Formulation for use in the prevention and treatment of carbohydrate induced diseases and conditions

Composition for preventing and treating diseases and disorders induced by sugar

Technical field

The present invention generally relates to compositions formulated for the prevention and treatment of diseases and disorders caused by high sugar intake. More specifically, the present invention relates to a novel composition containing a mixture of two specific types of compounds-unsubstituted B-ring flavonoids and flavans-which are used to prevent and treat weight gain and obesity And other diseases and disorders caused by high sugar intake. The diseases and conditions include, but are not limited to, hyperlipidemia, high cholesterol, arteriosclerosis, atherosclerosis, X syndrome (metabolic syndrome), hypertension, and systemic inflammation caused by obesity and diabetes.

Background technique

Energy is produced by ATP generated from "food". More specifically, when food is consumed, it is broken down into its constituent parts, mainly composed of simple and complex sugars, fats, proteins, and indigestible fibers such as cellulose. Then, these sugars, fats and proteins are further broken down into their basic units: sugars are broken down into monosaccharides, proteins are broken down into amino acids, and fats are broken down into fatty acids and glycerol. The body then uses these basic units to produce the substances needed for its growth, maintenance, and energy production. Sugar, protein, and fat can all be metabolized to provide energy in the form of ATP. However, sugar is the main substrate for the body to generate ATP through glycolysis and the tricarboxylic acid cycle.

According to the size of the molecule, sugars are classified as simple sugars or complex sugars. Simple sugars are small molecular compounds, especially monosaccharides and disaccharides such as glucose, fructose, galactose, and sucrose. Complex sugars or polysaccharides are composed of long chains of simple sugars. The most important polysaccharides are starch, glycogen and cellulose, all of which are glucose polymers with different connections between glucose molecules. Glycogen is the energy reservoir of animals, starch is the energy reservoir of plants, and cellulose is the main structural component of plants. Although most forms of starch are digestible, humans lack the enzymes necessary to digest cellulose, so starch becomes our dietary fiber (dietary fiber).

More than half of the sugar consumed by humans is traditionally derived from starch sources such as bread and cereals. Starch is a mixture of amylose and amylopectin. Amylose is a linear polysaccharide composed of glucose molecules covalently bonded with α-1,4 bonds. Amylopectin is a branched polysaccharide composed of glucose molecules covalently bonded with one α-1,6 bond for every thirty α-1,4 bonds. Starch can be rapidly hydrolyzed by alpha-amylase secreted by salivary glands and pancreas. During hydrolysis, amylose is broken down into small linear oligosaccharides, such as maltose (two glucose molecules with α-1, 4 bonding) and maltotriose (three glucose molecules with α-1, 4 bonding) ). Amylopectin is broken down into small linear oligosaccharides and branched oligosaccharide α-dextrins (several glucose molecules connected by α-1,4 bonds and α-1,6 bonds). These sugars are further broken down into glucose monomers by maltase and β-amylase.

Other sugars consumed in our diet are simple sugars such as the monosaccharide glucose and fructose and disaccharide sucrose. Glucose is present at low levels in most natural foods, while fructose is mainly obtained from processed foods, sweeteners and, to a lesser extent, fruits and certain vegetables. Fructose can be synthesized by the enzymatic isomerization of dextrose (Bhosale et al. (1996) Microbiol. Rev. 60:280-300). Another well-known sweetener, sucrose, consists of glucose and fructose linked by an α-1, 2 bond between glucose C1 and fructose C2. Sucrose is hydrolyzed by sucrase in the intestinal mucosa to obtain glucose and fructose (Dahlqvist (1972) Acta Med. Scand. Suppl. 542: 13-18).

Insulin is a hormone secreted by the beta cells of the pancreas, which enables the body to use glucose to produce energy. One of the key metabolic effects of the hormone insulin is to control blood sugar levels by promoting the uptake of glucose into fat and muscle cells. In short, when glucose is stimulated by insulin to enter cells, the GLUT4 glucose transporter, which accumulates on the cell surface of muscle and fat cells, is upregulated (Furtado et al., (2002) Biochem.Cell.Biol. 80:569 -578). Then the increased level of GLUT4 on the cell surface promotes more glucose uptake from the circulation and storage in fat and cell tissues. With reference to Figure 1, it can be seen that once glucose is ingested, it is converted into glucose-6-phosphate by the enzyme-hexokinase D, also known as glucokinase-that is, the phosphoryl group from ATP is added to C6 of glucose to produce ADP And glucose-6-phosphate. Then glucose-6-phosphate is converted into fructose-6-phosphate, and fructose-6-phosphate is converted into fructose-1,6-diphosphate under the action of fructose phosphokinase, that is, another ATP molecule adds a phosphoryl group to On C1. Then, aldolase B (aldolase) converts fructose-1,6-diphosphate into dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Glyceraldehyde-3-phosphate is a suitable substrate for final conversion to pyruvate. , And then pyruvate is converted into acetyl-CoA when it enters the tricarboxylic acid cycle. Other glyceraldehyde-3-phosphates are produced from dihydroxyacetone phosphate by triose phosphate isomerase. Therefore, in the process of glycolysis, each molecule of glucose initially consumes two ATPs, and then two ATP and one molecule of NADH are generated in the process, and this molecule of NADH is finally converted into a net product of three ATP molecules.

When the amount of glucose in the blood exceeds the current energy requirement, it can be stored as glycogen in the liver and muscle, or it can be converted into triglycerides in the liver and transferred to adipose tissue for storage. Lipogenesis includes the process of fatty acid synthesis and subsequent triglyceride synthesis. Conversely, when the blood glucose level becomes low, the process is reversed and glucose is produced from acetyl-CoA and pyruvate. Gluconeogenesis refers to the process of producing glucose from acetyl-CoA and pyruvate, which is essentially the reverse process of glycolysis. Similar to adipogenesis, gluconeogenesis mainly occurs in the liver, and gluconeogenesis is a method by which glucose is produced and secreted into the bloodstream for the body's cells.

Fructose is a component of sucrose, and is the main sweetener and preservative added to processed foods in the last two decades (Hanover and White (1993) Am.J.Clin.Nutr.58(Supp.):724S-732S) ; Park and Yeltley (1993) Am. J. Clin. Nutr. 58 (Supp.): 737S-747S), which can also be used as the main source of body energy, but it enters the glycolytic pathway through a different mechanism. Unlike glucose, fructose does not require insulin and is shunted directly into the glycolytic pathway. (Elliott et al. (2002) Am. J. Clin. Nutr. 76: 911-922). When fructose enters the bloodstream, most of it (about 70%) is absorbed by the liver through the portal vein. (Toppings and Mayes (1971) Nutr. Metab. 13: 331-338; Mayes (1993) Am. J. Clin. Nutr. 58 (Supp.): 754S-765S). Fructose is mainly transformed in the liver via the fructose-1-phosphate pathway. With reference to Figure 1, it can be seen that the first step of this pathway is the phosphorylation of fructose into fructose-1-phosphate under the action of fructokinase, that is, a phosphoryl group on ATP is added to C1 of fructose to generate ADP and fructose-1-phosphate. . (Hers(1952)Biochim.Biophys.Acta 8: 416-423). Fructose-1-phosphate is then converted into glyceraldehyde and dihydroxyacetone phosphate. This alditol cleavage is catalyzed by a specific fructose-1-phosphate aldolase. Then a second ATP molecule is required to convert glyceraldehyde into glyceraldehyde-3-phosphate to enter the glycolytic pathway. In addition, dihydroxyacetone phosphate is converted into glyceraldehyde-3-phosphate by the action of triose phosphate isomerase to enter the glycolytic pathway. This process requires two ATP molecules. Fructose can be taken up and used selectively and rapidly through the liver because there is fructokinase that is lacking in most other tissues (ie fat and muscle) in liver cells. (Van den Berghe (1986): Metabolic Effects of Dietary Carbohydrates. Progress in Biochemical Pharmacology (Mcdonal & Vrana, eds), 21: 1-32, Karger, Basel, Switzerland; Hallfrisch (1987): Metabolic Effects of Dietary fructose (Reiser & Hallfrisch, eds), pp. 25-40, CRC Press, Boca Raton, FL).

Alternatively, fructose can be phosphorylated fructose-6-phosphate by hexokinase, which mainly occurs in the kidney, adipose tissue, and skeletal muscle. It is estimated that about 20% of the ingested fructose is immediately converted by the kidneys, while about 10% is rapidly absorbed by adipose tissue and skeletal muscle. (Froesch and Ginsberg (1962) J. Biol. Chem. 237: 3317-3324; Bergstrom and Hultman (1967) Acta Med. Scand. 182: 93-107). Most fructose in the kidney, adipose tissue and skeletal muscle is metabolized by fructose-6-phosphate. Therefore, at any given time, the concentration of fructose circulating in the bloodstream is very low. (Macdonald and Turner (1968) Lancet 1:841-843; Crossley and Macdonald (1970) Nutr. Metab. 12:171-178). The intestinal absorption of fructose is less than the intestinal absorption of glucose or sucrose, but glucose stimulates a strong fructose absorption response in the intestinal mucosa. (Truswell et al. (1988) Am. J. Clin. Nutr. 48: 1424-1430). High fructose levels do not seem to cause any significant increase in circulating glucose levels. (Schwarz et al., 1992; Tounian et al., (1994) Am. J. Physiol. 267: E710).

Calories caused by fat consumption have steadily decreased in the past thirty years (Kennedy et al. (1999) J. Am. Coll. Nutr. 18: 207-212), while the intake of dietary starch or complex sugars is still fairly stable. However, contrary to these trends, the use of added sugar in the food and beverage industry has increased substantially. Coupled with the increase in the number of calories from sugar obtained from outside the home, the main calories in modern, industrialized diets come from sugar and complex sugar sources. (Krebs-Smith (2001) J. Nutr. 131: 527S-535S; Nielsen et al. (2002) Prev. Med. 35: 107-113).

The use of sugar cane and sugar beet sweeteners has been reduced by approximately 40% in the past three decades. (Kanter (1998) "A dietary assessment of the US Food Supply: Comparing per capita food consumption with food guide pyramid serving recommendations," from the Food and Rural Economics Division, Economics Research service, USDepartment of Agriculture, Agricultural Economic Report no.772). In other words, the use of high fructose corn sweeteners has increased by about 300%. The primary reason for switching from glucose to fructose as the main sweetener in the Western world may be economic. Fructose is sweeter than sucrose and glucose and its production is easier and cheaper. In the past two decades, fructose corn sweetener has become the main food additive in the American diet. Soft drinks and fruit juice beverages account for 43-44% of added fructose. (Kanter(1998) "A dietary assessment of the US Food Supply: Comparing per capita food consumption with foodguide pyramid serving recommendations, "from the Food and Rural Economics Division, Economics Research Service, USDepartment of Agriculture, Agricultural Economic Report no.772). The total sugar consumption is still rising, which is entirely due to High fructose corn syrup is added to the food. (Krebs-Smith (2001) J. Nutr. 131: 527S-535S).

The health effects of added sugar, especially fructose, were not recognized before its application in food processing became ubiquitous. The use of fructose has coincided with a significant increase in obesity and diabetes in the last two decades. (Flegal et al. (1998) Int. J. Obes. 22: 39-47). As discussed in detail below, studies have revealed that the chronic, long-term effects of increased consumption of sugar, especially fructose, can be extremely harmful to sugar utilization, purine metabolism, premature aging and lipid metabolism. In addition, the resulting obesity is accompanied by the production of pro-inflammatory cytokine (TNFα), interleukin-6 (IL-6) and C-reactive protein (CRP) in the body. Produces a sustained inflammatory effect.

The production of TNFα and IL-6 is regulated by the transcription factor NFκB. NFκB plays an important role in regulating systemic inflammation and its relationship with sugar-induced obesity and the occurrence of subsequent diseases (Lebovitz (2003) Int.J.Clin.Pract.Suppl.134:18- 27). The activation of NFκB is part of the stress response activated by several biological processes including growth factors, lymphokines, cytokines, UV rays, pharmacological substances, and diet. (Spencer et al. (1997) Int. Immunol. 9: 1581-1588). In the inactivated form, NFκB is mainly contained in the cytoplasm and binds to inhibitory proteins of the IκB family. Dietary changes such as increased sugar intake can activate NFκB and cause phosphorylation of IκB, thereby releasing NFκB that allows molecules to move into the nucleus. In the nucleus, NFκB binds to the consensus sequence (5'GGGACTTTCC-3') of various genes to activate their transcription. In the systemic inflammation caused by sugar-induced obesity, this causes an increase in the expression of TNFα and IL-6. The increase in these pro-inflammatory proteins subsequently leads to an increase in CRP.

It has been reported that continued large-scale use of fructose as an energy source disrupts sugar metabolism and leads to reduced utilization of starch and glucose by many tissues. (Bender and Thadini (1970) Nutr. Metab. 12: 22-39; Tuovinen and Bender (1975) Nutr. Metab. 19: 161-172). Specifically, the continuous consumption of fructose leads to the down-regulation of hexokinase and the up-regulation of glucose-6-phosphate kinase in the liver (see Figure 1; Freedland and Harper (1957) J. Biol. Chem. 228:743- 751). This adaptive response leads to a reduction in the conversion of glucose to liver glycogen. (Vrana et al. (1978) Nutr. Metab. 22: 262-268; Vrana et al. (1978) Nutr. Metab. 22: 313-320). In the pancreas, due to the low concentration of the fructose transporter GLUT5 protein in β cells, fructose does not up-regulate insulin production. (Grant et al. (1980) Diabetologia 19: 114-117; Curry (1989) Pancreas 4: 2-9; Sato et al. (1996) Tissue Cell 28: 637-643). This leads to increased blood sugar over time and insulin insensitivity, which is the main cause of type II diabetes. The liver can use fructose to produce glycogen through an adaptive enzyme reaction, but the level is much lower than that of glucose that can be converted into glycogen. (Freedland and Harper (1957) J. Biol. Chem. 228:743-751). In addition, the increased and long-term consumption of sucrose or fructose leads to an increase in the liver's ability to synthesize fatty acids, which results in a decrease in liver glycogen reserves due to the inability of glucose to be converted by glycolysis. (Vrana et al. (1978) Metablism 27: 885-888). The diet also impairs the ability of fat and muscle tissue to use glucose to produce energy (Bender and Thadini (1970) Nutr. Metab. 12: 22-39; Kelsay et al., (1977) Am. J. Clin. Nutr. 30: 2016-2022 ).

When fructose is used as an energy source, the level of glycolysis intermediates increases, but the cost of producing these intermediates is very high. (Hers (1952) Biochim. Biophys. Acta 8: 416-423). As mentioned above, when fructose is consumed, most of it enters the liver and is converted into fructose-1-phosphate. Therefore, individuals who consume a large amount of fructose, which is basically a substitute for glucose, sequestering phosphate ions in other parts of the body through the action of fructokinase in the form of fructose-1-phosphate (Figure 1; Woods et al. (1970) Biochem. J.119) : 501-510). Because there is no spare phosphate ion, the oxidative phosphorylation of ADP is inhibited, resulting in a shortage of ATP in the liver. The conversion of glyceraldehyde to glyceraldehyde-3-phosphate by trioskinase provides a substrate for glycolysis and further depletes the phosphate pool. When the phosphate reserve becomes sufficiently low, a large amount of AMP that can be metabolized by AMP deaminase and 5'-nucleotidase is produced. (Mayes (1993) Am. J. Clin, Nutr. 58 (Suppl): 754S-765S). The metabolism of AMP leads to an increase in the level of inosine, which eventually leads to the formation of large amounts of uric acid and the possibility of hyperuricemia.

Hyperuricemia has attracted attention in studies in which fructose is given to normal children and children with inherited fructose intolerance. (Perheentup and Raivio (1967) Lancet 2: 528-31). An increased incidence of hyperuricemia can also be detected when fructose is given to diabetic and those suffering from gout. (Hallfrisch (1987): Metabolic Effects of Dietary Fructose (Reiser & Hallfrisch, eds), pp. 25-40, CRC Press, Boca Raton, FL). Even when healthy subjects consume about 18% of their energy requirements in the form of fructose, many people show signs of hyperuricemia, indicating that ordinary individuals are sensitive to reduced phosphate ions and ATP concentrations in the body. (Hallfrisch (1987): Metabolic Effects of Dietary Fructose (Reiser & Hallfrisch, eds), pp. 25-40, CRC Press, Boca Raton, FL).

Especially young men (14-18 years old) are most likely to suffer from hyperuricemia. According to the 1977-1978 U.S. Department of Agriculture Nationwide Food Consumption Survey (1977-1978 U.S. Department of Agriculture Nationwide Food Consumption Survey), men in this age group consume about 100 g of fructose, which is mainly derived from soda, every day. Since then, especially in the United States, fructose has also been introduced into the general food supply. Only two cans of soda contains about 50g of fructose. It is estimated that the average fructose consumption of adolescents has increased significantly in the last two decades, from about 64g per day to 150g per day. (Kanter (1998) "A dietary assessment of the US Food Supply: Comparing per capitafood consumption with food guide pyramid serving recommendations," from the Food and Rural Economics Division, Economics Research Service, USDepartment of Agriculture, Agricultural Economic Report no.772; Elliott et al. (2002) Am. J. Clin. Nutr. 76: 911-922). Therefore, a whole generation of men are currently experiencing a decrease in the available phosphate storage and ATP in the body. Generally speaking, in addition to the increased risk of hyperuricemia, it also has a significant effect on a number of in vivo processes, and in fact can cause protein and uric acid synthesis to be inhibited, leading to stunted growth in children, increased disease incidence during aging, and Premature aging. (Maenpaa et al. (1968) Science 161: 1253-1254; Bode et al. (1973) Eur. J. Clin. Invest. 3: 436-441).

A special effect of consuming large amounts of sucrose and/or fructose is that fructose promotes glycation or non-enzymatic cross-linking of macromolecules such as nucleic acids, proteins and lipoproteins with sugars ("Maillard Reaction"). Glycation was first published by Monnier, who proposed that the aging process in the body may be promoted by the Maillar reaction. (Monnier (1989) "Toward a Malliear reaction theory of aging: the Malliard reaction in Aging, Diabetes, and Nutrition," (Baynes, JW & Monnier VM, eds.), pp.1-22, Alan R. Liss, New York, NY). In the Maillar reaction, sugars such as glucose and fructose first form an unstable Schiff's base with the N-terminal amino group of a protein or nucleic acid, and then undergo rearrangement to form a more stable compound. Over time, the sugar moiety bound to the glycated protein/amino acid is chemically modified into a molecular structure called advanced glycation end product (AGE). AGE can interfere with the normal function of the protein to which it is attached. In addition, in the presence of reactive oxygen species (ROS), AGE can be covalently cross-linked with adjacent protein chains. Therefore, proteins are fully connected to other macromolecules through the formation of covalent bonds, forming large, complexes that must then be cleared by the body.

In theory, both aldose and ketose can participate in the Melat reaction (Yaylayan and Huyghues (1994) Crit. Rev. Food Sci. 34: 321-369), but it has been found that the reactivity of glucose is much lower than that of fructose (Bunn And Higgins (1981) Science 213: 222-4; McPherson et al. (1988) Biochemistry 27: 1901-1907). All molecules with free amino groups can undergo a Maillar reaction in the body. However, in general, the ε-amino group of lysine is the main reactant of saccharification. Other amino acids such as arginine, histidine, tyrosine, tryptophan, serine, and threonine are also involved in the Maillar reaction between proteins and other macromolecules (Monnier (1989) "Toward a Malliearreaction theory of aging: the Malliard Reaction in Aging, Diabetes, and Nutrition, "(Baynes, JW & Monnier VM, eds.), pp. 1-22, Alan R. Liss, New York, NY, 1989).

Diabetics who use fructose as a substitute for glucose in their diet are particularly sensitive to the effects of glycation. As mentioned above, fructose is usually present in low concentrations in the blood, but the concentration of fructose found in the corneal crystals and nerves of diabetic patients is equivalent or higher than that of glucose. (Jedziniak et al. (1981) Investig. Ophthalmol. Vis. Sci. 20:314-326; Mayhew et al. (1983) Diabetologia 24: 13-15). This high concentration of fructose leads to glycation between proteins in the lens and blindness. (McPherson et al. (1988) Biochemistry 27: 1901-1907). With the increase in fructose consumption in the past two decades (Park and Yeltley (1993) Am.J.Clin.Nutr.58(supp):737S-747S), blindness is the most common microvascular complication associated with diabetes. Not surprising. (Jochmann and Hammes (2002) Z. Arztl. Fortbild. Qualitatssich. 96:167-174).

Among all the problems associated with high fructose intake, none can match the effects of fructose on lipid accumulation, fat production, and weight gain. Low-fat and high-sugar diets are popular in developing countries because of thorough understanding of the dangers of fat and cholesterol in cardiovascular disease, recognition of fructose as a sweetener substitute for diabetic patients, and unawareness of the danger of fructose consumption. (Gerrits and Tsalikian (1993) Am. J. Clin. Nutr. 58 (Supp.): 796S-799S; Sonko et al. (1993) Acta Physiol. Scand. 147: 99-108). This leads to a tremendous increase in fructose consumption and is associated with the increase in weight gain and obesity in the past two decades for the reasons discussed below.

Lipogenesis occurs when simple sugars, especially fructose, are ingested to a level that exceeds the current energy requirement. (Kazumi et al. (1997) Endocrinol. J. 44(2): 239-245; Noguchi and Tanaka (1995) Obes. Res. 3(Supp. 2): 195S-198S). As mentioned above, lipogenesis includes the process of fatty acid synthesis and subsequent triglyceride synthesis. Due to the limited ability of higher animals to store polysaccharides, when simple sugars such as glucose and fructose are ingested to exceed the current energy requirements and storage capacity, they are converted into triacylglycerols and stored in fat or adipose tissue. To understand how the composition described here affects lipogenesis, one must first understand the way lipids are formed in the body. Figure 1 illustrates the interaction of fructose and glucose in the formation of acylglycerol, acetyl-CoA and finally very low density lipid (VLDL). There are two main ways to produce fat from sugar. First, the excess glucose is transferred to adipogenesis and fat regeneration through the accumulation of acetyl-CoA (De novo lipogenesis) transfer. Referring to Figure 1, excess acetyl-CoA is converted into malonyl-CoA by acetyl-CoA carboxylase, and then into acyl-CoA. Acyl-CoA enters adipogenesis through esterification and is then converted into acylglycerol by the action of glycerol-2-phosphate acyltransferase. Acylglycerol and cholesterol are then converted into very low density lipids (VLDL). Insulin has a positive effect on this process to promote the regeneration of fat. (Park et al. (1997) J. Lipid Res. 38: 2529-2536).

Second, excess fructose is transferred to adipogenesis through the accumulation of dihydroxyacetone phosphate that exceeds the energy requirement, where dihydroxyacetone phosphate is converted to glycerol-3-phosphate (see Figure 1). Glycerol-3-phosphate is esterified by glycerol-3-phosphate acyltransferase to produce acylglycerol, which combines with cholesterol to produce VLDL. Due to the high energy consumption of fructose processed through glycolysis, the fat production through VLDL represents the body's energy savings. The deposition of glycogen from glucose (2.5 mol ATP/mol glucose) is also more efficient than from fructose (3.5 mol ATP/mol fructose). (Tapp and Jequier (1993) Am. J. Clin. Nutr. 58: 766S). However, as mentioned above, fructose metabolism has a negative effect on glycolysis and glycogen deposition, that is, inhibit the key glycolytic enzyme gene and protein expression, remove phosphoric acid from the liver to reduce ATP production, and ultimately reduce The general metabolic output in fructose-induced obesity.

Several studies have shown that fructose is more likely to produce fat than glucose. Rats continuously fed a large amount of fructose showed increased levels of VLDL triglycerides in the blood. (Herman et al. (1970) Fed. Proc. 29: 1302-1307; Steiner et al. (1984) Am. J. Physiol. 246: E187-E192; Kazumi et al. (1986) Am. J. Physiol. 250: E325-E330) . When fructose is ingested, it immediately forms high levels of plasma triglycerides and increases the rate of glycerol and fatty acid formation. (Reiser (1987) "Lipogenesis and blood lipids,": Metabolic effects of dietary fructose, (Reiser S & Hallfrisch J, eds.), pp. 83-111, CRC Press, Boca Raton, FL; Hallfrisch (1990) FASEB J. 4: 2652-2660). Long-term consumption of fructose can also increase mRNA or enzymes involved in fatty acid production such as fatty acid synthase (Bruckdorfer et al. (1972) Biochem. J. 129:439-446) and glycerol-3-phosphate dehydrogenase (Borrebach et al., (1976) Circ Res. 38: 1-21; Declerecq et al. (1982) Biochem. J. 204: 247-256). Conversely, a long-term fructose diet will reduce the activity of many key glycolytic enzymes, including ATP citrate lyase (Moser and Berdamier (1974) J. Nutr. 104: 687-94; Shafir et al. (1975) Isr. J. Med. Sci. 11: 1150-1154; Winder et al. (1975) Proc. Soc. Exp. Biol. Med. 148: 1150-1154), acetyl-CoA carboxylase (Bruckdorfer et al. (1972) Biochem. J. 129:439-446; Winder et al. (1975) Proc. Soc. Exp. Biol. Med. 148: 1150-1154; Waterman et al. (1975) Proc. Soc. Exp. Biol. Med. 150: 220-225), Glucose-6-phosphate dehydrogenase, NADP malate dehydrogenase and pyruvate kinase. However, the absorption of starch will enhance the activity of these enzymes. (Vrana and Fabry (1983) World Res. Nutr. Diet 42: 56-101). Specifically, the introduction of glycerol-3-phosphate dehydrogenase results in an increase in the content of VLDL in the liver by converting dihydroxyacetone phosphate into glycerol-3-phosphate (refer to Fig. 1).

It has been demonstrated that long-term feeding of fructose (Christophe and Mayer (1968) Am. J. Physiol. 197: 55-59) or sucrose diet (Fabry et al. (1968) Nutr. Diet 10: 81-90; Tepperman and Tepperman (1970) Fed. Proc. 29: 1284-1293) increased fat production in the liver of rats. Rats fed a high-fructose diet also showed increased lipid content and a substantial increase in organ weight compared to rats fed a standard starch or glucose-supplemented diet. (Wapnir and Devas (1995) Am. J. Clin. Nutr. 61: 105-110). The kidneys of animals fed a high-fructose diet were less affected. The weight of the heart and testes are not affected. In these studies, the addition of high fat to a high-fructose diet resulted in little increase in total kidney weight, indicating that fructose-induced lipogenesis is the main way of fat production. (Wapnir and Devas (1995) Am. J. Clin. Nutr. 61: 105-110).

Most of the studies conducted so far have been done on rats that can strictly control their diet. However, a lot of evidence shows that the increase in fructose consumption is blaming humans. Several tests have shown that fructose has a definite effect on weight gain in men and women. For example, when fourteen middle-aged men with diabetes added 50-60g of fructose or roughly the equivalent of two cans of soda sweetened with high-fructose corn syrup to their diets, they all showed Out of net weight gain. (Anderson et al. (1989) Diabetes Care 12: 337-344). In another study, overweight individuals who consumed artificial sweeteners or sucrose (50% fructose) that consumed about 28% of their energy requirements were compared. Individuals who consumed sucrose supplements showed body weight and fat mass over a 10-week period. And blood pressure increases. (Atrup et al. (2002) Am. J. Clin. Nutr. 75 (Suppl): 405S (abstract)). In addition, Raben et al. pointed out that individuals who were given starch lost weight during 14 days, while those who were given sucrose (50% fructose) had no change in weight. (Raben et al. (1997) Int. J. Obes. Relat. Metab. Disord. 21:846-859). These observations are consistent with the experiment done by Schwarz et al., showing that fructose-induced lipogenesis increased in thin or obese subjects. (Schwarz et al. (1995) J. Clin. Invest. 96: 2735-2743).

Certain pro-inflammatory markers are associated with weight gain and obesity. Among these are TNFα, IL-6 and CRP. TNFα is expressed and secreted by adipocytes, and has been shown to be directly related to obesity and BMI, but not necessarily directly related to insulin insensitivity and hyperinsulinemia. (Hotamisligil et al. (1993) Science 259: 87-91; Ronnemaa et al. (2000) J. Clin. Endocrinol. Metab. 85: 2728-2732; Berberoglu (2001) J. Pediatr. Endocrinol. Metab. 14: 543-547) .

Body mass index (BMI) is a measure of body fat based on height and weight. BMI is used as a measure of overall obesity and is one of many factors involved in the potential for the development of chronic diseases such as heart disease, cancer or diabetes. Other important factors for assessing the risk of an individual developing chronic diseases include diet, physical activity, waist circumference, blood pressure, blood sugar level, cholesterol level, and family history. BMI is calculated as follows:

An individual with a BMI between 25-29.9kg/m2 will be considered overweight, and an individual with a BMI 30kg/m2 will be considered obese. Individuals with a BMI 25 are considered to have normal weight.

There are three types of obesity in the body-subcutaneous, internal organs and organs. (Cinti (2000) EatWeight Disord. 5: 132-142). Liver (organ) obesity has been shown to be highly correlated with fructose consumption (Wapnir and Devas (1995) Am. J. Clin. Nutr. 61: 105-110). On the other hand, visceral obesity is related to the intake of various fats and sugars, including fructose/sucrose sources. (Tarui et al. (1991) Int. J. Obes. 2 (Suppl): 1-8; Keno et al. (1991) Int. J. Obes. 15:205-211). The higher the amount of visceral obesity, the higher the amount of TNFα produced by the body. (Tsigos et al. (1999) Metabolism 48: 1332-5; Vgontzas et al. (2000) J. Clin. Endorcrinol. Metab. 85: 1151-1158). It has been found that high sucrose/fructose diets increase visceral obesity.

TNFα induces adipose tissue to secrete IL-6, which leads to glucorticoid-induced lipolysis, thereby releasing a higher concentration of unesterified fatty acids in the circulation that can be used as an additional pool for fat re-synthesis (Patton et al. (1986) ) Proc. Natl. Acad. Sci. USA 83: 8313-8317; Fried et al. (1998) Endocrinol. Metab. 83: 847-850). In LDL-receptor-deficient mice fed high fructose, atherosclerotic damage contains high levels of TNFα, which induces transcription factors to promote gene expression of proteins that can cause greater damage (Goetze et al. (2001) Atherosclerosis 159: 93-101).

The acute phase represents a state of injury or inflammation in which IL-6 induces gene and protein expression of specific inflammatory proteins such as CRP and fibrinogen (Heinrich et al. (1990) Biochem. J. 265: 621-636) . No other cytokines can perform this function in the acute phase. Although it is known that there is a close correlation between increased BMI and serum fibrinogen concentration (Krobot et al. (1991) Arterioscler. Thromb. 12: 780-788), the increase was not known before the discovery of the production and secretion of IL-6 by abdominal fat cells There is a clear link between BMI and IL-6 (Mohamed-Ali et al. (1997) J. Clin. Endocrinol. Metab. 82: 4196-4200). Therefore, the level of IL-6 and BMI in the body are directly related to visceral obesity.

The side effects of IL-6 production may be even more damaging than those mentioned above. IL-6 secreted from visceral fat cells is mainly absorbed by the portal vein, and thus mainly acts on hepatocytes. Similar to TNFα, IL-6 also promotes lipolysis in the liver, but it specifically increases the expression of fibrinogen gene, leading to increased blood fibrinogen concentration and increased incidence of cardiovascular disease (McCarty (1999) Medica Hypotheses 52: 465-477).

Related to its gene expression activity is the effect of IL-6 on the increase in circulating CRP levels. IL-6 regulates the transhepatic synthesis of CRP (Heinrich et al. (1990) Biochem. J. 265:621-636; Bataille and Klein (1992) Arthritis Rheum. 35:982-983). Because increased BMI is related to the concentration of TNFα and IL-6, researchers have looked for a correlation with CRP. It has been found that there is a direct correlation between CRP serum concentration and BMI. (Visser et al. (1999) JAMA 282:2131-2135). In fact, there is almost a linear relationship between BMI and CRP concentration.

Compared with TNF and IL-6, it is found that there is a stronger correlation between visceral obesity and CRP concentration, (Forouhi et al. (2001) Int.J.Obes.Relat.Metab.Disord.25:1327-1331), so that it can It is the main predictor of brachial artery endothelial function during acute phase cardiovascular disease (Brooks et al. (2001) Am. J. Cardiol. 88:1264-9). Excessive intake of fructose and sucrose will undoubtedly lead to an increase in CRP, because increased secretion of TNFα induces an increase in IL-6 levels, which subsequently induces the expression of CRP. The cytokine cascade, which is initially caused by the up-regulation of fructose-induced adipogenesis in the liver, is responsible for the basic protein determinants in obesity, cardiovascular disease, diabetes, and many other diseases. (McCarty (1999) Medical Hypotheses 52:465-477). Reducing fructose and sugar-induced weight gain is essential to prevent these inflammation-based diseases.

"Starch blockers" are compounds derived from plants that can partially inhibit the action of α-amylase, thus causing starch to pass through the digestive tract without being fully utilized as a source of glucose. Specifically, it has been found that phaseolamin, an extract isolated from kidney beans, can effectively block the breakdown of sugars by inhibiting alpha-amylase. (Marshall and Lauda (1975) J. Biol. Chem. 250: 8030-8037). Many "starch blockers" isolated from various plant resources are currently commercially available. A comprehensive search of the literature on plants and plant extracts that affect sugar-induced weight gain yielded only one result. Bofu-tsusho-san (BOF), a traditional Chinese medicine, can inhibit the synthesis of triglycerides in the liver and enhance the lipolysis of fat cells. (Morimoto et al. (2001) Nippon Yakurigaku Zasshi 117:77-86). However, analysis of the extract revealed that it contains ephedrine and d-pseudoephedrine, which can inhibit the activity of phosphodiesterase (PDE) in adipocytes, which may explain its effect of inhibiting weight gain. (Yoshida et al. (1995) Int. J. Obes. Relat. Metab. Disord. 19: 717-722). Mu Huang, another well-known plant extract for weight loss, also contains ephedrine. (Boozer et al. (2002) Int. J. Obes. Relat. Metab. Disord. 26: 593-604; Boozer et al. (2001) Int. J. Obes. Relat. Metab. Disord. 25: 316-324). Although several short-term clinical studies have shown that the combination of Mu Huang, caffeine and guarana is safe, recent reports suggest that weight loss preparations containing ephedra have serious cardiac side effects.

The composition described here does not contain alpha-amylase inhibitors that affect starch breakdown and ephedrine, ephedrine or pseudoephedrine that affect sugar metabolism and weight gain. This extract reduces the weight gain caused by high-sugar diet by reducing the utilization of fructose and reducing the pro-inflammatory cytokines related to obesity, and helps to reduce weight.

Flavonoids or bioflavonoids are widely distributed natural products, which are reported to have antibacterial, anti-inflammatory, anti-allergic, anti-mutagenic, anti-viral, anti-tumor, anti-thrombic and vasodilator activities. The general structural unit of this group of compounds includes two benzene rings located on both sides of the 3-carbon ring, as shown in the following general structural formula:Various combinations of hydroxyl, sugar, oxygen, and methyl groups connected to this general tricyclic structure produce various types of flavonoids, including flavanols, flavonoids, flavan-3-ols (catechins), and anthocyanins Glycosides (anthocyanins) and isoflavones.

Unsubstituted B-ring flavonoids and flavonols are a special kind of flavonoids, which have no substituents on the aromatic B ring, as shown by the following general formula:

Wherein R1, R2, R3, R4 and R5 are independently selected from the following groups: -H, -OH, -SH, -OR, -SR, -NH2, -NHR, -NR2, -NR3+X-, carbon, Oxygen, nitrogen or sulfur, single or multiple sugar combined glycosides, including but not limited to aldose, aldose methyl, aldose, ketohexose and their chemical derivatives; where R has 1 -10 carbon atoms; and X is selected from the group of pharmaceutically acceptable counter anions, which include but are not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate Wait.

Although flavonoids are widely distributed natural products, unsubstituted B-ring flavonoids are relatively rare. Among 9396 flavonoids synthesized or isolated from natural sources, only 231 unsubstituted B-ring flavonoids are known (The Combined Chemical Dictionary, Chapman & Hall/CRC, 5:1 edition June 2001). According to reports, unsubstituted B-ring flavonoids have various biological activities. For example, galangal extract (3,5,7-trihydroxyflavone) acts as an antioxidant and free radical scavenger, and is believed to be a promising anti-genotoxic and cancer chemopreventive agent (Heo et al. (2001) Mutat. Res. 488:135-150). It is a tyrosinase monophenolase inhibitor (Kubo et al. (2000) Bioorg.Med.Chem. 8: 1749-1755), a rabbit heart carbonyl reductase inhibitor (Imamura et al. (2000) J. Biochem. 127: 653) -658), has antibacterial activity (Afolayan and Meyer (1997) Ethnopharmacol. 57: 177-181) and antiviral activity (Meyer et al. (1997) J. Ethnopharmacol. 56: 165-169). Begain, galangal extract and two other unsubstituted B-ring flavonoids have antiproliferative activity on human breast cancer cells (So et al. (1997) Cancer Lett. 112(2): 127-133).

The activity of flavonoids is usually determined randomly based on the effectiveness of flavonoids. Occasionally, specific biological activity emphasizes the need for substitution on the B ring, such as high affinity binding to p-glycoprotein (Boumendjel et al. (2001) Bioorg.Med.Chem.Lett.11(1):75-77), Effect (Itoigawa et al. (1999) J. Ethnopharmacol. 65(3): 267-272), the protective effect on endothelial cells against linoleic acid hydrogen peroxide-induced toxicity (Kaneko and Baba (1999) Biosci. Biotechnol. Biochem. 63(2): 323-328), COX-1 inhibitory activity (Wang (2000) Phytomedicine 7: 15-19) and prostaglandin endoperoxidase synthase activity (Kalkbrenner et al. (1992) Pharmacology 44(1): 1- 12) Need to be substituted on the B ring. There are only a few publications mentioning the importance of the unsubstituted B-ring in unsubstituted B-ring flavonoids. One example is the use of 2-phenylflavonoids, which inhibit NAD(P)H quinone receptor oxidoreductase, as potential anticoagulants (Chen et al. (2001) Biochem Pharmacol. 61(11):1417-1427).

The Chinese medicinal plant Scutellaria baicalensis contains a large amount of unsubstituted B-ring flavonoids, including begain, bekalin, vogonin and baicalenoside. Traditionally, the plant has been used to treat many diseases, including clearing away heat, purging fire, dampness and heat; polydipsia caused by high fever; carbuncle, ulcers and other purulent skin infections; upper respiratory tract infections such as acute tonsillitis, pharyngitis and scarlet fever ; Viral hepatitis; Nephritis; Pelvic inflammatory disease (pelvitis); Dysentery; Hematemesis and epistaxis. The plant is also traditionally used to prevent miscarriage (see Encyclopedia of Chinese Traditional Medicine, Shanghai Science and Technology Press, Shanghai, China, 1998). Clinically, Scutellaria baicalensis is currently used for the treatment of diseases including pediatric pneumonia, pediatric bacterial diarrhea, viral hepatitis, acute cholecystitis, hypertension, local acute inflammation caused by wounds and surgical procedures, bronchial asthma and upper respiratory tract infection (Encyclopedia of Chinese Traditional Medicine, Shanghai Science and Technology Press, Shanghai, China, 1998). The pharmacological efficacy of the root of Scutellaria baicalensis in treating bronchial asthma is reported to be related to the presence of unsubstituted B-ring flavonoids and their inhibitory effects, which can inhibit the supplementation of eosinophils related to eosinophil chemokines (Nakajima et al. (2001)) Planta Med. 67(2): 132-135).

So far, many naturally occurring unsubstituted B-ring flavonoids have been commercialized for various purposes. For example, liposome formulations of Scutellaria baicalensis extract are used for skin care (US Patent 5,643,598; 5,443,983). Because of its inhibitory effect on cancer-causing genes, Begalin is used to prevent cancer (US Patent 6,290,995). Bekalin and other compounds are used as antiviral, antibacterial and immunomodulatory agents (US Patent 6,083,921) and as natural antioxidants (Polish Patent Publication 9,849,256). Coyne is used because of its anxiety-reducing properties (US Patent 5,756,538). Anti-inflammatory flavonoids are used to control and treat anorectal and colon diseases (US Patent 5,858,371) and inhibit lipoxygenase (US Patent 6,217,875). These compounds are formulated with glucosamine collagen and other ingredients to repair and maintain connective tissue (Bath, US Patent 6,333,304). Flavonoid esters can constitute the active ingredients of cosmetic compositions (US Patent 6,235,294). Submitted on March 1, 2002, serial number 10/091,362, titled "Identification of Free-B-ring Flavonoids as Potent COX-2 Inhibitors" (Identification of Unsubstituted B-ring Flavonoids as Effective COX-2 Inhibitors) discloses the use of compositions containing unsubstituted B-ring flavonoids or unsubstituted B-ring flavonoids by administering to a subject in need The method of inhibiting cyclooxygenase COX-2 by the composition of the mixture. This is the first report linking the inhibitory activity of unsubstituted B-ring flavonoids with COX-2. The entire application is incorporated herein by reference.

Flavans include compounds represented by the following structural general formula:Wherein R1, R2, R3, R4 and R5 are independently selected from the following groups: -H, -OH, -SH, -OCH3, -SCH3, -OR, -SR, -NH2, -NRH, -NR2, -NR3 +X-, the esters of the substituents include but are not limited to gallic acid esters, acetates, cinnamoyl and hydroxycinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; carbon, oxygen, nitrogen or sulfur, single Or glycosides combined with multiple sugars, including but not limited to aldose, aldose methyl, aldose, ketulose and their chemical derivatives; dimerization, trimerization, and other polyflavans; Wherein R is an alkyl group having 1-10 carbon atoms; and X is selected from pharmaceutically acceptable counter anions, including but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, Carbonate and so on.

Catechin is a flavan, mainly found in green tea. It has the following structure:Catechin Catechin works alone and together with other flavonoids found in tea, and it has both antiviral and antioxidant activity. It has been confirmed that catechins are effective in the treatment of viral hepatitis. It has also been shown to prevent oxidative damage to the heart, kidneys, lungs, and spleen. Catechin can also inhibit the growth of gastric cancer cells.

Catechin and its derivatives derived from various plant sources, especially green tea seeds, are used to treat HPV-infected condyloma acuminata (Cheng, U.S. Patent 5,795,911) and to treat hyperplasia caused by papilloma virus (Cheng, U.S. Patent 5,968,973) And 6,197,808). Catechin and its derivatives are also used locally to inhibit mammalian tissues such as skin cancer, psoriasis, spider veins or under eye pupils. circle) and other angiogenesis (Anderson, U.S. Patent 6,248,341), used to combat UVB-induced tumorigenesis in mice (Agarwal et al. (1993) Photochem.Photobiol. 58:695-700), used to inhibit gene expression and enzyme activity levels Nitric oxide synthase (Chan, US Patent 5,922,756), and as a hair growth agent (Takahashi, US Patent 6,126,940). Catechin-based compositions are also formulated with other extracts and vitamins to treat acne (Murad, U.S. Patent 5,962,517), hardened digestive organs (Shi, U.S. Patent 5,470,589) and used in the treatment of androgen disorders related diseases And inhibit 5α-reductase activity in cancer (Liao, US Patent 5,605,929). Green tea extract was formulated with 7 other plant extracts to reduce inflammation by inhibiting the COX-2 enzyme without identifying any specific active ingredients (Mewmark, US Patent 6,264,995).

It has been confirmed that flavan, quercetin, and fisetin stimulate PDE activity in adipose tissue, contrary to inhibiting the activity of stimulating lipolysis. (Kuppusamy and Das (1994) Biochem. Pharmacol. 47:521-529). Quercetin and fisetin stimulate the activity of PDE in a dose-dependent manner. Regardless of the presence or absence of adrenaline, cyclic AMP accumulates in cells. In addition, the addition of the known lipolytic agent theophylline does not enhance its effect, while the addition of the specific β-adrenergic receptor agonist isoproterenol can inhibit its effect, which suggests that quercetin and fisetin are combined with β -Adrenaline receptor and adrenaline act synergistically. Catechin can also stimulate PDE activity, but cannot stimulate PDE activity in the presence of adrenaline, which suggests that it binds weakly to β-adrenergic receptors. (Kuppusamy and Das (1992) Bichem. Pharmacol. 44: 1307-1315). Moreover, in a one-month study in mice, green tea catechins caused an increase in acyl-CoA oxidase, medium-chain acyl-CoA dehydrogenase and β-oxidation activities in the liver, thereby increasing lipid catabolism . (Murase et al. (2002) Int. J. Obes. Relat. Metab. Disord. 26: 1459-1464).

The effect of catechins on fructose-induced obesity is unknown. There is also no evidence that Begain, Bekalin or Vogonin affect the metabolism of fructose. However, extracts containing two herbal compositions can indeed alter the metabolism of fructose, as described in detail below.

Acacia is a genus of legume trees and shrubs. The genus Acacia includes more than 1,000 species belonging to the legume family and the mimosa subfamily. Acacia is distributed all over the world, such as tropical and subtropical regions in Central and South America, Africa, parts of Asia, and Australia, which has the most endemic species. Acacia is mainly born in dry and arid areas where forests are often empty and spiny shrubs. The genus Acacia is divided into three subgenuses based on leaf morphology-Acacia, Aculiferum and Heterophyllum. However, based on the characteristics of mature tree leaves, Acacia can be divided into two "common" groups-typical bipinnate leaf varieties and petiole varieties. The petiole is an improved petiole expanded into a leaf-like structure without leaflets, which is an adaptation to the conditions of xerophytes.

Acacia has great economic significance. It provides raw materials for tannin, gum, wood, fuel and feed. Tannin is mainly isolated from the bark and is widely used in tanning leather and shredded leather. Some acacia bark is also used for flavoring local glutinous agents. Some native species such as Acacia vine also produce saponins, which are arbitrary plant polysaccharides, which when mixed with water and stirred to form soap-like foam. Saponins are used in detergents, foaming agents and emulsifiers. The fragrance of flowers of some species of the genus Acacia is therefore used in the production of perfumes. For example, Acacia perfume is obtained from A. ferrugenea. The heartwood of many acacias is used to make agricultural tools and is a source of firewood. Acacia gum is widely used in medicines and desserts, and as a styling and finishing material in the textile industry. Lac insects can grow on several species, including acacia acacia and catechu. Some species are used in wasteland afforestation, including acacia acacia, which can withstand flooding, and some areas of the same species have become bird refuges.

So far, about 330 compounds have been isolated from various Acacia species. Flavonoids are a class of water-soluble plant pigments, which are the main types of compounds isolated from Acacia. About 180 different flavonoids have been identified, 110 of which are flavans. Terpenoids are the second largest group of compounds isolated from Acacia species, and 48 compounds have been identified. Other types of compounds isolated from Acacia include alkaloids (28), amino acids/peptides (20), tannins (16), sugars (15), oxygen-containing heterocycles (15), and aliphatic compounds (10) (Buckingham, The Combined Chemical Dictionary, Chapman & Hall CRC, 5:2 edition, Dec. 2001).

All Acacia species have moderate to high concentrations of phenolic compounds, especially flavans (Abdulrazak et al. (2000) Journal Of Animal Sciences. 13:935-940). Historically, most plants and extracts of the Acacia genus have been used as astringents to treat gastrointestinal disorders, diarrhea, indigestion, and hemostasis (Vautrin (1996) Universite Bourgogne (France) European abstract 58-01C: 177; Saleem (1998) Hamdard Midicus. 41: 63-67). The bark and pods of A.Arabica Willd. contain a lot of tannins, so they are used as an astringent and expectorant (Nadkarni (1996) India Materia Medica, Bombay PopularPrakashan, pp. 9-17). It is reported that the diarylpropanol derivative isolated from the bark of A. tortilis from Somalia has a smooth muscle relaxation effect (Hagos et al. (1987) PlantaMedica. 53:27-31, 1987). It has also been reported that terpene glycosides isolated from Acacia victoriae (Hanausek et al. (2000) Proceedings American Association for Cancer Research Annual Meeting 41: 663) and induce cell apoptosis. (Haridas et al. (2000) 3 Proceedings American Association for Cancer Research Annual Meeting. 41:600). It is reported that the plant extract of Acacia acacia has spasmogenic, vasoconstrictor and antihypertensive effects (Amos et al. (1999) Phytotherapy Research 13: 683-685; Gilani et al. (1999) Phytotherapy Research 13:665-669), and anti-platelet aggregation (Shah et al. (1997) General Pharmacology. 29:251-255). Reports indicate that Acacia acacia has anti-inflammatory activity. It is speculated that flavonoids, polysaccharides and organic acids are possible active ingredients (Dafallah and Al-Mustafa (1996) American Journal of Chinese Medicine. 24:263-269). Submitted on March 22, 2002, serial number 10/104,477, titled "Isolation of a Dual Cox-2 and 5-Lipoxygenase Inhibitor from Acacia" (Isolation of dual Cox-2 and 5-lipoxygenase inhibitors from Acacia The U.S. application of the agent) is incorporated herein by reference in its entirety, which discloses dual inhibition of cyclooxygenase COX-2 and 5-fatty acid by administering a composition containing flavans or mixtures of flavans isolated from plants of the genus Acacia. Oxygenase (5-LO) method.

Summary of the invention

The present invention includes methods that can effectively inhibit sugar-induced weight gain and sugar-induced obesity. The method of inhibiting sugar-induced weight gain and inhibiting sugar-induced obesity includes administering to a subject in need a composition containing synthetic and/or unsubstituted B-ring flavonoids and flavan mixtures isolated from one plant or more plants (The trade name DIAFINTM is also used herein to refer to this composition). The ratio of unsubstituted B-ring flavonoids to flavans can range from 99.9:0.1 unsubstituted B-ring flavonoids to flavans to 0.1:99.9 unsubstituted B-ring flavonoids to flavans. In a preferred embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans in the composition is 80:20. In a preferred embodiment, the unsubstituted B-ring flavonoids are isolated from one or more Scutellaria plants, and the flavans are isolated from one or more Acacia plants.

The present invention also includes methods for preventing and treating other sugar-induced diseases and disorders. Methods of preventing and treating sugar-induced diseases and disorders include administering to a subject in need an effective dose of a composition comprising synthetic and/or unsubstituted B-ring flavonoids and flavans isolated from one or more plants And a pharmaceutically acceptable carrier. The ratio of unsubstituted B-ring flavonoids to flavans can range from 99.9:0.1 unsubstituted B-ring flavonoids to flavans to 0.1:99.9 unsubstituted B-ring flavonoids to flavans. In a preferred embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans in the composition is 80:20. In a preferred embodiment, the unsubstituted B-ring flavonoids are isolated from one or more Scutellaria plants, and the flavans are isolated from one or more Acacia plants.

The present invention also includes specific inhibition of key enzymes in fructose catalysis and glycolysis pathways, that is, methods for inhibiting specific fructose-1-phosphate aldolase. Referring to Figure 1, this inhibits the conversion of fructose-1-phosphate into glyceraldehyde and dihydroxyacetone phosphate by aldolase. This specific inhibition prevents the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate, which in turn is acylglycerol, and ultimately leads to the production of VLDL.

The present invention also includes a method for genomic reduction of the transcription factor NFκB that induces TNFα and IL-6 production. TNFα and IL-6 are the main markers of obesity and other inflammatory diseases. The method for reducing NFκB and thereby reducing TNFα and IL-6 includes administering to a subject in need an effective amount of a composition comprising synthetic and/or unsubstituted B-ring flavonoids isolated from one or more plants And a mixture of flavans. The ratio of unsubstituted B-ring flavonoids to flavans can be in the range of unsubstituted flavonoids: flavans 99.9:0.1 to unsubstituted B-ring flavonoids: flavans 0.1:99.9. In a preferred embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids: flavans in the composition is 80:20. In a preferred embodiment, the unsubstituted B-ring flavonoid is isolated from one or more plants of the genus Scutellaria, and the flavan is isolated from one or more plants of the genus Acacia.

The unsubstituted B-ring flavonoids that can be used according to the present invention-also referred to herein as unsubstituted B-ring flavonoids and unsubstituted B-ring flavonols-include compounds represented by the following general structural formulas:Wherein R1, R2, R3, R4 and R5 are independently selected from the following groups: -H, -OH, -SH, -OR, -SR, -NH2, -NHR, -NR2, -NR3+X-, carbon, Oxygen, nitrogen or sulfur, single or multiple sugar combined glycosides, including but not limited to aldose, aldose methyl, aldose, ketohexose and their chemical derivatives; where R has 1 An alkyl group of -10 carbon atoms; and X is selected from the group of pharmaceutically acceptable counter anions, including but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc. .

The flavans that can be used according to the following invention include compounds represented by the following general structural formulas:Wherein R1, R2, R3, R4 and R5 are independently selected from the following groups: -H, -OH, -SH, -OCH3, -SCH3, -OR, -SR, -NH2, -NHR, -NR2, -NR3 +X-, the esters of the substituents include but are not limited to gallic acid esters, acetates, cinnamoyl and hydroxycinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; its carbon, oxygen, nitrogen or sulfur, Single or multiple sugar combined glycosides, including but not limited to aldose, aldose methyl, aldose, ketulose and their chemical derivatives; dimerization, trimerization and other polyflavans ; Wherein R is an alkyl group having 1-10 carbon atoms; and X is selected from the group of pharmaceutically acceptable counter anions, including but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, Fluoride, carbonate, etc.

The method of the present invention can be used to treat and prevent many diseases and disorders related to obesity, including but not limited to sugar-induced obesity and diabetes, and hyperlipidemia, high cholesterol, arteriosclerosis, and atherosclerosis caused by hypertension. , X chromosome syndrome (metabolic syndrome) and systemic inflammation.

The unsubstituted B-ring flavonoids of the present invention can be synthesized by synthetic methods or from the family, including but not limited to Annonaceae, Asteraceas, Bignoniaceae, Gentlemenaceae, Compositae, Euphorbiaceae, Labiatae , Lauraceae, Leguminosae, Moraceae, Pinaceae, Pteriaceae, Chinese Pteridaceae, Ulmus and Pteridaceae. The unsubstituted B-ring flavonoids can be extracted, concentrated and purified from higher plant genus, including but not limited to Achyrocline, Achyrocline, Ophiopogon, Buchenavia, Coriander, Coriander (Cotula), Salvia, Helichrysum, Centaurea, Eupatorium, Baccharis, Ubera, Scutellaria, Molsa, Pseudocalyx, Stachys, Origanum, Newtonia, Lamia, Phyllanthus , Acacia, Rhododendron, Glycyrrhiza, Spatholobus, Pseudomonas, Gray Edamame, Woodbolt, Ficus, Pteridium, Cryptocystis, Pinus, Ulmus and Alpinia.

The flavans of the present invention can be obtained from one or more plants selected from the genus Acacia. In a preferred embodiment, the plant is selected from the following group: catechu, A. concmna, acacia, acacia, A. speciosa, arabia acacia, A. caesia, snake vine, vine acacia, black Wattle tree, A. picnantha, white powder acacia, large leaf Acacia, A. holoserecia and Ma Zhan Acacia.

The composition of the present invention can be administered by any method known to those of ordinary skill in the art. Modes of administration include, but are not limited to, enteral (oral) administration, parenteral administration (intravenous, subcutaneous and intramuscular) administration and topical administration. The treatment method of the present invention includes oral administration or external application to a subject in need of a therapeutically effective amount of synthetic and/or a mixture of unsubstituted B-ring flavonoids and flavans isolated from one or more plants.

It should be understood that the foregoing general description and the following detailed description are only for the purpose of illustration and explanation, and are not intended to limit the scope of the present invention as claimed.

Description of the drawings

Figure 1 shows a schematic diagram of the metabolism of fructose and glucose in the glycolysis and lipogenesis pathways in the liver.

Figure 2 illustrates the daily administration of a mixture of unsubstituted B-ring flavonoids and flavans to ICR female mice fed a normal diet, a diet supplemented with 65% fructose, or a diet supplemented with fat as described in Example 1 for 3 weeks. The role of fructose-induced weight gain and fat-induced weight gain. Mice on a normal diet served as controls. Both test groups were given either only 65% fructose or only fat. This figure shows that unsubstituted B-ring flavonoids and flavans can prevent excessive weight gain caused by fructose consumption.

Figure 3 illustrates the daily administration of unsubstituted B-ring to ICR mice fed a normal diet (control), a diet supplemented with 65% fructose and a diet supplemented with 65% glucose for 8 weeks as described in Example 3 daily The effect of a mixture of flavonoids and flavans on the weight gain induced by fructose versus glucose. Weigh every week and plot the average weight of each group. The mean standard deviation (SEM) of each group is calculated weekly.

Figure 4 shows the mixture of unsubstituted B-ring flavonoids and flavans (80:20) after exposure to lipopolysaccharide and different concentrations of unsubstituted B-ring flavonoids and flavan mixtures for 1 hour. Peripheral blood mononuclear cells (PBMC) The effect of lipopolysaccharide (LPS)-induced TNFα levels. The level of TNFα is expressed in pg/ml. Calculate the standard deviation of each data point.

Figure 5 shows the mixture of unsubstituted B-ring flavonoids and flavan (80:20) after exposure to lipopolysaccharide and different concentrations of unsubstituted B-ring flavonoids and flavan mixtures for 6 hours. The effect of lipopolysaccharide (LPS)-induced IL-6 levels in PBMC). IL-6 levels are expressed in pg/ml. Calculate the standard deviation of each data point.

Figure 6 shows the relative inhibition of NFκB gene expression by a mixture of unsubstituted B-ring flavonoids and flavans. Relative gene expression is measured in the presence of 0 to 100 μg/mL extract.

Figure 7 shows the relative inhibition of TNFα gene expression by a mixture of unsubstituted B-ring flavonoids and flavans. Relative gene expression is measured in the presence of 0 to 100 μg/mL extract.

Figure 8 shows the effect of unsubstituted B-ring flavonoids and flavans on the specific aldolase function of fructose-1-phosphate, which can catalyze the conversion of fructose-1-phosphate into glyceraldehyde and dihydroxyacetone phosphate. It showed a dose-specific inhibitory effect on this key enzyme within 10 minutes.

Figure 9 shows the effects of unsubstituted B-ring flavonoids and flavan extracts on weight loss in 13 individuals who took orally 250 mg daily for 90 days. Calculate the individual weight of each subject (beginning and last) at each measurement.

Figure 10 shows the effects of unsubstituted B-ring flavonoids and flavan extracts on weight loss of 13 individuals who took 500 mg orally daily for 90 days. Calculate the individual weight of each subject (beginning and last) at each measurement.

Figure 11 shows the effect of oral administration of placebo to 13 individuals on weight loss for 90 days. Calculate the individual weight of each subject (beginning and last) at each measurement.

Figure 12 shows the effects of unsubstituted B-ring flavonoids and flavan extracts on the daily oral administration of 250 and 500 mg relative to the body mass index (BMI) of individuals administered placebo on days 30 and 90. Calculate the SEM value of each group as well.

Figure 13 shows the effects of unsubstituted B-ring flavonoids and flavan extracts on daily oral administration of 250 and 500 mg versus placebo (baseline) on the changes in blood glucose of individuals on days 0, 30, and 90.

14 is a high performance liquid chromatography (HPLC) chromatogram of a mixture of unsubstituted B-ring flavonoids and flavans performed under the conditions described in Example 8. Under the conditions described, the unsubstituted B-ring flavonoids elute between 11 to 14 minutes, and the flavans elute between 3 to 5 minutes.

15 shows an HPLC chromatogram of an unsubstituted B-ring flavonoid and flavan mixture performed under the conditions described in Example 9. Under the conditions described, the two flavanoids (catechin and epicatechin) elute between 4.5 and 5.5 minutes, and the unsubstituted B-ring flavonoids (begain and begaline) elute between 12 and 13.5 minutes. Eluted between. Under the conditions described in Example 9, the separation is based on the difference in the molar absorption capacity of unsubstituted B-ring flavonoids and flavans.

detailed description

The present invention relates to a novel composition containing two special types of compounds-unsubstituted B-ring flavonoids and flavans-mixed mixtures, which are used for the prevention and treatment of weight gain and obesity and other causes caused by high sugar absorption Diseases and illnesses. The diseases and conditions include but are not limited to hyperlipidemia, high cholesterol, arteriosclerosis, atherosclerosis, X syndrome (metabolic syndrome) and systemic inflammation caused by obesity, diabetes and hypertension.

The various terms used herein relate to various aspects of the present invention. The following definitions are provided to help clarify the various components of the present invention.

It should be noted that the term "a" ("a" or "an") entity refers to one or more of that entity; for example, a flavonoid refers to one or more flavonoids. Likewise, the terms "a", "one or more" and "at least one" are interchangeable herein.

The "unsubstituted B-ring flavonoids" used here are a special kind of flavonoids, as shown in the following general structural formula, the aromatic B-ring has no substituents:Wherein R1, R2, R3, R4 and R5 are independently selected from the following groups: -H, -OH, -SH, -OR, -SR, -NH2, -NHR, -NR2, -NR3+X-, carbon, Oxygen, nitrogen or sulfur, single or multiple sugar combined glycosides, including but not limited to aldose, aldose methyl, aldose, ketohexose and their chemical derivatives; where R has 1 -10 carbon atoms; and X is selected from the group of pharmaceutically acceptable counter anions, which include but are not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, carbonate Wait.

"Flavan" is a special class of flavonoids, which can usually be represented by the following general structural formula:Wherein R1, R2, R3, R4 and R5 are independently selected from the following groups: -H, -OH, -SH, -OCH3, -SCH3, -OR, -SR, -NH2, -NRH, -NR2, -NR3 +X-, esters of substituents, including but not limited to gallic acid esters, acetates, cinnamoyl and hydroxycinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters; carbon, oxygen, nitrogen or sulfur, single or Glycosides combined with multiple sugars, including but not limited to aldose, aldose methyl, aldose, ketulose and their chemical derivatives; dimerization, trimerization, and other polyflavans; wherein R is an alkyl group having 1-10 carbon atoms; and X is selected from the group of pharmaceutically acceptable counter anions, including but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, and fluoride. , Carbonate and so on.

As used herein, "fructose-induced lipogenesis" refers to the specific pathway of lipid formation from fructose, as shown in Figure 1, which occurs through the production of the intermediate dihydroxyacetone phosphate, which is then converted into glycerol The -3-phosphate is then esterified to acylglycerol.

As used herein, "fat regeneration" refers to the specific way of forming lipids from fructose, which occurs through the production of the intermediate pyruvate, as shown in Figure 1, which is then converted into acetyl coenzyme in the carboxylation reaction A, Acetyl-CoA is converted into malonyl-CoA, and malonyl-CoA is then converted into acyl-CoA which is then esterified to acylglycerol.

As used herein, "sugar-induced lipogenesis" refers to both fructose-induced and secondary lipogenesis.

"Sugar" as used herein refers to both simple and carbohydrate, which includes but is not limited to monosaccharides, disaccharides and polysaccharides. Monosaccharides are simple sugars, which include, but are not limited to, glucose, fructose, and galactose. Disaccharides are disaccharides or sugars containing two molecules of simple sugars, such as sucrose. Sugar as used herein also refers to sugars derived from the decomposition or degradation of complex sugars. Complex sugars or polysaccharides are sugars containing three or more molecules of simple sugars. Complex sugars include starch and glycogen. The sugars produced by the degradation of complex sugars include, but are not limited to, maltotriose, α-dextrin, maltose and other disaccharides with metabolic activity.

"Treatment" as used herein includes treatment and/or prevention. When used, treatment refers to humans as well as other animals.

"Pharmaceutically or therapeutically effective dose or amount" refers to a dose level sufficient to induce the desired biological result. The result can be the alleviation of the symptoms, symptoms, or causes of the disease or any other desired changes in the biological system.

"Placebo" refers to the substitution of an inactive substance for a pharmacologically or therapeutically effective dose or amount that is sufficient to induce the desired biological result that can alleviate the symptoms, symptoms, or the cause of the disease.

The "subject" is the subject to which the composition of the present invention is administered, and is the survival target of a human or an animal.

Note that various citations are provided throughout this application. Each citation is fully incorporated herein as a reference.

The present invention includes methods for effectively inhibiting sugar-induced weight gain and sugar-induced obesity. The method of inhibiting sugar-induced weight gain and inhibiting sugar-induced obesity includes administering to a subject in need a mixture containing synthetic and/or unsubstituted B-ring flavonoids and flavans extracted from one or more plants. The ratio of unsubstituted B-ring flavonoids to flavans can range from 99.9:0.1 unsubstituted B-ring flavonoids to flavans to 0.1:99.9 unsubstituted B-ring flavonoids to flavans. In a specific embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans is selected from the following group: about 90:10, 80:20, 70:30, 60:40, 50:50, 40:60 , 30:70, 20:80 and 10:90. In a preferred embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans in the composition is 80:20. In a preferred embodiment, the unsubstituted B-ring flavonoids are isolated from one or more Scutellaria plants, and the flavans are isolated from one or more Acacia plants.

The present invention also includes methods of preventing and treating sugar-induced diseases and disorders. Methods of preventing and treating sugar-induced diseases and disorders include administering to a subject in need an effective amount of a mixture and a drug containing synthetic and/or unsubstituted B-ring flavonoids and flavanoids extracted from one or more plants The composition of a scientifically acceptable carrier. The ratio of unsubstituted B-ring flavonoids to flavans can range from 99.9:0.1 unsubstituted B-ring flavonoids to flavans to 0.1:99.9 unsubstituted B-ring flavonoids to flavans. In a specific embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans is selected from the following group: about 90:10, 80:20, 70:30, 60:40, 50:50, 40:60 , 30:70, 20:80 and 10:90. In a preferred embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans in the composition is about 80:20. In a preferred embodiment, the unsubstituted B-ring flavonoids are isolated from one or more Scutellaria plants, and the flavans are isolated from one or more Acacia plants.

The present invention also includes a method for genomically reducing NFκB and then reducing the levels of TNFα and IL-6, which are the main markers of obesity and other inflammatory diseases. The method for genomically reducing NFκB and subsequently reducing TNFα and IL-6 levels includes administering to a subject in need an effective amount of synthetic and/or unsubstituted B-ring flavonoids and flavanoids extracted from one or more plants The composition of the mixture. The ratio of unsubstituted B-ring flavonoids to flavans can range from 99.9:0.1 unsubstituted B-ring flavonoids to flavans to 0.1:99.9 unsubstituted B-ring flavonoids to flavans. In a specific embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans is selected from the following group: about 90:10, 80:20, 70:30, 60:40, 50:50, 40:60 , 30:70, 20:80 and 10:90. In a preferred embodiment of the present invention, the ratio of unsubstituted B-ring flavonoids to flavans in the composition is about 80:20. In a preferred embodiment, the unsubstituted B-ring flavonoids are isolated from one or more Scutellaria plants, and the flavans are isolated from one or more Acacia plants.

The unsubstituted B-ring flavonoids that can be used according to the present invention include compounds represented by the above general structural formula. The unsubstituted B-ring flavonoids of the present invention can be obtained by synthetic methods or extracted from the following plants, including but not limited to Annonaceae, Asteraceae, Bignoniaceae, Gentlemaniaceae, Compositae , Euphorbiaceae, Lamiaceae, Lauraceae (Lauranceae), Leguminosae, Moraceae, Pinaceae, Pteridophyteaceae, Chinese Pteridaceae, Ulmaceae and Zingiberaceae. The unsubstituted B-ring flavonoids can be extracted, concentrated and purified from higher plant genera, including but not limited to Achyrocline, Achyrocline, Ophiopogon, Buchenavia, Coriander, Coriander, Miria, Helichrysum , Knapweed, Eupatorium, Baccharis, Umpressia, Scutellaria, Molsa, Limonium, Stachys, Origanum, Newtonia, Mountain pepper, Phalanthus, Acacia Genus, Rhododendron, Glycyrrhiza, Spatholobus, Pseudomonas, Grey Edamame, Woodbolus, Ficus, Pteris, Cryptocystis, Pinus, Ulmus, and Alpinia .

The flavonoids are present in different parts of plants, including but not limited to stems, bark, twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowers and other reproductive organs, leaves and other aerial parts . No. 10/091,362 named "Identification of Free-B-ring Flavonoids as Potent COX-2 Inhibitors" submitted on March 1, 2002 The U.S. application describes a method for the separation and purification of unsubstituted B-ring flavonoids. The full text is hereby incorporated as a reference.

The flavans that can be applied according to the method of the present invention include the compounds represented by the above-mentioned general structural formulas. The flavans of the present invention can be obtained synthetically or extracted from one or more plants selected from plants of the genus Acacia. In a preferred embodiment the plant is selected from the group consisting of: catechu, A. concinna, acacia, acacia, A. speciosa, arabia acacia, A. caesia, snake vine, vine acacia, black wattle Tree, A. picnantha, White powder acacia, Acacia macrophylla, A. holoserecia, and Acacia ma Zhan.

The flavans are present in different parts of plants, including but not limited to stems, bark, stem, trunk bark, shoots, tubers, roots, root bark, shoots, seeds, rhizomes, flowers and other reproductive organs, Leaves and other aerial parts. No. 10/104,477 entitled "Isolation of aDual COX-2 and 5-Lipoxygenase Inhibitor form Acacia" submitted on March 22, 2002 The US application documents methods for separating and purifying flavans. The full text is hereby incorporated as a reference.

The method of the present invention can be used to treat and prevent many obesity-related diseases and disorders, including but not limited to hyperlipidemia, high cholesterol, arteriosclerosis, atherosclerosis, X chromosome synthesis caused by obesity, diabetes and hypertension. Signs (metabolic syndrome) and systemic inflammation.

The composition of the present invention can be administered by any method known to those of ordinary skill in the art. Modes of administration include, but are not limited to, enteral (oral) administration, parenteral administration (intravenous, subcutaneous and intramuscular) administration and topical administration. The treatment method of the present invention includes oral or external administration to a subject in need of a therapeutically effective amount of synthetic and/or a mixture of unsubstituted B-ring flavonoids and flavans isolated from one or more plants. In a preferred embodiment, the composition is administered at a dose selected from 0.01 to 200 mg/kg body weight.

The present invention adopts a series of in-vivo weight determination and in vitro biochemical, cell and gene expression screening combined strategies to identify the effect that is usually associated with the increase in lipid and fat content in the body, the enzymatic activity of metabolic enzymes, and the expression of mRNA genes. Active plant extracts that are involved in lipogenesis and can specifically inhibit fructose metabolism. The ability of unsubstituted B-ring flavonoids and flavans to inhibit fructose-induced obesity when administered by oral gavage was measured.

Example 1 describes a test designed to determine the effect of a mixture of unsubstituted B-ring flavonoids and flavans on weight gain caused by a 3-week diet supplemented with fructose or fat. The result is shown in Figure 2. With reference to Figure 2, it can be seen that the animals given fat and extract gained about the same amount of weight as those given only fat without extract. However, the mice given fructose and extract gained the same weight as the control group fed a normal diet. This result shows that a composition containing a mixture of unsubstituted B-ring flavonoids (60-90% by HPLC) and flavans (10-60% by HPLC) can effectively prevent excessive weight gain caused by fructose consumption. And without being limited by theory, it is believed that this result is due to changes in the utilization of fructose in the adipogenic pathway.

Example 2 describes a test designed to illustrate the effect of unsubstituted B-ring flavonoids and flavan mixtures on weight gain caused by an 8-week diet supplemented with fructose and glucose. The result is shown in Figure 3. With reference to Fig. 3, it can be seen that the rats given glucose and the extract gained about the same amount of weight as those given only glucose without the extract. However, the weight gain of rats given fructose and the extract was significantly lower than that of rats given only fructose without the extract. Rats given sucrose or commercial fructose syrup showed moderate weight gain under the conditions of this test (data not shown).

Example 3 illustrates the effect of a mixture of unsubstituted B-ring flavonoids and flavans on the secretion concentration of TNFα. The result is shown in Figure 4. Referring to Figure 4, it can be seen that the extract can significantly reduce the TNFα secreted into the cell culture supernatant in a wide range of concentrations from 2 to 100 μg/mL. Because TNFα is a marker of obesity, the extract has a significant effect because it can reduce the pro-inflammatory cytokine in primed inflammatory cells.

Example 4 illustrates the effect of a mixture of unsubstituted B-ring flavonoids and flavans on the secretion concentration of IL-6. The result is shown in Figure 5. Referring to Figure 5, it can be seen that the extract can significantly reduce the IL-6 secreted into the cell culture supernatant in a wide range of concentrations from 2 to 100 μg/mL. Because IL-6 is a marker of obesity, the extract has a significant effect on inflammatory cells that cause inflammation because it can reduce the pro-inflammatory cytokine.

Destruction of the transcription factor NFκB can lead to the decrease of TNFα and IL-6, because the promoters of both genes are activated by NFκB. In order to test this hypothesis, the effect of the unsubstituted B-ring and flavan mixture on the gene expression transcription factor NFκB was evaluated as described in Example 5. The results are shown in Figure 6, which illustrates the relative NFκB gene expression as a function of extract concentration. As can be seen in Figure 6, the expression of NFκB was down-regulated by 2.7 times when the extract concentration was the highest. A small change in NFκB can down-regulate the gene expression of other genes to a high degree.

Figure 7 illustrates the effect of a mixture of unsubstituted B-ring flavonoids and flavans on TNFα gene expression. As shown in the figure, the relative gene expression of TNFα is almost 10-fold down-regulated under the conditions described in Example 5. This result and the reduction of cytokines found by protein analysis all suggest that NFκB can be inhibited by the unsubstituted B-ring flavonoid/flavan extract.

In the metabolic pathways of fructose and glucose, there are two key enzymes that are affected by unsubstituted B-ring flavonoids/flavan extracts: aldolase A and aldolase B. Referring to Figure 1, it can be seen that aldolase B catalyzes the conversion of fructose-1-phosphate into glyceraldehyde and dihydroxyacetone phosphate, and aldolase A catalyzes the conversion of fructose-1,6-diphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone. Phosphoric acid. Triose isomerase catalyzes the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate, while triose kinase consumes a molecule of ATP to catalyze the conversion of glyceraldehyde into glyceraldehyde-3-phosphate, thereby connecting fructose catalysis and glycolysis pathways. Aldolase B is not commercially available, so as described in Example 6, the application of aldolase A assay extracts that can also catalyze the conversion of fructose-1,6-diphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate Potential effects on these enzymes in vitro. The results are shown in Figure 8, which is a graph of the absorbance of the reaction mixture versus time at various inhibitor concentrations. The graph illustrates that product formation decreases as the extract concentration increases. This result suggests that the extract can directly inhibit aldolase in the conversion of fructose-1-phosphate to glyceraldehyde and dihydroxyacetone phosphate. Therefore, while not being limited by theory, it is believed that the inhibition of aldolase in the fructose catalytic pathway is conducive to the mechanism that can reduce the weight gain of mice whose diet is supplemented with fructose and extracts.

Example 7 describes a human clinical trial to test the effectiveness of the extract for weight loss in humans. The result is shown in Figure 9-13. Figure 9 depicts the weight loss effects of unsubstituted B-ring flavonoids and flavan extracts on 13 individuals who were orally administered 250 mg daily for 90 days. Calculate the individual weight of each subject (beginning and last) at each measurement. With reference to Fig. 9, it can be seen that 13 of the 14 individuals remaining in the test after 90 days were observed to lose weight significantly at a daily dose of 250 mg. Only one subject (Subject No. 4) did not lose weight. The deviation of the weight data on day 0 and day 90 was statistically significant, p<0.001.

Figure 10 illustrates the weight loss effects of unsubstituted B-ring flavonoids and flavan extracts on 13 individuals who were orally administered 500 mg daily for 90 days. Calculate the individual weight of each subject (beginning and last) at each measurement. As shown in Figure 10, when the dose was increased to 500 mg daily, the weight loss showed different characteristics. Subjects with high BMI tend to lose more weight than those with low BMI. In some cases, stratification occurs, and subjects with lower body weight may not lose weight or even gain weight (see subjects 3, 15, 43, and 47). Thirteen subjects completed the study. The statistical significance of this data is p<0.011.

Figure 11 depicts the weight loss effect of oral administration of placebo to 13 individuals for 90 days. Calculate the individual weight of each subject (beginning and last) at each measurement. As can be seen in this figure, the weight changes of the 13 subjects who completed the trial in the placebo group were very small.

Figure 12 illustrates the effects of unsubstituted B-ring flavonoids and flavan extracts on the BMI of individuals who were orally administered 250 mg and 500 mg daily relative to placebo on the 30th and 90th days. As can be seen in Figure 12, when the average BMI values of all 3 groups are plotted, a significant difference can be observed. The daily 250 mg dose vs. placebo had p <0.075, while the daily 500 mg dose had p <0.005. Analysis between groups showed p<0.004 for a daily dose of 250 mg and p<0.051 for a daily dose of 500 mg.

Figure 13 illustrates the effect of unsubstituted B-ring flavonoids and flavan extracts relative to placebo on the blood glucose changes of individuals who were orally administered 250 and 500 mg daily on days 0, 30, and 90. Therefore, in addition to weight loss and favorable changes in BMI, the blood glucose levels of the 250 mg and 500 mg dose groups decreased, and the latter group changed the most. However, the blood glucose levels of those who received the placebo were relatively unchanged during the trial. Fasting serum glucose data showed significant differences from baseline data, with p<0.018 for a daily dose of 500 mg and p<0.014 for a daily dose of 250 mg. Fasting serum glucose data showed significant differences from baseline data, with p<0.018 for a daily dose of 500 mg and p<0.014 for a daily dose of 250 mg.

As described in Examples 8 and 9, two different methods were used to identify the purity and quality of unsubstituted B-ring flavonoids and flavan mixtures by HPLC analysis. The results are shown in Figures 14 and 15. With reference to Figure 14, it can be seen that after HPLC analysis, the elution area of unsubstituted B-ring flavonoids (mainly begain and begain) is >60% of the total elution area. The method described in Example 9 was applied, because flavan has a lower total adsorption capacity, about 3 times as much extract was loaded on the HPLC chromatographic column and eluted under isocratic conditions. Referring to Figure 15, after HPLC analysis, the elution area of flavans (catechin and epicatechin) was measured to be> 10% of the total elution area.

Use mice to test chronic and acute administrations containing high concentrations of unsubstituted B-ring flavonoids (60%-90% determined by HPLC) and flavans (10%-60% determined by HPLC) individually standardized extracts and containing unsubstituted Toxicity of the combined extract of B-ring flavonoid and flavan mixture. In the chronic dosing treatment regimen, mice were given a daily dose of 90 mg/kg (equivalent to 500 mg of human daily dose), 450 mg/kg (equivalent to 5 times the daily dose), and 900 mg/kg (equivalent to each 10 times the daily dose) feed the test product. In terms of weight gain, appearance characteristics and behavior, the treated mice showed no side effects. The overall autopsy results showed no organ abnormalities, and the histology of the stomach and liver showed no difference compared with untreated control mice. The full blood work of measuring electrolytes, blood proteins, blood enzymes and liver enzymes did not show any abnormality compared with untreated rats.

In the acute dosing test protocol, the administration of 2 g/kg (equivalent to 20 times the daily dose) contains high concentrations of unsubstituted B-ring flavonoids (HPLC determination 60%-90%) and flavans (HPLC determination 10%- 60%) individually standardized extracts and extracts containing a combination of unsubstituted B-ring flavonoids and flavan mixtures. The weight gain, appearance, behavior, general autopsy organ appearance, stomach and liver histology or blood examination of the treated rats showed no abnormalities.

Based on the ability of unsubstituted B-ring flavonoids and flavans to directly inhibit sugar-induced obesity, and their genomically reduced NFκB, the key transcription factor of pro-inflammatory cytokine markers TNFα and IL-6 that regulate obesity- -Active, the composition described herein can effectively inhibit weight gain, glyco-lipogenesis and systemic inflammation. In addition, the ability of the unsubstituted B-ring flavonoids and flavans to directly inhibit the aldolase that converts fructose-1-phosphate into glyceraldehyde and dihydroxyacetone phosphate will result in re-supply and use of fructose-induced lipogenesis for the synthesis of fat The number of substrates is reduced.

The following examples are for illustrative purposes and are not intended to limit the scope of the present invention.

EXAMPLES Example 1 The effect of a mixture of unsubstituted B-ring flavonoids and flavans on the weight gain caused by a three-week diet supplemented with fructose or fat. Application of Scutellaria baicalensis Georgi with a ratio of 80:20 (unsubstituted flavonoids: flavans) The isolated unsubstituted B-ring flavonoids and the definitive plant extracts of flavans isolated from catechu have studied the effect of a combination of unsubstituted B-ring flavonoids and flavans on weight gain. The composition was submitted on April 30, 2003, the serial number is 10/427,746, and the name is "Formulation With DualCox-2 And 5-Lipoxygenase Inhibitory Activity," (with dual Cox-2 and 5-lipoxygenase inhibition Active formulations) are formulated as described in the U.S. application, which is incorporated herein by reference in its entirety.

Six-week-old ICR female mice (10 per test group) (Harlan Laboratories) were gavaged with the extract in water at a therapeutic dose of 100 mg/kg. A group of mice were given a normal diet and given a 65% fructose solution as their drinking water and drank ad libitum. The second group of mice were given a diet supplemented with fat and ate ad libitum. The two test groups were given 65% fructose in their water (without extract) or their dietary supplements with fat (without extract). The result is shown in Figure 2.

Example 2 The effect of a mixture of unsubstituted B-ring flavonoids and flavans on the weight gain caused by a diet supplemented with fructose or glucose for eight weeks. The subjects six-week-old ICR female mice (10 per test group) (Harlan Laboratories) and as submitted on April 30, 2003, serial number 10/427,746, named "FormulationWith Dual Cox-2 And 5- Lipoxygenase Inhibitory Activity, "(a preparation with dual Cox-2 and 5-lipoxygenase inhibitory activity) described in the U.S. application of an 80:20 mixture of unsubstituted B-ring flavonoids: flavans, the application has been hereby The full text is incorporated as a reference. The result is shown in Figure 3.

With reference to Figure 3, it can be seen that the "control" group was fed a balanced diet as suggested by the vendor. The second "control" group was also given 100mg/kg of extract via gavage. The two experimental groups received the control diet plus 65% fructose added randomly to the water. One of these groups received 100 mg/kg of the extract daily by gavage. The last two groups of test groups received a control diet plus 65% glucose randomly added to the water. One of these groups received 100 mg/kg of the extract daily by gavage. After eight weeks, plot the average weight and the average standard deviation is shown on the graph.

Example 3 Effect of the mixture of unsubstituted B-ring flavonoids and flavans on the concentration of TNFα Histopaque gradient (Sigma) was used to isolate peripheral blood mononuclear cells (PBMC) from human blood donors. Then, in the presence of various concentrations of 80:20 unsubstituted B-ring flavonoids: flavan extracts, the cells were treated with 10 mg/mL lipopolysaccharide (LPS) to supplement 1% before inducing inflammation during 1 hour of incubation. Incubate bovine serum albumin in RPMI 1640 for approximately 12 hours. The result is shown in Figure 4.

Example 4 Effect of the mixture of unsubstituted B-ring flavonoids and flavans on IL-6 concentration Histopaque gradient (Sigma) was used to separate peripheral blood mononuclear cells (PBMC) from human blood donors. The cells were then supplemented with 1% before being treated with 10 μg/mL lipopolysaccharide (LPS) to induce inflammation in the presence of various concentrations of 80:20 unsubstituted B-ring flavonoids: flavan extracts in the six-hour incubation. Incubate bovine serum albumin in RPMI 1640 for approximately 12 hours. The result is shown in Figure 5. The extract can significantly reduce the IL-6 secreted into the cell culture supernatant in a wide concentration range from 2 to 100 μg. Since IL-6 is a marker of obesity, the extract has a significant impact because it can reduce pro-inflammatory cytokines in inflammatory cells that cause inflammation.

Example 5 The effect of the mixture of unsubstituted B-ring flavonoids and flavans on the transcription factor NFκB gene expression and TNFα gene expression. 1mg/mL LPS was used to induce PBMC for 18 hours, and the unsubstituted B-ring flavonoids: flavans in increasing amounts were induced The extracts are co-cultured. The RNA was then isolated (Qiagen), reverse transcribed into DNA, and PCR was performed using pre-validated primers (pre-validated) for both NFκB and TNFα in the TaqMan system and quantitative PCR (ABI). Relative gene expression is measured in the presence of 0-100 μg/mL extract. The results are shown in Figures 6 and 7.

Example 6 The effect of a mixture of unsubstituted B-ring flavonoids and flavans on aldolase A At 25°C, 1 unit/μL of aldolase A (Sigma) was added to 4mM fructose-1-phosphate containing 100μM EDTA And 3.5 mM hydrazine sulfate and pH 7.5 (Jagannathan et al. (1956) Biochem. J. 63: 94-105) and the extract concentration range of 0 to 65 μg/mL in a buffer solution. The reaction was initiated by the addition of enzyme, and the reaction was monitored at 240 nm for 10 minutes. The results are shown in Figure 8, which is a plot of the absorbance of the reaction mixture against time at various inhibitor concentrations.

Example 7 The effect of unsubstituted B-ring flavonoids and flavanoids on human weight loss. A 90-day, IRB-reviewed, double-blind, placebo-controlled trial was conducted on 15 subjects in each group in three different groups : 1) Placebo; 2) 250 mg daily (125 mg twice daily); and 3) 500 mg daily (250 mg twice daily). The subjects are of the same age and sex. The subjects were orally administered a placebo or extract in the form of a concealed pill, and their body weight, BMI, and blood glucose level were measured on days 0, 30, 60, and 90. No advice was given to patients about whether they are receiving weight loss products or that they should change their eating or activity habits. The result is shown in Figure 9-13.

Example 8 Quantification of the mixture of unsubstituted B-ring flavonoids and flavans by reversed-phase high performance liquid chromatography (HPLC) (Method 1) The mixture of unsubstituted B-ring flavonoids and flavans (20mL 1.13mg/mL) Standardized extract) in 80%: 20% methanol: tetrahydrofuran was loaded onto a Phenomenex Luna C-18 column (250×4.6mm, 5mm particle size), at 35°C, at a flow rate of 1.0mL/min, linearly 80% Gradient elution from %A to 20%A (A=0.1% (v/v) phosphoric acid; B=acetonitrile) for 19 minutes. As can be seen in Figure 14, under these conditions, the unsubstituted B-ring flavonoids (begain and bekalin) eluted as the main peak between 11 and 14 minutes, while the flavans (catechin and epicatechin) ) As a minor peak eluted in about 3 to 5 minutes. Quantify unsubstituted B-ring flavonoids and flavans by measuring the area under each curve and comparing with known standards.

Example 9 Quantification of a mixture of unsubstituted B-ring flavonoids and flavans by reverse phase isocratic HPLC (Method 2) A mixture of unsubstituted B-ring flavonoids and flavans (20mL 3.55mg/mL standardized extract) The solution in 80%:20% methanol:water was loaded on a Phenomenex Luna C-18 chromatographic column (250×4.6mm, 5mm particle size), at a flow rate of 1.0mL/min, 80%A (A=0.1%) at 35°C (v/v) phosphoric acid; B=acetonitrile) eluted isocratically. As can be seen in Figure 15, under these conditions, the two flavanoids (catechin and epicatechin) eluted between 4.5 and 5.5 minutes, while the non-substituted B-ring flavonoids (begain and begain) eluted between 4.5 and 5.5 minutes. ) As the main peak eluted between 12 and 13.5 minutes. The quantification of the flavan peak was performed as described in Example 8.