Method for efficiently generating mammal lignans through ruminant intestinal flora biotransformation effect

Abstract

The invention relates to a method for efficiently generating mammalian lignans through ruminant intestinal flora biotransformation, and belongs to the technical field of raw material medicine production. In order to solve the problem that the existing preparation method cannot be practically applied to the production of raw material medicines of enterodiol and enterolactone, the invention provides a method for efficiently generating mammal lignans through the biological transformation effect of intestinal flora of ruminants. The method comprises the following steps: mixing an organic material containing a plant lignan precursor into daily ration of a milk-producing ruminant according to a certain addition amount to prepare mixed daily ration, feeding the milk-producing ruminant by using the mixed daily ration in a conventional mode, collecting milk of the milk-producing ruminant, and extracting mammal lignan-enterodiol and / or enterolactone from the milk. The transformation method and the extraction method are simple and efficient, the production cost is low, the production period is short, strict bacteria in-vitro anaerobic culture conditions do not need to be established and controlled, and the obtained mammal lignans are high in yield, stable in activity and suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of raw material drug production, and in particular relates to a method for efficiently generating mammalian lignans through biotransformation of ruminant intestinal flora. Background Art

[0002] Mammalian lignans enterodiol (END) and enterolactone (ENL) are similar in structure to the estrogen estradiol, so they can bind to estrogen receptors and show estrogenic and anti-estrogenic effects. When the estrogen level in the body is too low, enterodiol and enterolactone play a certain estrogen-like role by binding to estrogen receptors, which is of great significance to menopausal women; when the estrogen level in the body is too high, enterodiol and enterolactone compete with natural estrogen receptors and play an estrogen antagonistic role, which is crucial for the prevention and treatment of estrogen-related cancers such as ovarian cancer, breast cancer, and uterine cancer.

[0003] Enterodiol and enterolactone can be synthesized by chemical methods, but chemical synthesis has problems such as complex process, high cost, and severe environmental pollution. The products of chemical synthesis are difficult to meet the standards of natural products in terms of purity and structural consistency, which limits their wide application in the fields of medicine and health care products.

[0004] The microbial transformation method uses microbial cells or enzyme systems as catalysts, which can achieve the modification and transformation of complex chemical structures under mild conditions, and has the advantages of high efficiency, environmental protection, and sustainability. However, the microbial transformation method also faces many technical bottlenecks in the process of producing enterodiol and enterolactone. Under in vitro experimental conditions, the microbial transformation method converts lignans into enterodiol and enterolactone, which requires a system engineering of collaborative operations among multiple microorganisms, involving multiple links such as deglycosylation, demethylation, and dehydroxylation, and each link requires different microorganisms. Although many laboratories in the world have successfully isolated bacteria that play a role in a certain link, it is still a huge challenge to isolate all the required bacteria and open up the entire metabolic pathway. There is no successful case so far. Most functional bacteria will lose their transformation function soon after passage under non-natural conditions in vitro. Moreover, the in vitro culture conditions are not easy to control, and a slight difference can cause low conversion or no conversion at all. Therefore, the problems of unstable yield of strains, low conversion efficiency, and difficulty in product separation and purification in the microbial transformation method make it difficult to obtain sufficient yields of enterodiol and enterolactone, and cannot be actually applied to the production of raw materials for enterodiol and enterolactone. Summary of the invention

[0005] In order to solve the problem that the existing preparation methods cannot be actually applied to the production of raw materials of enterodiol and enterolactone, the present invention provides a method for efficiently producing mammalian lignans through the biotransformation of ruminant intestinal flora.

[0006] The technical solution of the present invention:

[0007] A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, comprising mixing an organic material containing plant lignan precursors into a diet of a lactating ruminant in a certain amount to prepare a mixed diet, feeding the lactating ruminant with the mixed diet in a conventional manner, collecting milk from the lactating ruminant and extracting mammalian lignans therefrom.

[0008] Furthermore, the mammalian lignans are enterodiol and / or enterolactone.

[0009] Furthermore, the organic material containing plant lignan precursors includes one or a combination of flaxseed, sesame seed, rapeseed, rye bran, wheat bran, barley bran, oat bran, corn bran, burdock, sorghum, buckwheat, coix seed, soybean, red bean, soybean, mung bean, kidney bean, black bean, potato, sweet potato, taro, yam or seaweed.

[0010] Furthermore, the milk-producing ruminant includes cattle, yaks, buffaloes or camels.

[0011] Furthermore, the addition amount of the organic material containing plant lignan precursors is 12% to 86% of the mass of the mixed diet.

[0012] Furthermore, when extracting mammalian lignans, milk produced by lactating ruminants on the second day or later after consuming the mixed diet described in claim 2 is collected.

[0013] Furthermore, the method for extracting mammalian lignans from the milk of lactating ruminants is as follows: the milk is mixed with ethanol having a mass concentration of 95% to obtain a sample solution, the sample is loaded onto a macroporous resin chromatographic column, and eluted with an ethanol aqueous solution with a certain mass concentration, and the eluate containing mammalian lignans is collected and concentrated by rotary evaporation to obtain mammalian lignans.

[0014] Furthermore, the elution is a gradient elution with ethanol aqueous solution having a mass concentration of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50% in sequence.

[0015] Furthermore, when extracting mammalian lignans, the ethanol mass concentration of the sample solution is 30% to 40%; the eluate with an ethanol mass concentration of 20% to 50% is collected and concentrated by rotary evaporation to obtain mammalian lignans.

[0016] Furthermore, if the organic material containing plant lignans used in the mixed diet is oil-containing plant seeds, it needs to be defatted, and the method is to use mechanical hot oil pressing method or solvent extraction method or low temperature cold pressing method to press the oil from flax seeds, and then detoxify the defatted plant lignans obtained for the preparation of the mixed diet. The detoxification treatment can be carried out by extrusion method, microbial method, microwave method, autoclave method, boiling method or solvent method.

[0017] Beneficial effects of the present invention:

[0018] The present invention converts plant lignan precursors into mammalian lignans through bacteria in the bodies of milk-producing ruminants. The conversion method and extraction method are simple and efficient, with low production cost and short production cycle. There is no need to establish and control harsh in vitro anaerobic bacterial culture conditions. The obtained mammalian lignans have high yield and stable activity, are suitable for large-scale production, can fill market gaps, and meet the society's urgent needs in the fields of medical care, health, wellness, food, scientific research, etc.

[0019] The plant lignans-rich mixed diet of the present invention is used for the method of efficiently generating mammalian lignans through biotransformation of ruminant intestinal flora, wherein the weight (mass) proportion of the plant lignans-rich food in the mixed diet is 12% to 86%, and the food processing temperature is 58 to 121°C.

[0020] The method of efficiently generating mammalian lignans from plant lignans by intestinal flora in vivo conversion of plant lignans of the present invention is established in accordance with natural laws and according to natural conditions, and has incomparable advantages over in vitro methods. In vivo conversion is based on the symbiotic relationship between intestinal flora and the host, and both parties benefit, so the process is spontaneous, without the need for external force to intervene. The specific biochemical mechanism system includes multiple metabolic steps such as deglycosylation, demethylation, and dehydroxylation, and the final result is that the host provides habitats and food (plant lignans, etc.) for these bacteria, and these bacteria provide beneficial metabolites (mammalian lignans, etc.) for the host. The in vivo conversion process is complete, thorough, and efficient.

[0021] The method of efficiently generating mammalian lignans from plant lignans through intestinal flora biotransformation of the present invention, wherein the main component of the raw material plant lignans is flax lignan secoisolariciresinoldiglucoside (SDG), which mostly exists in the form of oligomers, forms ester bonds with 3-hydroxy-3-methylglutaric acid (methylglutaricacid) and cinnamic acid, and also exists in the form of glucoside with other phenolic compounds. The intestinal flora can metabolize flax lignans into mammalian lignans through biotransformation, and the main steps include hydrolyzing and deglycosylation to produce secoisolariciresinol (SECO), and then demethylating and dehydroxylating to produce mammalian lignans enterodiol and enterolactone.

[0022] The method of efficiently generating mammalian lignans from plant lignans through intestinal flora biotransformation of the present invention can produce mammalian lignans in milk of milk-producing ruminants 12 hours after consumption, and reach a peak of transformation after 72 hours. Every 100g of plant lignans in the mixed diet can be transformed into 0.6-0.9g of mammalian lignans, which is difficult to achieve with any previous technical process.

[0023] The method of efficiently generating mammalian lignans from plant lignans through intestinal flora biotransformation of the present invention is that when the milk-producing ruminant ingests the plant lignans, the flora involved in the biotransformation becomes active and the abundance increases dramatically. Therefore, the relative abundance of the flora involved in the in vivo transformation is dynamic and depends on the intake of the plant lignans, and the method of the present invention has achieved the best result. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The HPLC chromatogram of the mammalian lignan enterodiol detected in the milk of dairy cows after ingestion of plant lignans;

[0025] Figure 2 This is a bar graph comparing the inhibition of ovarian cancer ES-2 cell survival rate by different concentrations of mammalian lignan enterodiol;

[0026] Figure 3 This is a bar graph comparing the inhibition of ovarian cancer ES-2 cell survival rate by different concentrations of mammalian lignan enterolactone;

[0027] Figure 4 This is a comparison of HE staining of ovarian cancer tissues in the PBS group, enterolactone 1 mg / kg group and normal control group;

[0028] Figure 5 The immunohistochemical images of MMP-2 expression in PBS group, mammalian lignan enterolactone and normal tissues;

[0029] Figure 6 The immunohistochemical images are of MMP-9 expression in PBS group, mammalian lignan enterolactone and normal tissues. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0031] Example 1

[0032] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein flaxseed is mixed into the diet of a milk-producing ruminant to prepare a mixed diet, wherein the amount of flaxseed added is 30% of the weight of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0033] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0034] In this embodiment, after the dairy cows ingested the mixed diet containing plant lignans, the conversion product mammalian lignans appeared in the milk 12 hours later, and the conversion peak was reached at 72 hours. Figure 1 High performance liquid chromatogram shown.

[0035] In vivo conversion is a purely biological and natural process. The biochemical reaction conditions in the intestine are optimized after millions of years of evolution. Table 1 is a comparison of the in vivo and in vitro conversion of mammalian lignans:

[0036] Table 1 Comparison of the effect of defatted flaxseed on the conversion of animal lignans in vitro and in vivo

[0037]

[0038] Example 2

[0039] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein sesame seeds are mixed into the diet of milk-producing ruminants to prepare a mixed diet, wherein the amount of sesame seeds added is 40% of the weight of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0040] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0041] Example 3

[0042] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein rapeseed is mixed into the diet of a milk-producing ruminant to prepare a mixed diet, wherein the amount of rapeseed added is 50% of the mass of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0043] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0044] Example 4

[0045] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein rye bran, wheat bran and barley bran are mixed into the diet of a milk-producing ruminant to prepare a mixed diet, wherein the rye bran, wheat bran and barley bran are mixed in equal weights, and the total addition amount is 60% of the weight of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0046] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0047] Example 5

[0048] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein oat bran and corn bran are mixed into the diet of a milk-producing ruminant to prepare a mixed diet, wherein the oat bran and corn bran are mixed in equal weights, and the total addition amount is 70% of the weight of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0049] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0050] Example 6

[0051] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein soybeans are mixed into the diet of milk-producing ruminants to prepare a mixed diet, wherein the amount of soybeans added is 20% of the weight of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0052] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0053] Example 7

[0054] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein potatoes are mixed into the diet of milk-producing ruminants to prepare a mixed diet, wherein the amount of potatoes added is 25% of the weight of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0055] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0056] Example 8

[0057] This embodiment provides a method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, wherein sweet potatoes and yam are mixed into the diet of milk-producing ruminants to prepare a mixed diet, wherein the sweet potatoes and yam are mixed in equal weights, and the total addition amount is 35% of the weight of the mixed diet. The mixed diet is used to feed dairy cows in a conventional manner, and milk produced by the dairy cows on the second day of the mixed diet and thereafter is collected.

[0058] The latex is mixed with ethanol with a mass concentration of 95% to obtain a loading solution, which is loaded onto a D101 macroporous resin chromatographic column, and eluted in sequence with ethanol aqueous solutions with mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%, and the eluate with an ethanol mass concentration of 30% is collected and concentrated by rotary evaporation to obtain enterodiol; and the eluate with an ethanol mass concentration of 40% is collected and concentrated by rotary evaporation to obtain enterolactone.

[0059] The product prepared by the method of efficiently generating mammalian lignans through in vivo conversion of plant lignans by intestinal flora can be used to inhibit the level of vascular endothelial growth factor VEGF and angiogenesis, thereby inhibiting tumor growth and metastasis. Figure 2 and Figure 3 The inhibitory effects of different concentrations of enterodiol and enterolactone on the activity of ovarian cancer ES-2 cells are shown in Figure 2. The data of each group are from three independent experiments. * indicates P < 0.05 compared with the control group. Figure 2 , Figure 3 It can be clearly seen that when the concentration of enterodiol or enterolactone reaches 10 -3 mol / l, it can significantly inhibit the activity of ovarian cancer ES-2 cells, and the cell survival rates of enterodiol and enterolactone are 28.02% and 24.38%, respectively.

[0060] MMP-2 and MMP-9 are members of the matrix metalloproteinase (MMPs) family, which play an important role in tumor invasion and metastasis, and are also important indicators for detecting tumor metastasis ability. Part of the tumor tissue after nude mouse tumor formation was used for sequencing analysis, and the other part was fixed in 10% formalin for tissue sectioning. HE staining was used to compare the differences between the tumor structure and normal tissue structure in the enterolactone 1 mg / kg group and the PBS group, and immunohistochemistry was used to compare the expression levels of MMP-2 and MMP-9 genes in the enterolactone 1 mg / kg group (6 tumor tissues) and the PBS group (5 tumor tissues) as well as normal tissues. Figure 4 HE staining comparison group of PBS group, enterolactone 1mg / kg group and normal tissue; Figure 5 The immunohistochemical images of MMP-2 expression in PBS group, enterolactone 1 mg / kg group and normal tissues; Figure 6 The immunohistochemical images of MMP-9 expression in PBS group, enterolactone 1 mg / kg group and normal tissues. Figure 4-Figure 6 It can be clearly seen that enterolactone significantly inhibited the metastasis and growth of tumors.

Claims

1. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora, It is characterized in that The organic material containing plant lignan precursors is mixed into the diet of a lactating ruminant according to a certain addition amount to prepare a mixed diet, the lactating ruminant is fed with the mixed diet in a conventional manner, and the milk of the lactating ruminant is collected and mammalian lignans are extracted therefrom.

2. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 1, It is characterized in that The mammalian lignans are enterodiol and / or enterolactone.

3. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 1 or 2, It is characterized in that The organic material containing plant lignan precursors includes one or a combination of flaxseed, sesame seed, rapeseed, rye bran, wheat bran, barley bran, oat bran, corn bran, burdock, sorghum, buckwheat, coix seed, soybean, red bean, soybean, mung bean, kidney bean, black bean, potato, sweet potato, taro, yam or seaweed.

4. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 3, It is characterized in that The milk-producing ruminant includes cattle, yaks, buffaloes or camels.

5. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 4, It is characterized in that The added amount of the organic material containing the plant lignan precursor is 12% to 86% of the mass of the mixed diet.

6. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 5, It is characterized in that When extracting mammalian lignans, the milk produced by the lactating ruminants on the second day or later after consuming the mixed diet according to claim 2 is collected.

7. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 6, It is characterized in that The method for extracting mammalian lignans from the milk of lactating ruminants comprises: mixing the milk with ethanol with a mass concentration of 95% to obtain a sample solution, loading the sample onto a macroporous resin chromatographic column, eluting with an ethanol aqueous solution with a certain mass concentration, collecting the eluate containing the mammalian lignans, and concentrating it by rotary evaporation to obtain the mammalian lignans.

8. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 7, It is characterized in that The elution is performed in sequence by gradient elution with ethanol aqueous solution having mass concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%.

9. A method for efficiently producing mammalian lignans through biotransformation of ruminant intestinal flora according to claim 8, It is characterized in that When extracting mammalian lignans, the ethanol mass concentration of the sample solution is 30% to 40%; the eluate with an ethanol mass concentration of 20% to 50% is collected for rotary evaporation concentration to obtain mammalian lignans.

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