USES OF 5-METHYLTETRAHYDROFOLATE AND ITS COMPOSITION

Abstract

The present invention provides the uses of 5-methyltetrahydrofolate and its composition. The 5-methyltetrahydrofolate or its composition is used to treat, relieve or prevent diseases or symptoms caused by acute alcohol intoxication and chronic alcohol intoxication. The injuries or diseases caused by the acute alcohol intoxication include: headaches caused by drinking, negative emotions or depression caused by drinking, and hangover symptoms after drinking. The injuries or diseases caused by the chronic alcohol intoxication include: alcoholic fatty liver, central nervous system (CNS) inflammation, etc.

Claims

1. A pharmaceutical composition or health-care food composition comprising an effective amount of 5-methyltetrahydrofolate, and the composition is used to treat or relieve diseases or symptoms caused by drinking or alcohol, wherein the dosage of 5-methyltetrahydrofolate in the composition is 5-50 mg/day.

2. The composition according to claim 1, wherein it is used to treat, relieve or prevent injuries or diseases or symptoms caused by acute alcohol intoxication including headaches caused by drinking, negative emotions or depression caused by drinking, and hangover symptoms or combinations thereof after drinking.

3. The composition according to claim 1, wherein it is used to treat, relieve or prevent injuries or diseases caused by chronic alcohol intoxication including alcoholic fatty liver, central nervous system (CNS) inflammation and migraines and headaches caused by it.

4. The composition according to claim 1, wherein it is used to treat or relieve non-alcoholic fatty liver disease including non-alcoholic fatty liver (NAFL) and non-alcoholic steatosis hepatitis (NASH).

5. The composition according to claim 1, wherein it is used to shorten sobering up time or reduce risk of cardiovascular and cerebrovascular diseases caused by drinking.

6. The composition according to claim 1, wherein it can significantly reduce the levels of total cholesterol (TC), triglycerides (TG), and malondialdehyde (MDA) in serum and increase superoxide dismutase (SOD) level.

7. The composition according to claim 1, wherein it can treat fatty liver, transform pathological steatohepatitis into benign fatty liver, and prevent pathological process of liver fibrosis and cirrhosis.

8. The composition according to claim 2, wherein 5-methyltetrahydrofolate reduces the production of endogenous methanol and/or formaldehyde, and/or promotes the metabolism of formaldehyde and formate in brain tissue, thereby reducing the concentrations of formaldehyde and formate to prevent and treat hangovers.

9. The composition according to claim 2, wherein 5-methyltetrahydrofolate improves the secretion of 5-hydroxytryptamine (5-HT) to prevent and treat negative emotions or depression caused by drinking.

10. A method of using 5-hydroxytryptamine or the composition comprising an effective amount of 5-methyltetrahydrofolate, wherein the dosage of 5-methyltetrahydrofolate in the composition is between 5-50 mg/day to claim 1 in the preparation of medicines for preventing and treating the diseases or symptoms including diseases or symptoms caused by acute alcohol intoxication including headaches caused by drinking, negative emotions or depression caused by drinking, hangover symptoms or combinations thereof after drinking, injuries or diseases caused by chronic alcohol intoxication including alcoholic fatty liver, central nervous system (CNS) inflammation and migraines and headaches caused by it, non-alcoholic fatty liver disease including non-alcoholic fatty liver (NAFL) and non-alcoholic steatosis hepatitis (NASH), reduce risk of cardiovascular and cerebrovascular diseases caused by drinking, to reduce the levels of total cholesterol (TC), triglycerides (TG), and malondialdehyde (MDA) in serum and increase superoxide dismutase (SOD) level, to transform pathological steatohepatitis into benign fatty liver, and prevent pathological process of liver fibrosis and cirrhosis, to reduce the production of endogenous methanol and/or formaldehyde, and/or promote the metabolism of formaldehyde and formate in brain tissue, thereby reducing the concentrations of formaldehyde and formate, or to improve the secretion of 5-hydroxytryptamine (5-HT).

11. The composition according to claim 1, further comprising other active compounds that act alone or synergistically including an effective amount of curcumin, wherein the mass ratio of 5-methyltetrahydrofolate to curcumin is 3˜1:1˜100.

12. The composition of claim 11, wherein the mass ratio of 5-methyltetrahydrofolate to curcumin is 3˜1:1˜3.

13. The composition of claim 12, wherein the mass ratio of 5-methyltetrahydrofolate to curcumin is 1:1.

14. The composition of claim 4, wherein the composition protects intestinal barrier function, reduces serum endotoxin levels, and prevents production of alcoholic “permeable intestine” thus to reduce the flow of endotoxins into enterohepatic circulation.

15. The composition of claim 6, wherein it is h used to treat or prevent hyperlipidemia and diseases caused by hyperlipidemia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a diagram of liver tissue of rats in normal group in Experiment 4.

[0066] FIG. 2 is a diagram of liver tissue of rats in model control group in Experiment 4.

[0067] FIG. 3 is a diagram of liver tissue of rats in positive drug group in Experiment 4.

[0068] FIG. 4 is a diagram of liver tissue of rats in low dose group in Experiment 4.

[0069] FIG. 5 is a diagram of liver tissue of rats in medium dose group in Experiment 4.

[0070] FIG. 6 is a diagram of liver tissue of rats in high dose group in Experiment 4.

[0071] FIG. 7 is an ion chromatogram and an internal standard ion chromatogram of MR1 without administration in Experiment 6.

[0072] FIG. 8 is a broken line graph of urine formaldehyde average concentration and time in each group of rats after drinking in Experiment 10.

[0073] FIG. 9 is a broken line graph of urine formic acid average concentration and time in each group of rats after drinking in Experiment 10.

[0074] FIG. 10 is a diagram of oral dose fraction (small intestine permeability) of 5-hour urine lactulose in each group of alcohol-fed rats at week 8 in Experiment 12.

[0075] FIG. 11 is a diagram of oral dose fraction (permeability of whole intestine [small intestine+large intestine]) of 5-hour urine sucralose in each group of alcohol-fed rats at week 8 in Experiment 12.

[0076] FIG. 12 is a diagram of serum endotoxin levels in each group of alcohol-fed rats at week 8 in Experiment 12.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0077] Special Notes:

[0078] The serum folic acid proposed in the present invention refers to 5-methyltetrahydrofolate in the serum.

[0079] Folic acid, unless otherwise specified, refers to synthetic folic acid.

Embodiment 1

[0080] 100 g of 5-methyltetrahydrofolate calcium salt was mixed with 700 g of microcrystalline cellulose. After dry granulation, 1000 capsules were filled to make capsule preparations containing 100 mg of 5-methyltetrahydrofolate calcium each.

Embodiment 2

[0081] 200 g of superfine silica powder was added to 100 g of 5-methyltetrahydrofolate calcium salt, mixed evenly and compressed by tablet machine to form antialcoholic lozenges.

Embodiment 3

[0082] The weight of each bulk drug is as follows: 40 g of 5-methyltetrahydrofolate calcium, and 40 g of curcumin The bulk drugs were crushed and mixed with microcrystalline cellulose and croscarmellose sodium to make granules, then dried and filled to obtain capsules containing 20 mg of 5-methyltetrahydrofolate calcium and 20 mg of curcumin each.

[0083] The weight of each bulk drug is as follows: 40 g of 5-methyltetrahydrofolate calcium, 20 g of reduced glutathione particles, and 40 g of curcumin. The bulk drugs were crushed and mixed with microcrystalline cellulose and croscarmellose sodium to make granules, then dried and filled to obtain capsules containing 20 mg of 5-methyltetrahydrofolate calcium, 10 mg of reduced glutathione particles and 20 mg of curcumin each.

[0084] Experiment 1 Effect of 5-methyltetrahydrofolate on disappearance and recovery of righting reflex of alcohol drinking rats.

[0085] 30 SPF-grade SD rats were chosen in half male and half female, and divided into 5 groups with 6 rats in each group, including normal group, model group, positive drug group (Weihe Liver Protectant), 5-methyltetrahydrofolate administration group (4 mg.Math.Kg−1 medium dose group, 8 mg.Math.Kg−1 high dose group, respectively). After fasting for 12 h, the administration group was given Weihe Liver Protectant at 50 mg.Math.kg−1, 5-methyltetrahydrofolate (Jinkang Hexin product) at 4 mg.Math.Kg−1, 5-methyltetrahydrofolate (Jinkang Hexin product) at 8 mg.Math.Kg−1, respectively. After 30 min, the model group and the administration group were given Erguotou (batch number: 201603092, specification: 2L, place of origin: Beijing Shunxin Agriculture Co., Ltd.) at 9 ml.Math.Kg−1, and the normal group was given the same amount of normal saline. The intoxication in rats depends on righting reflex disappearing, that is, cover the head of the rat with gauze, and gently place the rat back down in the animal cage. If the front paw of the rat turns back within 1 min, it shows that no righting reflex has occurred, otherwise righting reflex has occurred. The disappearance time and recovery time of the righting reflex in rats after drinking were recorded. The experimental results are shown in the table below.

TABLE-US-00001 TABLE 1 Effect of 5-methyltetrahydrofolate on the time of righting reflex in rats (x ± s) Note: n = 6, compared with the model group: *p < 0.05, **p < 0.01 indicates data missing or illegible when filed

[0086] The above results indicate that 5-methyltetrahydrofolate has a significant antagonistic effect on rats with acute alcohol intoxication. Where, the recovery time of righting reflex of alcohol drinking rats in the medium and high dose groups with much lower dosage than the positive drug group is better than that of the positive drug group, which shows that 5-methyltetrahydrofolate has the effect of shortening the sober-up time.

[0087] Experiment 2 Effect of 5-methyltetrahydrofolate on homocysteine (Hcy) in plasma of alcohol drinking rats.

[0088] 30 SPF-grade SD rats were chosen in half male and half female, and divided into 5 groups with 6 rats in each group, including normal group, model group, positive drug group (Weihe Liver Protectant), 5-methyltetrahydrofolate administration groups (4 mg.Math.Kg−1 medium dose group, 8 mg.Math.Kg−1 high dose group, respectively). After fasting for 12 h, the administration groups were given Weihe Liver Protectant at 50 mg.Math.kg−1, 5-methyltetrahydrofolate (Jinkang Hexin product) at 4 mg.Math.Kg−1, 5-methyltetrahydrofolate (Jinkang Hexin product) at 8 mg.Math.Kg−1, respectively. After 30 min, the model group and the administration groups were given Erguotou (batch number: 201603092, specification: 2L, place of origin: Beijing Shunxin Agriculture Co., Ltd.) at 20ml.Math.Kg−1, and the normal group was given the same amount of normal saline. 6 h after drinking, blood was collected from the eye socket, treated with 0.5% EDTA-Na for anticoagulation (10 ml/L), immediately cooled in an ice bath, and centrifuged at 4° C. (3500r/min) for 15 min within 30 min. Plasma was collected and stored at −20° C. The plasma Hcy concentration was determined with high pressure liquid chromatography-fluorescence detection method. The experimental results are shown in the table below.

TABLE-US-00002 TABLE 2 Effect of 5-methyltetrahydrofolate on homocysteine in plasma of alcohol  (x ± s) rats. Disappearance Recovery Group Dosage time/min time/min Normal group / / / Model group / 17.4 ± 9.6  143.2 ± 13.5  Positive drug 50 mg .Math. Kg.sup.−1 37.4 ± 8.2** 108.5 ± 14.5* group 5-methyltetrahydrofolate 4 mg .Math. Kg.sup.−1 30.1 ± 14.4*  78.5 ± 14.5* (medium) 5-methyltetrahydrofolate 8 mg .Math. Kg.sup.−1  42.3 ± 15.6**  64.1 ± 14.5** (high) Hcy Group Dosage (μmol/L) Normal control — 2.52 ± 0.89 group Positive drug 50 mg .Math. Kg.sup.−1 5.53 ± 1.26 group Model group — 5.58 ± 2.23 Medium dose 4 mg .Math. Kg.sup.−1  3.13 ± 1.26** group High dose 8 mg .Math. Kg.sup.−1  2.32 ± 1.49** group Note: n = 6, compared with the model group: *p < 0.05, **p < 0.01 indicates data missing or illegible when filed

[0089] The results show that acute heavy alcohol intake can cause an increase in homocysteine in rats, and 5-methyltetrahydrofolate can significantly reduce the level of Hcy in serum of rats, and has a dose-effect relationship. Hcy may not only cause damage to the nervous system, but also cause ischaemic heart disease and ischemic stroke. Although these indexes return to normal levels with alcohol metabolism, bad conditions still be produced. Therefore, 5-methyltetrahydrofolate can also reduce the risk of cardiovascular diseases caused by alcohol drinking.

[0090] Experiment 3 Protection of 5-Methyltetrahydrofolate Calcium on Liver Injury Induced by Carbon Tetrachloride

[0091] 30 SPF-grade SD rats were chosen in half female and half male and randomly divided into vehicle control group (vegetable oil), model control group (1.5 ml.Math.kg−1, 20% carbon tetrachloride vegetable oil solvent, intraperitoneal injection every 3 days), 5-methyltetrahydrofolate calcium low-dose group (1mg.Math.Kg−1), 5-methyltetrahydrofolate calcium medium-dose group (2 mg.Math.Kg−1), and 5-methyltetrahydrofolate calcium high-dose group (4 mg.Math.Kg−1) according to their body weight. 20% carbon tetrachloride vegetable oil solvent was used as the inducer, and intraperitoneal injection was performed every 3 days for a total of 60 days. After successful modeling, the administration groups were given a dosage of 5-methyltetrahydrofolate calcium as prescribed above for gavage once a day, and the model group and the normal control group were gavaged once daily with purified water for 60 consecutive days. Blood was taken on the 61st day, and the levels of ALT and AST in serum were detected. The results are shown in the table below.

TABLE-US-00003 TABLE 3 Protection of 5-methyltetrahydrofolate calcium on liver injury induced by carbon tetrachloride Dosage ALT AST Group (mg .Math. kg.sup.−1 .Math. d.sup.−1) (IU/L) (IU/L) Normal control — 26.34 ± 12.31.sup.  .sup. 87.29 ± 35.25 group Model group — 182.56 ± 67.37*.sup.  .sup. 382.32 ± 87.94* Low dose 1 54.64 ± 20.52.sup.# 185.73 ± 63.46.sup.# group Medium dose 2 50.54 ± 20.12.sup.# 156.97 ± 50.35.sup.# group High dose 4 40.84 ± 16.31.sup.# 125.87 ± 43.84.sup.# group Note: 1. Number of animals: n = 6 in each group; 2. *Compared with the vehicle control group, each dose group, P < 0.05; .sup.#Compared with the model control group, in each dose group, P < 0.05.

[0092] Experiment 4 Prevention of a Certain Dose of 5-Methyltetrahydrofolate Calcium on Alcoholic Fatty Liver Damage

[0093] 96 SPF-grade SD rats were chosen in half female and half male with an average weight of 176-220 g at the beginning of the experiment. According to their weight, they were randomly divided into vehicle control group (purified water, ig), model control group (liquor, 10 ml.Math.Kg−1), positive control group (bicyclol, 50 mg.Math.Kg−1, ig), folic acid calcium salt high-dose group (4 mg.Math.Kg−1), folic acid calcium salt medium-dose group (2 mg.Math.Kg−1), folic acid calcium salt low-dose group (1 mg.Math.Kg−1), in half female and half male, and 16 rats in each group. Purified water was used as a negative control, and bicyclol (place of origin: Beijing Xiehe Pharmaceutical Factory, specification: 25 mg/tablet, batch number: H20040467) was used as a positive control drug. Route of administration: all were administered by oral gavage. Dosage of administration: 1 mg.Math.Kg−1, 2 mg.Math.Kg−1, and 4 mg.Math.Kg−1. Frequency of administration: once a day for 60 consecutive days. Erguotou (batch number: 201603092, specification: 2L, place of origin: Beijing Shunxin Agriculture Co., Ltd.) model establishing method: gavage with diluted 56-degree Erguotou twice a day for 60 consecutive days.

[0094] Method of administration: administration started at prescribed doses on the day of modeling for 60 consecutive days and ended at the 60th day. At the same time, rats in each group were weighed every other week during administration period to monitor their body weight changes. The changes in the weight gain of rats during administration period are shown in Table 4. On the 61st day, rats were dissected and serum was collected for determination of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), superoxide dismutase (SOD) and liver histopathologic examination. The results are shown in Table 5. The brain tissue of rats was taken out, and hippocampus tissue was separated on ice plate, prepared to homogenate with a mass fraction of 10% with purified water, then the homogenate was centrifuged to obtain supernatant. ELISA was used to detect the levels of inflammatory factors TNF-α and IL-1β in hippocampus tissue with continuous spectrum scanning microplate reader. The results are shown in Table 6.

TABLE-US-00004 TABLE 4 Changes in the weight gain of rats during the administration period.sup.(.sup.x± s) Dosage Weight gain (g) (mg .Math. kg.sup.−1 .Math. Week Week Week Week Week Week Week Week Week Group d.sup.−1) 0 1 2 3 4 5 6 7 8 Normal — 196.29 ± 236.66 ± 368.89 ± 397.12 ± 411.60 ± 429.28 ± 448.22 ± 478.01 ± 497.12 ± control 11.43 17.36 30.91 31.58 33.49 36.81 36.46 36.37 39.37 group Model — 189.21 ± 213.15 ± 297.55 ± 299.35 ± 308.43 ± 313.23 ± 344.95 ± 351.78 ± 397.12 ± group 12.41 12.36 36.46* 22.04* 25.43* 22.98* 44.01* 26.22* 30.57* Positive 50 188.19 ± 211.95 ± 279.31 ± 292.58 ± 317.80 ± 313.19 ± 333.57 ± 360.01 ± 380.37 ± drug 14.62 20.75 41.05* 41.80* 44.63* 45.51* 48.82* 52.79* 52.88* group Low dose 1 195.64 ± 215.16 ± 282.30 ± 299.34 ± 312.99 ± 317.48 ± 334.47 ± 355.89 ± 375.85 ± group 13.22 17.62 25.51* 26.51* 28.23* 29.75* 32.73* 38.97* 46.00* Medium 2 190.85 ± 219.44 ± 274.69 ± 293.12 ± 321.78 ± 328.10 ± 345.05 ± 352.90 ± 383.69 ± dose 12.05 10.44 28.72* 39.28* 26.22* 48.25* 24.98* 37.55* 39.66* group High dose 4 196.33 ± 218.24 ± 285.54 ± 296.05 ± 307.43 ± 321.49 ± 349.47 ± 372.49 ± 392.02 ± group 10.82 13.72 31.80* 30.67* 31.52* 32.43* 31.48* 37.88 45.54* Note: 1. Number of animals: n =16 in each group; 2. Compared with normal control group, in each dose group, *: P < 0.05.

[0095] The weight of rats in each group increased gradually, but the weight of rats in each model and administration group was significantly different from that in normal control group since the second week. The difference in weight between alcohol drinking model group and administration groups is not obvious, but the weight gain of high-dose group is higher than that of medium- and low-dose groups, that is, there is a certain relationship between weight gain and administration dosage of 5-methyltetrahydrofolate proposed in the present invention, indicating that 5-methyltetrahydrofolate relieves the loss of appetite caused by alcohol drinking or slow weight gain of rats caused by other factors.

TABLE-US-00005 TABLE 5 Improvement of 5-methyltetrahydrofolate calcium salt on alcoholic liver damage and fatty liver and changes in biochemical indexes of rats tested (x ± s) Dosage TG TC SOD MDA Group (mg .Math. kg.sup.−1 .Math. d.sup.−1) (mmol/L) (mmol/L) (U/mgprot) (nmol/mgprot) Normal control — 1.06 ± 0.51.sup.  1.29 ± 0.31 227.21 ± 22.34.sup.  48.75 ± 9.64.sup.  group Model group — .sup. 1.71 ± 0.35*  1.62 ± 0.67* .sup. 156.85 ± 22.52*  .sup. 72.36 ± 11.25* Positive drug 50 1.03 ± 0.34.sup.# 1.45 ± 0.43 167.47 ± 18.91.sup.   .sup. 70.41 ± 20.33* group Low dose 1 1.08 ± 0.34.sup.# 1.42 ± 0.36 253.20 ± 24.66.sup.# 45.20 ± 8.17.sup.# group Medium dose 2 1.03 ± 0.50.sup.# 1.50 ± 0.37 257.41 ± 31.28.sup.# 42.17 ± 8.65.sup.# group High dose 4 1.04 ± 0.43.sup.# 1.47 ± 0.44 248.87 ± 34.64.sup.#  47.96 ± 10.28.sup.# group Note: 1. Number of animals: n = 16 in each group; 2. *Compared with the vehicle control group, in each dose group, P < 0.05; .sup.#Compared with the model control group, in each dose group, P < 0.05.

[0096] The results show that: the indexes of rats in the model group are significantly different from those in the control group (P<0.05), and TG, MDA and SOD in three dosage group of test substance are significantly different from those in the model group (P<0.05). The 5-methyltetrahydrofolate calcium group at a low dose of 1 mg/kg/day has TG and TC indexes close to those of the positive drug group, and better SOD and MDA indexes than those of the positive drug group, indicating that 5-methyltetrahydrofolate has an excellent therapeutic or relieving effect on liver damage and fatty liver caused by drinking.

TABLE-US-00006 TABLE 6 Comparison of inflammatory factors in hippocampus tissue of rats in each group (x ± s) Dosage TNF-α IL-1β Group (mg .Math. kg.sup.−1 .Math. d.sup.−1) (μg .Math. g.sup.−1 prot) (μg .Math. g.sup.−1 prot) Normal control — 244.32 ± 29.81.sup.  60.32 ± 5.32 group Model group — .sup. 290.45 ± 51.90* 74.98 ± 7.43 Positive drug 50 .sup. 286.32 ± 43.79.* 73.93 ± 9.37 group Low dose 1 250.39 ± 32.40.sup.# 63.05 ± 5.31 group Medium dose 2 241.32 ± 29.32.sup.#  63.24 ± 13.44 group High dose 4 220.43 ± 24.34.sup.# .sup. 55.21 ± 3.59.sup.# group Note: 1. Number of animals: n = 16 in each group; 2. *Compared with normal control group, in each dose group, P < 0.05; .sup.#Compared with model control group, in each dose group, P < 0.05.

[0097] The results show that 5-methyltetrahydrofolate calcium can inhibit the production of inflammatory factors induced by chronic alcohol intake in the brain of rats. Individuals are greatly different from each other with large fluctuations in the data of the model group. Therefore, it is necessary to increase duration of alcohol intake and establish a “alcoholism” model to observe the effect of alcohol on the induction of brain inflammation in rats. However, it can be determined that 5-methyltetrahydrofolate can significantly inhibit the expression of inflammatory factors in the brain of rats, showing a certain dose-effect relationship. Therefore, 5-methyltetrahydrofolate can prevent headaches and migraines induced by alcohol.

[0098] Pathological Examination:

[0099] Control group: clear structure of hepatic lobules, no degeneration, necrosis and hyperplasia of hepatocytes, and no hyperplasia of interstitial connective tissue;

Model group: moderate hyperplasia of hepatic interstitial connective tissue and fatty degeneration of hepatocytes; low-dose group: a few fatty vacuoles in hepatocytes, and no obvious hyperplasia of interstitial connective tissue; positive drug group, medium-dose group and high-dose group: no hyperplasia of interstitial connective tissue and fatty degeneration of hepatocytes. (Refer to accompanying drawings 1-6 to the disclosure)

[0100] Experiment 5 Effect of 5-Methyltetrahydrofolate Calcium on Hyperlipemia Rats

[0101] 50 SPF-grade SD rats were chosen in half female and half male and fed with normal diet for 5 weeks, then randomly divided into normal diet feeding group (n=9) and model group (n=41), which were respectively fed with normal diet or high-fat diet consisting of 83% normal diet, 15% lard, and 2% cholesterol. 7 weeks later, one rat in the control group and one rat in the model group were sacrificed for liver tissue section to check fatty degeneration of hepatocytes (see Table 7) to confirm whether modeling was successful. The remaining 40 modeling rats were randomly divided into low-dose (1 mg.Math.Kg−1) group, medium-dose (2 mg.Math.Kg−1) group, high-dose (4 mg.Math.Kg−1) group, positive drug group, and model group.

[0102] Bicyclol (place of origin: Beijing Xiehe Pharmaceutical Factory, specification: 25 mg/tablet, batch number: H20040467) was used as a positive drug.

[0103] After successful modeling, rats were administered drugs with dosages as prescribed for 60 consecutive days, continued to be fed with high-fat diet, and dissected on the 61st day. Liver was harvested and weighed, liver index was calculated, right lobe tissue was taken to prepare frozen sections, and fat staining was performed with Sudan III for pathological scoring according to guidelines for diagnosis and treatment of NAFLD. Another liver tissue was taken to prepare homogenate, and total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), and superoxide dismutase (SOD) in serum were detected. The results are shown in Table 8.

TABLE-US-00007 TABLE 7 Comparison of body mass, liver mass and liver index of rats in five groups (x ± s) Pathological Number Body mass Liver mass state of cases (g) (g) Liver index (NASH) Model group 8 475.7 ± 36.5 20.6 ± 3.5  4.3 ± 0.5  4 cases Positive drug 8 453.8 ± 34.7 14.6 ± 4.5  3.2 ± 0.4* 2 cases group Low dose 8 445.89 ± 39.0 15.3 ± 3.5* 3.4 ± 0.3* 1 case group Medium dose 8 452.93 ± 37.5  11.6 ± 2.9**  2.6 ± 0.2** 0 case group High dose 8 472.49 ± 37.8  10.9 ± 2.5**  2.3 ± 0.5** 0 case group Normal group 8 452.83 ± 37.5 13.6 ± 2.8* 3.0 ± 0.4* 0 case Note: *Compared with model group, in each dose group, P < 0.05; **Compared with model group, in each dose group, P < 0.01.

[0104] The above data indicate that 5-methyltetrahydrofolate calcium can prevent and treat fatty liver caused by hyperlipidemia, restore the liver to its normal form, and prevent fatty liver in case-control state from changing to nonalcoholic steatohepatitis (NASH). All rats in the medium- and high-dose 5-methyltetrahydrofolate groups exhibited a benign or normal liver.

TABLE-US-00008 TABLE 8 Effect of 5-methyltetrahydrofolate calcium on biochemical indexes of (x ± s) Dosage TG TC SOD MDA Group (mg .Math. kg.sup.−1 .Math. d.sup.−1) (mmol/L) (mmol/L) (U/mgprot) (nmol/mgprot) Normal control — 1.08 ± 0.53.sup.  1.27 ± 0.29 230.11 ± 21.24.sup.  49.75 ± 10.54  group Model group — .sup. 1.70 ± 0.27*  1.92 ± 0.47* .sup. 158.73 ± 21.48* 74.26 ± 12.05* Positive drug 50 1.23 ± 0.35.sup.# 1.45 ± 0.63 157.41 ± 16.45.sup.  72.39 ± 19.23* group Low dose 1 1.07 ± 0.33.sup.# 1.45 ± 0.38 234.20 ± 21.36.sup.# 45.20 ± 8.17.sup.#   group Medium dose 2 1.03 ± 0.55.sup.# 1.57 ± 0.36 257.41 ± 30.29.sup.# 42.26 ± 9.35.sup.#   group High dose 4 1.05 ± 0.63.sup.# 1.50 ± 0.34 260.97 ± 30.04.sup.# 40.98 ± 11.37.sup.#  group Note: Compared with normal control group, in each dose group, *P < 0.05; .sup.#Compared with model control group, in each dose group, P < 0.05.

[0105] Table 8 shows that the results are similar to those in Table 5 of Embodiment 4. The liver is an important organ in the metabolism of folic acid, and there is a complex and direct relationship between liver damage and folic acid metabolism. The inventors have found that 5-methyltetrahydrofolate can prevent benign fatty liver from transforming into pathological fatty liver, and can produce therapeutic effect under a certain dosage.

[0106] Experiment 6 Concentration of Folic Acid in Plasma of Humans after Drinking Alcohol

[0107] Three adults, based on a complete understanding of the experimental protocol, volunteered to participate in the study, with basic information as shown in Table 9.

[0108] JK001 capsule: synthetic folic acid, source: Zhengzhou Yuhe Food-Additive Co., Ltd., purity: 99.8%, molecular weight: 441.4, conversion factor: 1. 42 g of synthetic folic acid was taken and mixed evenly with microcrystalline cellulose, filled into 1000 capsules to make JK001 capsules (containing 42 mg folic acid); JK002 capsule: 5-methyltetrahydrofolate calcium, source: Lianyungang Jinkang Hexin Pharmaceutical Co., Ltd., purity: 99.9%, molecular weight: 497.5, conversion factor: 0.8872. 47.34 g of 5-methyltetrahydrofolate calcium was taken and mixed evenly with microcrystalline cellulose, filled into 1000 capsules to make JK002 capsules (containing 42 mg folic acid, conversion value);

[0109] JK003 capsule: microcrystalline cellulose, filled into 1000 capsules to make JK003 capsules.

[0110] The above capsules have the same model and specifications and cannot be distinguished from each other by the appearance.

[0111] Without having any breakfast, the three volunteers took capsules immediately after drinking 400 ml of alcohol in stages (within half an hour), and then ate and drank normally during the 24 h.

TABLE-US-00009 TABLE 9 Basic information of volunteers Weight Group Gender (kg) Age Health condition Capsule Mr1 Male 64 23 Normal liver function, JK001 no other diseases Mr2 Male 60 25 Normal liver function, JK002 no other diseases Mr3 Male 72 25 Normal liver function, JK003 no other diseases

[0112] Blood was collected from the vein at 30 min before drinking, 0 min before administration, and 0.25, 0.5, 1, 2, 4, 6, 8, 24 h after administration.

6, 8, 24 h. The collected blood samples were centrifuged at 5000 rpm for 5 min at 4° C., and plasma obtained was transferred to a 1.5 mL centrifuge tube, and then stored in a refrigerator at −20° C. The concentration of folic acid in the blood samples was detected according to the analysis and detection conditions in Table 9.

TABLE-US-00010 TABLE 10 Analysis and detection conditions Liquid phase Liquid phase: Agilent 1200 HPLC-CTC autosampler conditions Mobile phase: Solution A: methanol-water-formic acid (10:90:0.1, v/v/v) Solution B: methanol-formic acid (100:0.1, v/v) Time (min) A(%) B(%) 0.00 100.0 0.0 0.10 100.0 0.0 1.00 10.0 90.0 2.00 10.0 90.0 2.10 100.0 0.0 4.00 100.0 0.0 Mass Injection volume: 5 μL spectrometry Flow rate: 0.40 mL/min conditions Chromatographic column: Grace Alltima HP C18 (2.1*50 mm) is equipped with in-line filter AB API4000 tandem mass spectrometer Polarity: Cationic model Compound-related parameters Parameter DP EP CE CXP 6S-5- 80 8 25 9 methyltetrahydrofolate Carbamazepine (IS) 80 8 28 7 Source-related parameters GS1 (gas 1) 50 GS2 (gas 2) 50 TEM (temperature) 450 CUR (curtain gas) 25 IS (spray voltage) 5500 CAD (collision gas) 4 ihe (interface heater): On Internal standard Carbamazepine Analyte MRM Object to be tested: [M + H]+ m/z 460.2.fwdarw.313.2 (quantitative) [M + H]+ m/z 460.2.fwdarw.331.1 (qualitative) Internal standard: [M + H].sup.+ m/z 237.1.fwdarw. 193.9 Linear range Plasma 5~10000 ng/mL Sample Take 50 μL of blank plasma, add 5 μL of standard working processing solution of each concentration (add 5 μL of working diluent to unknown samples and blank samples), vortex for 10 secs, and then add 5 μL of stabilizer B (DTT and Vc mixed aqueous solution, 5 mg .Math. mL − 1, this operation is limited to standard curve), vortex for 10 secs, and then add 300 μL of methanol precipitant (containing internal standard carbamazepine at 40 ng .Math. mL − 1, no internal standard carbamazepine is added to double blank samples) (containing DTT and Vc at the same time, with a concentration of 1 mg .Math. mL − 1), vortex for 1 min, centrifuge at 12000 rpm for 10 min, take 20 μL of the supernatant and mix with 80 μL of stabilizer C (DTT and Vc mixed aqueous solution, 1 mg .Math. mL − 1), then take 5 μL of the solution for LC-MS/MS quantitative analysis.

[0113] The results are shown in the table.

TABLE-US-00011 TABLE 11 Plasma drug concentration at different blood sampling time Blood Blood concentration sampling (ng .Math. mL.sup.−1) time (h) Mr1 Mr2 Mr3 −0.5 13.2152 12.6531 10.1152  0 12.1243 11.3575 9.5745 0.25 13.1254 16.1863 8.6253 0.5 14.2134 39.3437 5.6423 1 12.0134 45.0376 BLOQ 2 9.4556 49.7386 BLOQ 4 BLOQ 43.1819 BLOQ 6 BLOQ 17.1690 BLOQ 8 BLOQ 10.4056 BLOQ 24 5.1434 9.1884 5.7857 Note: Linear range: 5~10000 ng/mL; LLOQ: 5 ng/mL; BLOQ: below the limit of quantification

[0114] The results show that: heavy drinking can sharply decrease serum folic acid in a short time to the level of LLOQ, which is beyond our expectation. The liver itself can store about 5-20 mg of folic acid, but this portion of folic acid does not enter the bloodstream immediately after drinking alcohol. 5-methyltetrahydrofolate can solve the decrease of serum folic acid caused by heavy drinking in a short time while synthetic folic acid cannot solve this problem.

[0115] Combined with the above animal experiment data, it shows that 5-methyltetrahydrofolate calcium has a good protective effect on alcoholic liver injury models, indicating that the lack of folic acid in the body inevitably affects its protective function. This experimental data is consistent with the animal experiment data, which can mutually confirm and complement each other.

[0116] Experiment 7 Effect of Sober-Up Capsule on Hangover Symptoms

[0117] In order to prove the effect of the present invention on adverse reactions caused by hangovers after drinking, the capsule prepared in Embodiment 3 was used as a sample, and 10 volunteers, aged 20 to 40, male, were randomly divided into two groups, of which one group used the sample, and the other group used negative sample. The two groups took the sober-up sample or negative sample before drinking, and then drank alcohol in a prescribed amount. Before 8:00 am on the next day, they were asked to fill out the questionnaire on whether they had excessive thirst, nausea, drowsiness, sweating, anorexia, fuzzy cognition, migraine and other symptoms. The results are shown in the table.

TABLE-US-00012 TABLE 12 Human hangover reaction test results Drowsi- Sweat- Anor- Fuzzy Thirst Nausea ness ing exia cognition Group (case) (case) (case) (case) (case) (case) Negative 4 4 3 1 1 0 group Test 3 0 0 0 0 0 group Note: They are mainly subjective evaluations on corresponding symptoms, i.e. statistical results, which show that, the composition disclosed in the present invention can improve adverse physiological reactions after drinking, especially have an obvious effect on nausea and drowsiness, and can significantly maintain the mental state of drinkers to improve their mood and reduce their drowsiness after drinking.

[0118] Experiment 8 Tail Suspension Test and Swimming Test in Mice after Drinking

[0119] Clean-grade male Kunming mice, weighing 18-22 g, were chosen. The test was carried out at 23° C. with a humidity of 50%. The mice ate and drank freely, adapting to the environment for 5 days before the test. Then they were divided into 5 groups: model group, normal group, high-dose 5-MTHF-Ca group, medium-dose 5-MTHF-Ca group and low-dose 5-MTHF-Ca group (16 mg.Math.Kg−1, 8mg.Math.Kg−1, 4mg.Math.Kg−1),

10 mice in each group. The model group was given (by gavage) Erguotou (batch number: 201603092, specification: 2L, place of origin: Beijing Shunxin Agriculture Co., Ltd.) at 18 ml.Math.Kg−1, and calcium folate (Lianyungang Jinkang Hexin Pharmaceutical Co., Ltd.). The high-dose, medium-dose and low-dose groups were also given the solution containing Erguotou and calcium folate. The mice were tested after 24 h. The tail suspension test is carried out by fixing the tail of a mouse so that it is suspended head down in a state of desperately struggling to escape but unable to escape. After a period of time, record the immobility time of the mouse in a desperate state in this environment to observe the therapeutic effect after administration. The head of the mouse suspended upside down was 10 cm from the bottom of the box, and two mice were suspended at a time, which were separated by a partition to avoid collision. The mice were suspended for 10 min, and the accumulated immobility time of the suspended mice was counted within the latter 5 min. The results are as follows:

TABLE-US-00013 TABLE 13 Tail suspension test in mice (x ± s) Group Suspension immobility time (S) Model group 89.54 ± 5.58  Normal group  55.23 ± 1.53** High dose 63.53 ± 2.98* group Medium dose 68.32 ± 3.46* group Low dose 72.42 ± 4.52  group Note: n = 10 *Compared with the model group, p < 0.05, **Compared with the model group, p < 0.01

[0120] The mice were placed in a plastic swimming box with a length of 50 cm, a width of 40 cm and a height of 40 cm. The time was counted for 10 min after the mice were placed in the water. The accumulated immobility time of the mice with only slight limb movements and no struggling, and remaining afloat was recorded within the latter 5 min.

TABLE-US-00014 TABLE 14 Swimming test in mice (x ± s) Group Swimming immobility time (S) Model group 143.24 ± 6.53  Normal group  97.35 ± 3.45** High dose 104.64 ± 5.24** group Medium dose 110.42 ± 2.64** group Low dose 123.56 ± 3.64*  group Note: n = 10 *Compared with the model group, p < 0.05, **Compared with the model group, p < 0.01

[0121] The results show that the application of the present invention can significantly improve the mental state of mice after drinking alcohol, such as improving the performance of depression in mice.

[0122] Experiment 9 Inhibition of 5-Methyltetrahydrofolate and Curcumin on LPS-Induced Inflammatory Response in Astrocytes

[0123] The cerebral cortex of Kunming mice at 1˜2 d was taken out under aseptic conditions, washed 3 times with cold PBS after removing residual meninges and blood vessels, prepared into single cell suspension by blowing, centrifuged, the supernatant was discarded, and the precipitant was resuspended in AST basic medium (high-sugar DMEM containing 10% FBS). The suspension was inoculated into a culture flask coated with poly-L-lysine (0.1 mg/ml), and cultured in a CO2 incubator. After 24 h, tissue debris and non-adherent cells were removed by replacing the medium. Afterwards, the medium was replaced every 3 days until the cells reached 80% confluence, then the flask was placed on a constant temperature rotary shaker, where the suspension was centrifuged at 180 r/min, 37° C. for 18 h to remove oligodendrocytes and microglia from the upper layer. Then trypsin digestion was used for subculture. The second-generation AST was inoculated into a 6-well plate, and mice in the experiment was divided into: blank group with PBS; control group:

LPS (1 μg.Math.ml-1), LPS (1 μg.Math.ml-1)+curcumin (10 μg.Math.ml-1), LPS (1 μg.Math.ml-1)+5-MTHF-Ca (10 μg.Math.ml-1); experimental group: LPS (1 μg.Math.ml-1)+curcumin (5 μg.Math.ml-1)+5-MTHF-Ca (5 μg.Math.ml-1). After 24 h, the culture supernatant was collected, and the cytokines IL-6, IL-1β and TNF-α were detected by ELISA. The results are shown in Table 15.

TABLE-US-00015 TABLE 15 Effects of curcumin and 5-methyltetrahydrofolate on LPS-stimulated release of inflammatory factors from microglia (x ± s) IL-6 IL-β TNF-α Group (pg .Math. ml.sup.−1) (pg .Math. ml.sup.−1) (pg .Math. ml.sup.−1) PBS group .sup. 854.82 ± 21.43.sup.# 673.52 ± 13.22.sup.#  483.97 ± 9.38.sup.# LPS group 1178.43 ± 73.02* 831.93 ± 15.21*  .sup. 570.25 ± 10.56* LPS + Cur group 1003.97 ± 60.33* 742.12 ± 10.33* 492.32 ± 8.92.sup.# 5-MTHF-Ca group  993.24 ± 52.05* 721.45 ± 8.79.sup.#    489.21 ± 10.71.sup.# LPS + Cur +  .sup. 982.13 ± 45.32*.sup.# 702.53 ± 9.53.sup.#   452.43 ± 8.47.sup.# 5-MTHF-Ca group Note: n = 6, compared with normal control group, in each dose group, *P < 0.05; .sup.#Compared with model control group, in each dose group, P < 0.05.

[0124] The results show that both 5-methyltetrahydrofolate and curcumin inhibit the lipopolysaccharide-induced release of inflammatory factors from neuroglia, especially for tumor necrosis factor alpha (TNF-α). The combined use of them surprisingly reduces the level of TNF-α below the normal control group, indicating that curcumin and 5-methyltetrahydrofolate have a synergistic effect. This composition inhibits alcohol-induced inflammation in human nervous system, and plays a role in preventing headaches.

[0125] Experiment 10 Effects of Drinking and 5-Methyltetrahydrofolate on the Formate in the Urine of SD Rats

[0126] In order to study the effects of 5-methyltetrahydrofolate and ethanol on methanol metabolism in rats, SPF-grade female SD rats, fasted overnight, were divided into 4 groups: (1) ethanol group, with intragastric administration of ethanol at a dose of 1 g/kg respectively at 1, 2, 3 h, n=7; (2) control group, with intragastric administration of glucose at a dose of 1.6 g/kg (equivalent to alcohol calories) respectively at 1, 2, and 3 h, n=5; (3) 5-methyltetrahydrofolate group, with intragastric administration of 5-methyltetrahydrofolate calcium at a dose of 4 mg/kg at 0 h, followed by glucose at a dose of 1.6 g/kg (equivalent to alcohol calories) respectively at 1, 2, and 3 h, n=5; (4) 5-methyltetrahydrofolate alcohol group, with intragastric administration of 5-methyltetrahydrofolate calcium at a dose of 4 mg/kg at 0 h, followed by ethanol at a dose of 1 g/kg respectively at 1, 2, and 3 h, n=5.

[0127] Food grade alcohol was used, with a methanol content of less than 5 mg/L. Urine was collected, from 0 to 24 h, into a test tube containing 0.1 mL of thioethanol and stored at −70° C. The urine samples were divided into three parts, of which gas chromatographic method, fluorescence method and HPLC-DNPH derivative method were used to detect ethanol and methanol contents (see Table 16), formate content, and formaldehyde content (see Table 17) in urine, respectively.

[0128] The method for detecting formate is as follows: 0.1 mL of urine was mixed with 0.1 mL of 10 mmol/L NAD+, 0.1 mL of potassium phosphate buffer (pH 7.4, 20 mmol/L) and 50 μL of formate dehydrogenase, then 0.1 mL of diaphorase (4 U/mL), 50 μL of resazurin solution (0.2 mg/ml) and 0.5 ml of phosphate buffer (pH 6.00, 200 mmol/L) were added. The mixture was incubated at 37° C. for 5 min, then immersed in boiling water for 3 min, and then cooled to room temperature. Fluorescence spectrophotometry was used to determine the content of formate in the mixture, with an emission wavelength of 590 nm and an absorption wavelength of 565 nm.

[0129] The method for detecting formaldehyde is as follows: 0.1 ml of urine was taken and filtered through filtering membrane, 0.05 ml of 2,4-dinitrophenylhydrazine (DNPH, 0.1 g/L) and 0.25 ml of trifluoroacetic acid were added to it. The sample was vortexed for 30 s, centrifuged, supernatant 60° C. water bath. HPLC was used for analysis, with a detection wavelength of 355 nm, column temperature of 35° C., and mobile phase of 65% acetonitrile.

TABLE-US-00016 TABLE 16 Effects of drinking and 5-methyltetrahydrofolate on ethanol and methanol contents in urine of rats 3 h 4 h 8 h 12 h 16 h 24 h Group (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) Ethanol Ethanol group 11.2 ± 1.2  23.3 ± 1.9  25.8 ± 2.5  10.3 ± 1.8  4.2 ± 1.0 1.7 ± 0.8 Content 5- 0.6 ± 0.4 0.8 ± 0.5 1.5 ± 0.4 1.5 ± 0.3 1.7 ± 0.5 1.6 ± 0.4 methyltetrahydrofolate group 5- 11.6 ± 1.3  25.2 ± 2.2  24.1 ± 2.3  11.8 ± 2.0  4.3 ± 0.8 1.7 ± 0.7 methyltetrahydrofolate ethanol group Control group 0.7 ± 0.3 0.8 ± 0.4 1.4 ± 0.6 1.7 ± 0.6 1.4 ± 0.5 1.5 ± 0.5 Methanol Ethanol group 1.7 ± 0.3 3.3 ± 0.6 7.8 ± 2.1 5.6 ± 1.3 5.2 ± 1.0 4.4 ± 0.7 content 5- 1.0 ± 0.3 1.1 ± 0.4 1.2 ± 0.4 1.1 ± 0.4 1.3 ± 0.5 1.1 ± 0.3 methyltetrahydrofolate group 5- 1.6 ± 0.3 2.5 ± 0.4 6.4 ± 1.0 4.4 ± 0.9 3.7 ± 0.9 1.9 ± 0.7 methyltetrahydrofolate ethanol group Control group 0.9 ± 0.2 1.0 ± 0.3 1.1 ± 0.2 1.1 ± 0.2 1.0 ± 0.3 1.0 ± 0.2

[0130] The results show that the content of ethanol in the urine of rats is highest about 8 hours after drinking, and then decreases rapidly. Surprisingly, the content of methanol in the blood of rats after drinking also increases rapidly, consistent with the increasing trend of ethanol. However, the elimination rate of methanol is much slower than that of ethanol. It shows that the metabolism of endogenous methanol is inhibited by ethanol, which leads to an increase in blood methanol concentration. Surprisingly, 5-methyltetrahydrofolate reduced the concentration of methanol in urine.

TABLE-US-00017 TABLE 17 Effects of drinking and 5-methyltetrahydrofolate on formaldehyde and formate contents in urine of rats 3 h 4 h 8 h 12 h 16 h 24 h Group (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) Formaldehyde Ethanol group 0.8 ± 0.6 1.8 ± 0.7 4.3 ± 1.8 6.1 ± 1.8 4.2 ± 1.7 1.8 ± 0.7 Content 5- 0.8 ± 0.4 0.8 ± 0.5 0.7 ± 0.4 0.8 ± 0.3 0.8 ± 0.5 0.8 ± 0.4 methyltetrahydrofolate group 5- 0.8 ± 0.4 1.7 ± 0.6 2.9 ± 1.2 3.1 ± 1.0 1.9 ± 0.9 1.0 ± 0.5 methyltetrahydrofolate ethanol group Control group 0.8 ± 0.3 0.9 ± 0.4 0.9 ± 0.5 0.9 ± 0.6 0.9 ± 0.4 1.0 ± 0.5 Formate Ethanol group 230 ± 38  276 ± 41  550 ± 68  648 ± 72  411 ± 52  248 ± 34  content 5- 215 ± 33  237 ± 42  198 ± 36  187 ± 32  190 ± 41  188 ± 36  methyltetrahydrofolate group 5- 241 ± 42  255 ± 30  345 ± 43  351 ± 45  247 ± 48  211 ± 39  methyltetrahydrofolate ethanol group Control group 210 ± 36  247 ± 32  243 ± 36  217 ± 32  236 ± 47  226 ± 45 

[0131] The results show that the intake of 5-methyltetrahydrofolate can significantly reduce the concentrations of formaldehyde and formate in urine of rats, and promote the elimination of methanol metabolites.

[0132] Experiment 11 Effects of Drinking and 5-Methyltetrahydrofolate on the Concentrations of Formaldehyde and Formate in the Brain of Rats

[0133] According to the results of Experiment 10, the majority of alcohol in rats is metabolized 24 hours after drinking. The concentrations of formaldehyde and formate, as well as those of serotonin and dopamine in brain tissue of rats were detected at this time. SPF-grade female SD rats, fasted overnight, were divided into 4 groups.

[0134] (1) Ethanol group, with intragastric administration of ethanol at a dose of 1 g/kg respectively at 1, 2, 3 h, n=5; (2) control group, with intragastric administration of glucose at a dose of 1.6 g/kg (equivalent to alcohol calories) respectively at 1, 2, and 3 h, n=5; (3) 5-methyltetrahydrofolate group, with intragastric administration of 5-methyltetrahydrofolate calcium at a dose of 4 mg/kg at 0 h, followed by glucose at a dose of 1.6 g/kg (equivalent to alcohol calories) respectively at 1, 2, and 3 h, n=5; (4) 5-methyltetrahydrofolate alcohol group, with intragastric administration of 5-methyltetrahydrofolate calcium at a dose of 4 mg/kg at 0 h, followed by ethanol at a dose of 1 g/kg respectively at 1, 2, and 3 h, n=5.

[0135] Food grade alcohol was used, with a methanol content of less than 5 mg/L. At 24 h after drinking (the results of Experiment 10 show that the concentration of ethanol in urine of rats is close to zero after 24 h), eyeballs were removed and blood was collected, rats were sacrificed by spinal dislocation, brain tissue was taken and stored in a freezer at −80° C. Gas chromatography was used to detect methanol and ethanol concentrations in blood of rats, and HPLC was used to detect formaldehyde concentration in brain tissue. 0.1 g of brain tissue was taken and 0.5 ml of SDN lysate was added to make homogenate, then 0.5 ml of trifluoroacetic acid was added. The sample was centrifuged at 4° C., 0.4 ml of supernatant was collected, 0.1 ml DNPH (1 g/L) was added, mixed and incubated in 60° C. water bath for 30 min, centrifuged at 4° C., and supernatant was collected to detect formaldehyde concentration. Fluorescence spectrometry was used to detect formate concentration in brain. Serotonin and dopamine enzyme-linked immunosorbent reagent was used to detect neurotransmitters in brain in strict accordance with the instructions.

[0136] The results are as follows:

TABLE-US-00018 TABLE 18 Contents of formaldehyde, formate, serotonin, and dopamine in brain tissue of rats 24 h after drinking 5- Formaldehyde Formate hydroxytryptamine Dopamine Group (mg .Math. L.sup.−1) (mg .Math. L.sup.−1) (μg .Math. L.sup.−1) (ng .Math. L.sup.−1) Ethanol group .sup. 3.87 ± 0.61* 32.2 ± 4.7*.sup.  .sup. 0.07 ± 0.02* 32 ± 3 5-methyltetrahydrofolate 0.39 ± 0.11.sup.# 2.1 ± 0.2.sup.# 0.12 ± 0.04.sup.# 35 ± 3 group 5-methyltetrahydrofolate  1.14 ± 0.31*.sup.#  5.5 ± 0.4*.sup.# 0.10 ± 0.1.sup.#  27 ± 4 ethanol group Control group 0.48 ± 0.12.sup.# 2.6 ± 0.4.sup.# 0.11 ± 0.03.sup.# 29 ± 4 Note: n = 5, compared with normal control group, in each dose group, *P < 0.05; .sup.#Compared with ethanol control group, in each dose group, P < 0.05.

[0137] The results show that 5-methyltetrahydrofolate can promote the metabolism of formaldehyde and formate in brain tissue, and reduce the concentrations of formaldehyde and formate, thereby preventing and treating hangovers. In addition, the level of serotonin in brain tissue of rats after drinking decreased, and 5-methyltetrahydrofolate can improve the secretion of serotonin.

[0138] Experiment 12 Effect of 5-Methyltetrahydrofolate on Intestine Induced by Chronic Alcohol

[0139] 96 SPF-grade SD rats, half female and half male, were chosen with an average weight of 176-220 g at the beginning of the experiment, and randomly divided into vehicle control group (glucose, 10 g.Math.Kg−1) and model control group (liquor, 6 g.Math.Kg−1), 5-methyltetrahydrofolate calcium group (folic acid, 4 mg.Math.Kg−1, glucose, 10 g.Math.Kg−1), half female and half, 16 rats in each group. During the experiment, each rat was given alcohol or isocaloric glucose daily by gavage, and the dose was gradually increased every day. The day on which the dose of alcohol reached 6g.Math.Kg−1 was defined as the first day of modeling. The intestinal permeability of rats was measured at Weeks 2 and 8, then 5 rats were sacrificed at random, blood and liver tissues were taken.

[0140] 8 weeks later, rats in the remaining groups received fecal microbiota transplantation from patients with alcoholic steatohepatitis, and were sacrificed at Week 10, then blood and liver tissues were taken.

[0141] An oral sugar test was carried out to evaluate intestinal permeability. After fasting for 8 h, the rats were given 2.0 ml of sugar solution,

with lactulose (107 mg/kg), mannitol (30 mg/kg), sucralose (15 mg/kg) and sucrose (570 mg/kg). The rats were individually housed in metabolic cages, and urine was collected. The sugar concentration in the urine was determined by gas chromatography. Blood samples were used to analyze endotoxin in serum with Kinetic-QLC kit.

[0142] The intestinal permeability detection results are shown in FIGS. 3 and 4. The results show that alcohol at a dose of 6 g/kg destroys the intestinal barrier function of rats, and urine lactulose (small intestinal permeability index) of chronic alcohol-fed rats is significantly higher than that of glucose-fed rats at Week 8. Sucralose (whole intestinal permeability index) in urine of alcohol-fed rats also increased, and the difference was significant at Week 8.

[0143] The blood endotoxin detection results are shown in FIG. 5. Endotoxin was detected in blood serum obtained from sacrificed rats. Throughout the study, the value of endotoxin in serum of glucose-fed rats was very low, and drinking led to an increase in serum endotoxin level. The level of endotoxin in the serum of the ethanol group at Week 8 increased by about 3 times compared with that in the second figure.

[0144] Fatty degeneration was detected in the liver of sacrificed rats at the earliest within 2 weeks, and alcoholic steatohepatitis (inflammatory cell infiltration, spot necrosis and hepatocyte necrosis) was not seen. Typical symptoms of steatohepatitis were seen in sacrificed rats at Week 8, but still few. It has been determined that endotoxin is an important factor that causes severe liver injury and promotes the development of hepatitis. Endotoxin produced by bacteria in intestinal lumen penetrates into the hepatic portal circulation and then reaches the liver.

[0145] 60 g of stools were collected from patients with alcoholic steatohepatitis, mixed with 300 mL of normal saline and filtered, and the bacteria solution was stored in an anaerobic bag at 4° C. for later use. After 8 weeks, the remaining rats in all groups were anesthetized with ether, and catheter was used to slowly inject the bacteria solution (2 mL) into their colon, and the rats continued to be fed with the same doses the next day. At Week 10, all rats were sacrificed, liver tissues were stained with H&E and sectioned for disease assessment. At least three different sections of each rat were studied to reasonably assess liver lesions. In order to reasonably assess fatty degeneration, necrosis, inflammation, and fibrosis of liver, liver lesions were graded into different degrees. The proportion of fatty liver cells was <50%, 50-75% and >75%, respectively, corresponding to the severity of fatty degeneration. Focal necrosis was also quantified (number of necrotic foci/mm2), and dense inflammatory infiltrates were also graded. Alcoholic steatohepatitis (ASH) is defined as the presence of inflammatory cell infiltration, spot necrosis and stem cell necrosis in the liver.

[0146] The results are as follows:

TABLE-US-00019 TABLE 19 Indexes of hepatitis and stem cell damage after transplantation of intestinal bacteria in different groups Ethanol group 5-methyltetrahydrofolate Control group Case percentage group Case percentage Week Week Case percentage Week Week 8 10 Week 8 Week 10 8 10 Check item (n = 5) (n = 6) (n = 5) (n = 6) (n = 5) (n = 6) Fatty Percentage of fatty 20%  100% 0% 0% 0% 0% degeneration liver cells > 75% Percentage of fatty 40%   0% 20%  33.3%   20%  20%  liver cells 50-75% Percentage of fatty 40%   0% 80%  66.7%   80%  80%  liver cells < 50% Necrosis foci >3 pcs/mm.sup.2  0% 66.7% 0% 0% 0% 0% 1-3 pcs/mm.sup.2 20% 33.3% 0% 0% 0% 0% <1 pc/mm.sup.2 80%   0% 100%  100%  100%  100%  Liver fibrosis  0% 1 case 0% 0% 0% 0% of mild peri- sinusoid fibrosis Inflammatory >4 pcs/mm.sup.2  0%   50% 0% 0% 0% 0% foci 1-4 pcs/mm.sup.2 40%   50% 0% 0% 0% 0% <1 pc/mm.sup.2 60%   0% 100%  100%  100%  100% 

[0147] The results support that intestinal endotoxin infiltration is involved in the occurrence of hepatic necrosis and inflammation, which is consistent with previous conclusions. Alcohol and endotoxin exert a synergistic damaging effect on the liver. Oral non-absorbable antibiotics or lactic acid bacteria can reduce liver injury in rats caused by alcohol. Experiments have also proved that endotoxemia precedes steatohepatitis.

[0148] The transplantation of intestinal microbiome from patients with severe alcoholic steatohepatitis was used to humanize normal rats to induce liver injury in rats. The results show that 5-methyltetrahydrofolate can improve intestinal barrier function in the presence of alcohol and prevent liver injury caused by harmful bacteria.