Use of propolis and / or propolis extract in reducing bioavailability of heavy metals in food

Abstract

The present application belongs to the technical field of heavy metal pollution removal, and relates to application of propolis and / or propolis extract in reducing bioavailability and / or toxicity of heavy metals in food. The present application develops a product containing propolis and / or propolis extract for reducing bioavailability and toxicity of lead in food. The propolis and / or propolis extract not only can supplement nutritional elements without side effects, but also has good effect in reducing toxicity, and has wider application prospect compared with traditional chemical heavy metal chelating agents. The propolis and / or propolis extract can reduce bioavailability and / or toxicity of heavy metals in food, and reduce potential health risks of heavy metals to human body. The present application has important significance for reducing and preventing heavy metal hazards of agricultural products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of heavy metal pollution removal, and particularly relates to application of propolis and / or propolis extract in preparation of products for reducing bioavailability and / or toxicity of heavy metals in food. BACKGROUND

[0002] At present, heavy metal pollution in farmland in southern China is very serious, leading to the problem of excessive heavy metals in agricultural products, which has become an important factor endangering public health. In particular, some crop (rice and vegetable, etc.) varieties have the characteristics of absorbing heavy metals, and the safety problem of agricultural products has become a hot spot of public concern. Diet is considered to be the main way of human exposure to heavy metals, which poses a serious threat to human health.

[0003] Traditional heavy metal antidotes usually use chemical chelating agents such as dimercaptosuccinic acid DMSA and CaNa2EDTA, which can inhibit the absorption and toxicity of Pb to a certain extent, but have certain side effects. Since the actual absorption level of heavy metals in agricultural products by the human body is affected by many factors, other dietary materials or ingredients that enter the gastrointestinal tract at the same time may interact, and if there is a certain food material or food ingredient that can effectively reduce the availability and accumulation of heavy metals during the common digestion process, it can play a detoxification role. Therefore, finding certain specific dietary ingredients to regulate the availability and absorption of heavy metals in the body is another effective way to solve the harm of heavy metals in agricultural products to the human body. SUMMARY

[0004] The purpose of the present application is to provide application of propolis and / or propolis extract in preparation of products for reducing bioavailability and / or toxicity of heavy metals in food. Developing products for reducing bioavailability and toxicity of lead in food has important significance for reducing and preventing harm of heavy metals in agricultural products.

[0005] The present application provides application of propolis and / or propolis extract in preparation of products for reducing health risks of heavy metals in food.

[0006] The present application also provides application of propolis and / or propolis extract in preparation of products for reducing toxic effects after heavy metal poisoning in animals.

[0007] Preferably, the heavy metal is lead or cadmium.

[0008] Preferably, the propolis extract comprises one or more than two of kaempferol, quercetin and caffeic acid phenethyl ester.

[0009] Preferably, the product comprises a dietary supplement or a drug, and the drug comprises a heavy metal antidote.

[0010] The present application also provides a product for reducing the bioavailability and / or toxicity of heavy metals in food or reducing the toxic effects after heavy metal poisoning in animals, which comprises propolis and / or propolis extract and excipients.

[0011] The present application also provides a method for reducing the bioavailability and / or toxicity of heavy metals in food for non-disease treatment purposes, which comprises the following steps: mixing food with propolis and / or propolis extract for consumption; or consuming propolis and / or propolis extract before and / or after consuming food.

[0012] Preferably, the dosage of propolis and / or propolis extract is 3.5-33 mg / kg of food.

[0013] The present application also provides a method for evaluating the health risks of heavy metals in food, which comprises an in vitro simulated gastrointestinal digestion evaluation method or an animal model evaluation method; the in vitro simulated gastrointestinal digestion evaluation method is used to detect the concentration of heavy metals in digestive juice and food and calculate the bioavailability; the animal model evaluation method is used to detect the content of heavy metals in mouse organs and calculate the bioavailability; and the bioavailability and bioavailability are combined to comprehensively evaluate the health risks of heavy metals in food.

[0014] Preferably, the heavy metal is lead or cadmium.

[0015] The present application provides the use of propolis and / or propolis extract in the preparation of a product for reducing the bioavailability and / or toxicity of heavy metals in food. The present application finds that propolis and / or propolis extract not only can supplement nutritional elements without side effects, but also can reduce the bioavailability and / or toxicity of heavy metals in food, and is used for reducing the toxic effects after heavy metal poisoning in animals, reducing heavy metal toxicity and damage, and providing a new technical measure for solving heavy metal poisoning in the public.

[0016] Propolis is widely used in food additives or dietary supplements in daily life, and has no side effects on human body within a proper additive amount. The present application can play the role of competition or adsorption of propolis itself on Pb and Cd ions in contaminated food, effectively reduce the bioavailability of Pb and Cd in food, reduce the accumulation of Pb and Cd in animal body and reduce the risk of toxicity. Compared with other chelating agent therapies for treating Pb or Cd poisoning, the method of the present application has the characteristics of low cost, easy availability, low toxicity and good effect. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings.

[0018] Figure 1 The result graph of the influence of adding propolis and the like on the bioavailability of Pb and Cd in the middle intestine stage of rice 1 provided by the present application;

[0019] Figure 2 The result graph of the influence of adding propolis and the like on the bioavailability of Pb and Cd in the middle intestine stage of rice 2 provided by the present application;

[0020] Figure 3 The result graph of the influence of other several propolis extracts on the bioavailability of Pb and Cd in the middle intestine stage of rice 1 provided by the present application;

[0021] Figure 4 The result graph of the influence of adding propolis and the like on the bioavailability of Pb and Cd in rice 1 provided by the present application;

[0022] Figure 5 The result graph of the influence of adding propolis and the like on the health risk entropy of Pb and Cd after eating rice 1 and rice 2 provided by the present application. DETAILED DESCRIPTION

[0023] The present application provides the use of propolis and / or propolis extract in the preparation of a product for reducing the bioavailability and / or toxicity of heavy metals in food. In the present application, the heavy metal is preferably lead or cadmium, more preferably lead. In the present application, the propolis is preferably free of heavy metals. The present application does not have special limitations on the source of the propolis, and a conventional commercially available purified propolis powder free of heavy metals known to those skilled in the art can be used. Although the chemical composition of propolis from different sources is different, it contains a large amount of flavonoids such as chrysin, kaempferol, quercetin, myricetin, and gingerol. In the present application, the propolis extract preferably includes one or more of kaempferol, quercetin, and caffeic acid phenethyl ester. The results of the examples of the present application show that kaempferol, quercetin, and caffeic acid phenethyl ester all have a reducing effect on the bioavailability of lead. The present application does not have special limitations on the source of the propolis extract, and a conventional commercially available purified propolis product can be used. In the present application, the product preferably includes a dietary supplement or a drug, and the drug preferably includes a heavy metal detoxifier. Propolis has no side effects on the human body, and has complexation, competition, or adsorption effects on Pb or Cd ions in contaminated food, which can effectively reduce the bioavailability of Pb and Cd in food, reduce their accumulation in the animal body, and reduce the risk of toxicity. In addition, propolis and its propolis extract can scavenge free radicals as an antioxidant substance, effectively restore the level of reduced glutathione (GSH), and play a protective role on the liver by inhibiting Pb and Cd-induced oxidative stress.

[0024] The present application also provides the use of propolis and / or propolis extract in the preparation of a product for reducing the toxic effects after heavy metal poisoning in animals. In the present application, the heavy metal is preferably lead or cadmium, more preferably lead. Compared with other treatments for heavy metal poisoning such as chelator therapy, the method of the present application using a dietary ingredient (propolis and / or propolis extract) has the characteristics of low cost, easy availability, low toxicity, and good effect. In the present application, the propolis extract preferably includes one or more of kaempferol, quercetin, and caffeic acid phenethyl ester. In the present application, the propolis is preferably free of heavy metals. The present application does not have special limitations on the source of the propolis, and a conventional commercially available purified propolis powder free of heavy metals known to those skilled in the art can be used. In the present application, the product preferably includes a dietary supplement or a drug, and the drug preferably includes a heavy metal detoxifier.

[0025] The present application also provides a product for reducing the bioavailability and / or toxicity of heavy metals in food or reducing the toxic effects after heavy metal poisoning in animals, which includes propolis and / or propolis extract, and excipients. In the present application, the heavy metal is preferably lead or cadmium, more preferably lead. The present application mixes propolis and / or propolis extract with food, especially food with excessive heavy metals, which has a detoxification effect. The food in the present application preferably includes rice or vegetables.

[0026] The present application also provides a method for reducing the bioavailability and / or toxicity of heavy metals in food for non-disease treatment purposes, comprising the following steps: mixing the food with propolis and / or propolis extract for consumption; or consuming propolis and / or propolis extract before and / or after consuming the food. In the present application, the heavy metal is preferably lead or cadmium, and more preferably lead. In the present application, the dosage of propolis and / or propolis extract is preferably 3.5-33 mg / kg of food, i.e. the amount of propolis and / or propolis extract added to the food is preferably 3.5-33 mg / kg.

[0027] The present application also provides a method for evaluating the health risk of heavy metals in food, comprising an in vitro simulated gastrointestinal digestion evaluation method or an animal model evaluation method; detecting the concentration of heavy metals in the digestive juice and food using the in vitro simulated gastrointestinal digestion evaluation method to calculate the bioavailability; detecting the content of heavy metals in the organs of mice using the animal model evaluation method to calculate the bioavailability; and combining the values of bioavailability and bioavailability to comprehensively evaluate the health risk of heavy metals in food. The present application does not have special restrictions on the simulated gastrointestinal digestion evaluation method or the animal model evaluation method, and conventional evaluation methods known to those skilled in the art can be used.

[0028] Specifically, the in vitro simulated gastrointestinal digestion evaluation method of the present application preferably comprises the following steps:

[0029] Mixing propolis and / or propolis extract, food and simulated gastric juice, simulating gastric digestion, centrifuging to obtain supernatant, filtering to obtain filtrate;

[0030] Mixing the filtrate with gastric juice, trypsin and bile salt, adjusting the pH value to 7, simulating intestinal digestion, centrifuging to obtain supernatant, filtering to obtain digestive juice;

[0031] Detecting the content of heavy metals in the digestive juice, and calculating the bioavailability of heavy metals according to formula I;

[0032] Bioavailability (%) = (soluble concentration of heavy metals in digestive juice (mg / L) x volume of digestive juice (L)) / (concentration of heavy metals in food (mg / kg) x weight of food sample (kg)) x 100, formula I;

[0033] The animal model evaluation method comprises the following steps:

[0034] The mice are divided into a treatment group, a blank control group and a standard control group for feeding, the treatment group is fed with heavy metal contaminated feed, and is fed with propolis and / or propolis extract by gavage; the blank control group is fed with uncontaminated feed; the standard control group is fed with heavy metal standard solution plus food; the weight of the supplied and remaining food is accurately recorded every day, after 10 days of steady state exposure, the mice are fasted overnight, weighed, and then humanely sacrificed, and liver and kidney samples are collected; the samples are freeze-dried to a constant weight, the content of heavy metals in the liver and kidney of the mice is detected, and the bioavailability of heavy metals is calculated according to the formula III;

[0035] Bioavailability (%) = (treatment group / blank control group organ heavy metal content (mg / kg)) / (standard control group organ heavy metal content (mg / kg)) x (standard dose (mg)) / (food sample dose (mg)) x 100, formula III.

[0036] In the present application, the heavy metal is preferably lead or cadmium, and more preferably lead. The present application combines the values of bioavailability and bioavailability to achieve comprehensive evaluation of the health risk of heavy metals in food. The evaluation method of the present application is simple to operate, easy to control test conditions, less polluting, economical and accurate. Bioavailability and bioavailability of heavy metals are two commonly used and most useful indicators for evaluating health risks. The evaluation method of the present application is suitable for evaluating the health risk of heavy metals in various foods such as cereals and vegetables.

[0037] In order to further illustrate the present application, the application of propolis and / or propolis extract in the preparation of products for reducing the bioavailability and / or toxicity of heavy metals in food provided by the present application is described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0038] Example 1

[0039] This embodiment provides a method for evaluating the bioavailability of heavy metals during digestion, including the following steps: The bioavailability of Pb and Cd in rice is determined using an internationally accepted in vitro simulated gastrointestinal digestion method (physiologically based extraction test). 1) Stomach stage: Prepare 1 L of simulated gastric juice, adjust the pH to 1.5 ± 0.05 with 37% HCl, add 1.25 g of pepsin, and thoroughly dissolve and mix. Mix the food with propolis and / or propolis extract in a reactor (50 ml centrifuge tube) at a ratio of 1:100 to the gastric juice. Place in a constant temperature water bath at 37℃ and shake for 1 h, centrifuge, then collect the supernatant and filter for later use. Each treatment is repeated 3 times, with a blank control. 2) Small intestine stage: Add pancreatic enzymes and bile to the gastric juice, adjust the pH of the gastric stage reaction solution to 7 with saturated NaHCO3 solution, shake for 4 h, maintaining the pH at 7.0 ± 0.05, then centrifuge and filter for analysis. The contents of Pb and Cd in the digestion fluid were detected separately, and the bioavailability of Pb and Cd was calculated according to the formula shown in Equation I. The evaluation results were obtained based on the bioavailability of heavy metals.

[0040] Bioavailability (%) = (Soluble concentration of heavy metals in digestive fluid (mg / L) × Volume of digestive fluid (L)) / (Concentration of heavy metals in food (mg / kg) × Weight of food sample (kg)) × 100, Equation I.

[0041] In an in vitro simulated gastrointestinal digestion method, three representative substances were selected from propolis extract: kaempferol, quercetin, and phenethyl caffeate. Using highly contaminated rice (rice 1, with Pb and Cd contents of 7.14 mg / kg and 0.82 mg / kg, respectively), low, medium, and high doses (1%, 3%, and 6%, respectively, where % refers to mass percentage, the same below) of propolis, kaempferol, phenethyl caffeate, and quercetin significantly reduced Pb bioavailability in the intestinal stage of digestion. The reduction was most pronounced with low, medium, and high doses of propolis, reaching 40%, 57%, and 67%, respectively. Furthermore, kaempferol also showed a strong inhibitory effect, reducing Pb bioavailability by 37%, 43%, and 45% at low, medium, and high doses, respectively. Figure 1 The results indicate that the inhibitory effect is relatively stable. Although low-dose quercetin showed the weakest inhibitory effect on Pb bioavailability, it still achieved a 15% reduction. For Cd, neutralizing high doses of propolis reduced Cd bioavailability by 34% and 41%, respectively. High-dose caffeic acid phenethyl ester also showed some effect in inhibiting Cd, indicating that propolis, by combining multiple active ingredients, can better inhibit Cd bioavailability in food.

[0042] Example 2

[0043] This study used another low-pollution rice variety, Rice 2 (Pb and Cd contents were 3.02 mg / kg and 0.71 mg / kg, respectively), to further verify the inhibitory effect of a moderate dose of propolis and its extracts used in Example 1 on the bioavailability of heavy metals in the digestive tract. The dosage was 3%. Using the above-mentioned in vitro simulated gastrointestinal method, it was found that propolis, quercetin, kaempferol, and phenethyl caffeate significantly reduced Pb bioavailability, with reductions of 53%, 25%, 21%, and 24%, respectively. Figure 2 Regarding Cd, only propolis significantly reduced Cd bioavailability, by as much as 47%. Quercetin, kaempferol, and phenethyl caffeate did not inhibit Cd bioavailability during the simulated intestinal digestion process. These results validate that the effects of propolis and its extracts on Pb and Cd bioavailability are consistent regardless of whether the food is highly or lightly contaminated. Notably, the reduction effect of propolis and its extracts on Pb bioavailability is significantly better than that on Cd, with reductions exceeding 20%.

[0044] Example 3

[0045] Based on the heavy metal bioavailability results of Examples 1 and 2 and the internationally accepted target hazard factor method (Equation II), the health risk entropy (THQ) of Pb and Cd after an adult consumes 326g of m1 or m2 was estimated. Figure 3 The effects of propolis and its extracts on the health risk entropy of Pb and Cd were shown. The results showed that the addition of propolis and its extracts reduced the Pb-THQ of rice 1, but the value remained above 1, indicating a potential health risk (due to the high Pb content in rice 1). For rice 2 (low concentration), the addition of propolis and its extracts reduced the Pb-THQ to below 1, demonstrating that the addition of propolis, quercetin, kaempferol, and phenethyl caffeate is beneficial in reducing the health risk of Pb after consuming rice. Propolis effectively reduced the Cd-THQ in both rice 1 and 2, but the effect of its extracts on reducing Cd-THQ in both rice 1 and 2 was not ideal.

[0046] THQ = (exposure frequency (d / year) × exposure duration (year) × average daily intake (g / d) × heavy metal content in food (mg / kg)) / (internationally stipulated daily allowable level (mg / kg·d) × body weight (kg) × average time (d)), Equation II.

[0047] Comparative Example 1

[0048] To compare the inhibitory effects of propolis and its extracts on the bioaccessibility and availability of heavy metals, propolis extract was added to the high-pollution rice (Rice 1) at a low, medium, and high dose (1%, 3%, and 6%, respectively) and compared with calcium carbonate. The results, as shown in Table 1, show that propolis extract and calcium carbonate can significantly reduce the bioaccessibility of Pb and Cd, with the reduction rates reaching 66% and 37%, respectively. Figure 2 In comparison, although quercetin, kaempferol, and caffeic acid phenethyl ester have a slightly weaker effect on the bioaccessibility of Pb than calcium carbonate, propolis has a significantly stronger effect than calcium carbonate. Similarly, propolis has a much stronger effect on the bioaccessibility of Cd than calcium carbonate, indicating that propolis detoxifies the potential of Pb and Cd in food.

[0049] Comparative Example 2

[0050] To compare the inhibitory effects of propolis and its extracts on the bioaccessibility and availability of heavy metals, propolis extract was added to the high-pollution rice (Rice 1) at a low, medium, and high dose (1%, 3%, and 6%, respectively) and compared with calcium carbonate. The results, as shown in Table 1, show that propolis extract and calcium carbonate can significantly reduce the bioaccessibility of Pb and Cd, with the reduction rates reaching 66% and 37%, respectively. Figure 4 In comparison, although quercetin, kaempferol, and caffeic acid phenethyl ester have a slightly weaker effect on the bioaccessibility of Pb than calcium carbonate, propolis has a significantly stronger effect than calcium carbonate. Similarly, propolis has a much stronger effect on the bioaccessibility of Cd than calcium carbonate, indicating that propolis detoxifies the potential of Pb and Cd in food.

[0051] Experimental Example 1

[0052] To explore the actual effect of propolis and its extracts on reducing the health risk of heavy metals, an in vivo animal model was adopted to analyze the inhibition of propolis and its extracts on the bioavailability of heavy metals, including the following steps: a mouse animal model was established, Balb / C female mice (about 6 weeks old, 16-20 g) were selected, the mice were given basic feed, and after adaptive feeding for 7 days, 3 mice per cage were randomly allocated to metabolic cages, and 3 replicates were set for each treatment. The treatment group was fed with contaminated rice (rice 1) for multiple times, and the mice were given propolis and / or propolis extracts by gavage, 2 ml each time, once a day, the dose of propolis was 3.5 mg / kg of food, the doses of kaempferol, quercetin and caffeic acid phenethyl ester were 33 mg / kg of food, and the dose of calcium carbonate was 20 mg / kg of food. The mice fed with uncontaminated rice were used as the blank control group, and the mice fed with rice added with Pb or Cd standard solution (0 mg / kg, 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg and 1.6 mg / kg, respectively) were used as the standard control group. The food intake was accurately recorded every day. After 10 days of steady-state exposure, the mice were fasted overnight, and liver and kidney samples were collected. Freeze-drying was performed to constant weight for standby. The content of heavy metals in the liver and kidney of mice was detected, and the bioavailability of heavy metals was calculated according to the formula III.

[0053] Bioavailability (%) = (treatment group / blank control group organ heavy metal content (mg / kg)) / (standard control group organ heavy metal content (mg / kg)) x (standard dose (mg)) / (food sample amount (mg)) x 100, formula III.

[0054] After 10 days of exposure of the mice to rice 1, quercetin and caffeic acid phenethyl ester effectively reduced the accumulation of Pb in the kidney and liver; but propolis and its extracts had no significant effect on the accumulation of Cd in the liver and kidney of mice (p>0.05), as shown in Table 1. Propolis, kaempferol, quercetin and caffeic acid phenethyl ester all significantly reduced the Pb bioavailability of rice 1, and the reduction amplitude from high to low was quercetin (37%)> caffeic acid phenethyl ester (31%)> propolis (26%)> kaempferol (23%), as shown in Figure 5 The inhibition of propolis, quercetin and caffeic acid phenethyl ester on the bioavailability of Pb was obviously better than that of calcium carbonate (23.5%). Therefore, compared with the recognized heavy metal passivator calcium carbonate, propolis and its extracts have better effect on reducing the bioavailability of heavy metals (especially Pb) in food.

[0055] Table 1 Content of Pb and Cd in the organs of mice in the rice 1 group with dietary ingredients added (mg / kg, dry weight, mean ± standard deviation, n=3)

[0056]

[0057] Note: Different lower case letters in the same column indicate that there are significant differences (p < 0.05) in the content of Pb and Cd in the organs of mice in the rice 1 group with the addition of dietary ingredients.

[0058] The propolis, kaempferol, quercetin and caffeic acid phenethyl ester of the present application can effectively reduce the bioavailability of Pb in contaminated rice in the in vitro simulated gastrointestinal digestion process at low, medium and high doses, and reduce the solubility of heavy metals in the gastrointestinal tract. The propolis, kaempferol, quercetin and caffeic acid phenethyl ester of the present application have the ability to complex heavy metals, prevent metal ions from catalyzing the chain reaction in the autoxidation reaction, and have better ability to inhibit the bioavailability of Pb in animals than calcium carbonate, and perform more excellent in reducing the accumulation of Pb in the organs of the body. The health risk entropy estimated based on the bioavailable content of Pb in the simulated gastrointestinal tract can well reflect that the propolis, kaempferol, quercetin and caffeic acid phenethyl ester can reduce the risk of heavy metals after eating contaminated food to a certain extent. The propolis of the present application can effectively reduce the bioavailability of Cd in contaminated rice in the in vitro simulated gastrointestinal digestion process and the bioavailability of Cd in animals, and significantly reduce the risk of Cd after eating rice. In addition, the propolis, kaempferol, quercetin and caffeic acid phenethyl ester of the present application not only can supplement the required nutritional elements of the body without side effects, but also have strong antioxidant properties to improve the oxidative damage of heavy metals to the body, and have good application prospect in ensuring the safe consumption and health risk of agricultural products.

[0059] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. Use of a propolis extract in the manufacture of a product for reducing the bioavailability and / or toxicity of lead in food; the propolis extract being one or both of kaempferol and caffeic acid phenethyl ester.

2. Use of a propolis extract in the manufacture of a product for reducing the toxic effects following heavy metal lead poisoning in an animal; the propolis extract being one or both of kaempferol and caffeic acid phenethyl ester.

3. Use according to claim 1 or 2, characterized in that, The product comprises a dietary supplement or a medicament, the medicament comprising a heavy metal antidote.

4. A product for reducing the bioavailability and / or toxicity of heavy metal lead in food or reducing the toxic effects after heavy metal lead poisoning in animals, characterized in that, The product comprises the propolis extract, being one or both of kaempferol and caffeic acid phenethyl ester, and an excipient. The product comprises the propolis extract, being one or both of kaempferol and caffeic acid phenethyl ester, and an excipient.

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