopropanol, and prepare before use.

1.8 Preparation of bamboo shoot crude polysaccharide stock solution (100mg/ml)

Bamboo shoot crude polysaccharide powder 40g→PBS400ml→in ice cubes, stir and mix for 30min→ultrasonic treatment (ice bath)→centrifuge at 15000rpm at 4°C for 10min→take supernatant, filter with 10 layers of gauze→0.45μm filter membrane suction filtration→sterile 0.22μm Membrane suction filtration→storage at 4°C→prepared to 33.33mg/ml and 11.11mg/ml respectively before use

2. Preparation of splenocyte suspension:

The mice were killed, soaked in 75% Alc, cut open, and the spleen was aseptically removed, placed in a small plate filled with an appropriate amount of sterile Hanks solution, and the spleen was gently torn up with tweezers to make a single cell suspension. Filter through a 200 mesh screen, wash with Hanks solution, centrifuge for 10min (1500r/min), add 1mlNH ₄ Cl for 3.5min, add Hanks solution, centrifuge for 10min (1500r/min), then centrifuge for 10min with Hanks solution (1500r/min) wash once. Then the cells were suspended in 2 mL of complete culture medium, and the number of living cells (should be above 95%) was counted by trypan blue staining, and the cell concentration was adjusted to 2×10 ⁶ cells/Ml.

3. Lymphocyte proliferation response:

Add the cell suspension to the 24-well culture plate in separate holes, 1Ml per well. Then add the corresponding reagents.

Blank control PBS50μl High-concentration freeze-dried bamboo shoots crude polysaccharide powder 50μl High-concentration vacuum-dried bamboo shoots crude polysaccharide powder 50μl ConA control 50 μl Low-concentration freeze-dried bamboo shoots crude polysaccharide powder 50μl High-concentration vacuum-dried bamboo shoots crude polysaccharide powder 50μl ConA synergy control PBS50μl+ConA25μl High-concentration freeze-dried bamboo shoots crude polysaccharide powder 50μl+ConA25μl High-concentration vacuum-dried bamboo shoots crude polysaccharide powder 50μl+ConA25μl

Incubate at 37°C for 72 hours in 5% CO2. 4 hours before the end of the culture, gently aspirate 0.7 mL of supernatant from each well, add 0.7 mL of RPMI1640 culture medium without calf serum, and add MTT (5 mg/mL) 50 ul/well at the same time, and continue to culture for 4 hours. After culturing, add 1mL of acidic isopropanol to each well, and mix well by pipetting to completely dissolve the purple crystals. Then it was divided into 96-well culture plates, and each well was divided into 3 wells as parallel samples, and an enzyme-linked immunosorbent assay was used. Optical density values were measured at a wavelength of 570 nm.

IV: Experimental results and judgments:

1. Experimental results:

2. Judgment:

It can be seen from the experimental results that the OD ₅₇₀ value of the vacuum-dried bamboo shoot crude polysaccharide powder test solution is significantly higher than the values of other items. From the results of this experiment, the vacuum-dried bamboo shoot crude polysaccharide powder test solution has the function of promoting cellular immunity.

Two: Acute toxicity test of crude polysaccharides from bamboo shoots:

1. Experimental materials and methods

1.1 Experimental animals Kunming white mice 18-22g, half male and half male, ready for use after quarantine.

1.2 Experimental method Mice were divided into 6 dose groups according to body weight, 20 in each group. Fasting without food and water for 12 hours before the experiment, divided into six dosage groups according to the pre-test results: 11.03g/Kg, 13.00g/Kg, 14.25g/Kg, 16.00g/Kg, 18.40g/Kg, 20.75g/Kg , the ratio of adjacent doses is 0.88, dilute it and give it to the mice, and observe it once every 4h, 24h, 48h, 72h, and 7 days after the sample is given. After 7 days, the toxic reaction and death number of the mice were recorded.

2. Test results:

Dosage (g/Kg) Number of animals (only) Number of dead animals (only) (♂:♀) mortality rate(%) 11.03 20 0 (0:0) 0 13.00 20 0 (0:0) 0 (but with symptoms of poisoning) 14.25 20 10 (2:3) 50 16.00 20 12 (3:3) 60 18.40 20 16 (3:5) 80 20.75 20 20 (5:5) 100

After seven consecutive days of observation, there was no further death. No obvious pathological changes were found in the autopsy of dead animals. From this experiment, it can be concluded that the LD50 of this sample is 14.25g/Kg (90% credible limit). Judging by the acute toxicity grading standard, the sample is actually non-toxic.

In summary, the crude polysaccharides from bamboo shoots have good effects in promoting cellular immunity in vitro, and have little toxic and side effects, so they have good development and application prospects.

The raw materials used in the bamboo shoot crude polysaccharide of the present invention are old roots or bamboo shoots of bamboo shoots and their dried products, rather than dried shoots with high commercial value, which will improve the effective utilization rate of bamboo shoots to a certain extent. The method of the invention has simple process, meets the requirements of the Chinese Pharmacopoeia, and does not need special equipment. The crude polysaccharide of bamboo shoots in the present invention is prepared by methods suitable for industrial production such as common water extraction, membrane concentration or low-pressure film concentration, and alcohol precipitation extraction. The obtained bamboo shoot crude polysaccharide is light grayish yellow powder.

The steps of the above-mentioned method for extracting crude polysaccharides from bamboo shoots are as follows: calculate in parts by weight, weigh 0.1 to 20 parts of dried bamboo shoots, add 0.5 to 200 parts of water, heat and reflux for extraction for 3 to 5 hours, and control the temperature at 60 to 105° C. The temperature can be set higher, as long as the temperature is maintained in the later stage), the extract is concentrated in vacuum (vacuum degree -0.4-0.01Mpa, temperature 55-70°C), concentrated to a concentration of 25-55 degrees (by refractometer) ,cool down. Under stirring, slowly add the concentrated solution to 3-15 times the volume of ethanol or slowly add 3-15 times the volume of ethanol to the concentrated solution that is stirred (stirring speed is 30-400 rpm), and precipitate 0.2- After 10 hours, vacuum-dry or freeze-dry the precipitate to obtain crude polysaccharide from bamboo shoots.

Crude polysaccharides from bamboo shoots have obvious immune regulation function, and are tasteless and odorless. They can be easily compatible with other food raw materials, or used as the main ingredient alone to develop products, so that the active ingredients of bamboo shoots can be fully utilized, thus filling the gap in the utilization of bamboo shoots in my country. A blank item has practical value.

Detailed ways

The following examples are used to further illustrate the present invention, but do not limit the scope of the present invention thereby.

Example 1:

Calculated by weight, weigh 500 grams of dried bamboo shoots, put them into a reflux extraction tank, add 2500 milliliters of water, turn on the heater for heating, and connect condensed water in the reflux condenser, when the feed liquid boils, turn off the heat Make the feed liquid boil slightly, extract for 4 hours, and obtain a dark brown extract after cooling; put the extract into a vacuum thin film evaporator, control the vacuum degree at -0.08, and the temperature is 65 ° C, and evaporate the feed liquid to 40 degrees ( With refractometer), obtain about 200 milliliters of concentrates; Put 1000 milliliters of 95% edible ethanol into 2000 milliliters of beakers, insert a stirrer, start stirring, adjust the rotating speed at 100 rev/min, concentrate the concentrate at 20 milliliters/minute Add it into 95% edible ethanol at a high speed, continue stirring for more than 30 minutes after adding the material, so that the polysaccharide is fully precipitated; stop stirring, precipitate for 4 hours, suck out the supernatant, filter the precipitate with filter paper, and filter the precipitate with 50 ml Wash twice with water and ethanol, and vacuum-dry (temperature 55 ℃, vacuum degree-0.08Mpa), obtain about 5 grams of off-white bamboo shoot crude polysaccharide powder.

Example 2:

Calculated by weight, take 50 kilograms of dried bamboo shoots, put them into a multifunctional extraction tank, add 300 kilograms of purified water, open the jacket steam valve for heating, and connect condensed water in the reflux condenser, when the feed liquid boils , close the steam valve, keep the material above 70°C, extract for 6 hours, extract the slag with 200 kg of purified water, combine the extracts, and obtain a dark brown extract after cooling; put the extract into a vacuum concentration tank , control the vacuum degree at -0.08, the temperature is 70 ° C, evaporate the feed liquid to 38 degrees (by refractometer), and obtain about 60 liters of concentrated liquid; put 400 liters of 95% edible ethanol into a 500 liter recovery tank, open Stir, adjust the speed at 50 rpm, add the concentrated solution to 95% edible ethanol at a rate of 1 liter/min, continue stirring for 60 minutes after adding the material, so that the polysaccharide is fully precipitated; stop stirring, and precipitate for 4 hours, Suck out the supernatant, pour the lower layer into a three-legged centrifuge, use industrial filter cloth as the filter medium, filter out the precipitate, collect the precipitate, wash the precipitate twice with 10 liters of absolute ethanol, and vacuum dry the precipitate after centrifugation (45 ℃ of temperature, vacuum tightness-0.09Mpa), obtain about 650 grams of off-white bamboo shoots crude polysaccharide powder.

Claims (1)

1, bamboo shoot polysaccharide has application in the health food of immunoloregulation function in preparation.