阅读说明：本技术 一种靶向血管内皮细胞防治心脑血管疾病的罗布麻茶复配功能饮料 (Apocynum venetum tea compound functional beverage for preventing and treating cardiovascular and cerebrovascular diseases by targeting vascular endothelial cells ) 是由 贺小星 王文娟 于 2020-06-03 设计创作，主要内容包括：本发明公开了一种通过靶向血管内皮细胞来防治心脑血管疾病的罗布麻茶(以总黄酮终浓度3-100mg/L计算原液量)复配功能饮料,额外添加成分为：5-羟基色氨酸(4-200mg/L)、二氢硫辛酸(4-100mg/L)、泛酸(1-10mg/L)和烟酸(2-20mg/L)。配方存在良好协同作用,在罗布麻茶抑制NADPH氧化酶的情况下,补充烟酸不易产生活性氧簇(ROS)损伤,也防止了一氧化氮与ROS形成活性氮诱导硝化应激损伤血管内皮细胞,本发明的添加成分通过诱导内皮细胞高一氧化氮低ROS状态增强罗布麻茶生理效果并降低副作用。另外本配方开发上利用高通量的标准化细胞实验和随机双盲对照多中心志愿者实验,精确探明了配方成分的作用范围和毒性阈值,在使用剂量上有一定宽容度和极强安全性,适合作为产品开发的核心配方。(The invention discloses a apocynum venetum tea (the amount of a raw material is calculated by 3-100 mg/L) compound functional beverage for preventing and treating cardiovascular and cerebrovascular diseases by targeting vascular endothelial cells, which is additionally added with 5-hydroxytryptophan (4-200 mg/L), dihydrolipoic acid (4-100 mg/L), pantothenic acid (1-10 mg/L) and nicotinic acid (2-20 mg/L), wherein the formula has good synergistic effect, and under the condition that the apocynum venetum tea inhibits NADPH oxidase, the supplemented nicotinic acid is not easy to generate Reactive Oxygen Species (ROS) damage, and the phenomenon that nitric oxide and ROS form reactive nitrogen to induce nitration stress to damage vascular endothelial cells is also prevented.)

1. Apocynum venetum tea (the original solution amount is calculated according to the final concentration range of 3-100 mg/L of total flavonoids) is used for inhibiting the activity of NADPH oxidase and relieving the vascular endothelial cell injury induced by active oxygen clusters, but the general preparation process of the Apocynum venetum tea is not in the protection range.

2. A compound formula based on herba Apocyni Veneti tea for preventing and treating vascular endothelial cell injury comprises 5-hydroxytryptophan (4-200 mg/L), dihydrolipoic acid (4-100 mg/L), pantothenic acid (1-10 mg/L), and nicotinic acid (2-20 mg/L).

3. The ingredients of the formulation of claim 2 are required only for food and pharmaceutical grade purity, no special requirements are imposed on the preparation method, and the preparation process of the relevant components is out of the scope of this patent.

Technical Field

The invention belongs to the technical field of biology, and relates to a apocynum venetum tea compound functional beverage for preventing and treating cardiovascular and cerebrovascular diseases by targeting vascular endothelial cells.

Background

Cardiovascular and cerebrovascular diseases are the leading causes threatening the health of modern people, and have the characteristics of high disability rate, high recurrence rate and high death rate, and information released by the world health organization shows that in the ten death causes in the past decade, coronary heart disease accounts for 12.9%, stroke accounts for 11.4%, and the total accounts for 24.3% of the death causes; data published by the Weijian committee of China also show that death caused by cardiovascular and cerebrovascular diseases is the top of people in China, and with high adiposity of people's dietary structure, risk factors of people's cerebrovascular diseases still show a remarkable rising trend.

Vascular endothelial cells (vascular endothelial cells) are the core targets of cardiovascular and cerebrovascular diseases, and have been widely studied and paid attention from the foundation to the clinic. Related pathological studies show that microvascular injury is usually manifested by endothelial cell necrosis at the injury site, which in turn causes phenomena such as microvascular barrier dysfunction, capillary permeability increase, inflammatory response, microcirculation vasospasm and microvascular perfusion defect. Endothelial cells are used as important targets of metabolic substances and hemodynamic signals for regulating microcirculation during vascular injury, and damaged endothelial cells stimulated by stress can release various vasoactive substances and dilate blood vessels to cause microcirculation disturbance; simultaneously, the thrombin tissue factor and inflammatory mediators are released to act on inflammatory cells to generate inflammatory reaction, and fibrinogen and platelets are activated to form thrombus, so that serious consequences are generated.

The specific molecular mechanism of vascular endothelial cell injury is mainly Nitric Oxide (NO) injury and Reactive Oxygen Species (ROS) injury in two paths, the insufficient NO content of vascular endothelium causes the body to fail to exert a vasodilating effect when stimulated by a large amount of vasoconstriction, causing endothelial cell dysfunction and blood vessel damage, but excessive NO also causes severe ROS injury, eNOS is an upstream regulatory factor of NO and is an important target in the blood vessel wall influencing the function of endothelial cells, tetrahydrobiopterin (BH 4) is a key determinant of eNOS activity and function, BH4 inactivation causes eNOS dissociation and NO re-catalysis of L-arginine oxidation and NO synthesis, so that the generated progenitor NO is reduced, causing the endothelial cell dysfunction, whereas regulation at the BH4 level can effectively mediate eNOS coupling, improve the growth and proliferation of vascular endothelial cells, enhance the capacity of vascular endothelial cell repair and circulating endothelial cells after vascular injury, and further, the intracellular signaling of NO modified endothelial cells is balanced by SNNOS, the formation of endogenous nitric oxide-CoA, the endogenous nitric oxide-CoA-mediated, the endogenous nitric oxide-CoA-forming a dynamic signaling pathway.

ROS of vascular endothelial cells are derived from NADPH oxidase, mitochondrial respiratory chain leakage and cytochrome oxidase, which are three ROS signal paths which are mutually compensated and have different cell physiological functions. Normally, the level of pro-oxidant and antioxidant in the body are in a dynamic balance, but the generation and existence of ROS have cellular regionality and important difference between polar phase and non-polar phase, and the elimination of ROS also has two modes of metabolic free energy driving and reaction self free energy driving, thus often causing pathological phenomena such as ROS local runaway and the like. The biological function of ROS depends on intracellular concentration, moderate concentration of ROS has signal transduction effect, but high concentration of ROS acts on basement membrane of vascular endothelial cells to damage the body antioxidant system, and reduce the activities of antioxidase such as superoxide dismutase, catalase and glutathione S-transferase, etc., and lipid in the ROS can increase the permeability of the vascular endothelial cells, so that protein passes through a barrier and is abnormally deposited in the basement membrane to damage the structure and function of the vascular endothelial cells. ROS can also oxidize key cell signals such as tyrosine phosphatase to increase NF- κ B activity, reduce the synthesis of endothelial heparan sulfate proteoglycan, destroy endothelial cell glycoproteins, thicken basement membrane, and cause endothelial cell dysfunction. Meanwhile, ROS and NO are rapidly combined to form peroxynitrite and other active nitrogen, the speed of the peroxynitrite is 3-4 times faster than that of the superoxide generated by Nox, and the peroxynitrite can induce to generate nitration stress to increase the proinflammatory load of ROS and seriously damage vascular endothelial cells.

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, Nox) reduces in-vivo oxygen molecules into superoxide anions through NADPH-dependent single-electron reduction, is the only enzyme which directly generates ROS in vivo, and is also an important reason for ROS damaging vascular endothelial cells, so that the nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a core target for resisting ROS damaging the vascular endothelial cells, and therefore, a specific and selective Nox inhibitor needs to be found.

The currently published preventive care scheme for relieving vascular endothelial cell injury generally has the problems of incomplete technology and the like, for example, the application number 201910015995.1 and other patents utilize RNA to relieve vascular endothelial injury, which causes difficulty in the aspect of administration; the patent with application numbers of 201811150537.0 and 201880008458.8 and the like only provides a possible drug action target point of vascular endothelial cell injury, but no mature effective drug can be targeted at the target point; the patent with the application number of 201911011122.X and the like provides several widely questioned traditional Chinese medicine extracts such as resveratrol and the like, which are claimed to be capable of preventing and treating vascular endothelial injury, but the action mechanism and the side effect are not detailed. In conclusion, there is no safe and effective mature scheme for preventing and treating cardiovascular diseases by targeting vascular endothelial cells as a core target.

Disclosure of Invention

Apocynum venetum (with the scientific name of Apocynum venetum L.) is an upright shrub of Apocynum genus in Apocynaceae family, widely distributed in northern areas of China, has extremely strong stress resistance, and is particularly easy to grow in areas with harsh natural environments such as desert edges, saline-alkaline arid lands, and the like, for example, in some fields in Xinjiang, the salt content is as high as 14.5%, the salt content in 30 cm of the surface layer of soil in some fields in Kadsura basin is even as high as 50%, the Apocynum venetum can well grow, in northern Altai areas in Xinjiang, the extremely low temperature is-47 ℃, the snow accumulation period on the ground is 130 days, the Apocynum venetum can also naturally grow, the plant height can reach 70-120 cm, in Turpan basin, the highest temperature in summer can reach 47.8 ℃, the ground temperature can reach more than 52 ℃, and the Apocynum venetum can still well grow.

The dogbane leaf is a genuine medicinal material recorded in pharmacopoeia of the people's republic of China (2010 version one); the Mongolian medicine records that dogbane leaves or whole herbs treat dizziness, hypertension, palpitation and the like; uygur nationality recorded in "ethnic medicine Zhi san" records that dogbane leaf is used for dizziness, palpitation, hypertension and the like; the records of 'Western Water course Ji' and 'Western West recorded records' take folium Apocyni Veneti and flowers as tea for thousands of years, and wild Apocynum venetum tea is popular with Apocynum venetum L.and is known as 'Shencha'.

Early experimental research of the invention shows that the apocynum venetum tea is an effective Nox inhibitor, the Nox catalyzes NADPH to generate superoxide anion which is metabolized into hydrogen peroxide by superoxide dismutase and then generates Fenton reaction with iron ions in cells to generate hydroxyl free radicals with extremely strong reaction activity, and generates extensive ROS damage such as lipid peroxidation and DNA damage, and the apocynum venetum tea can protect vascular endothelial cells and prevent related diseases by inhibiting the generation of superoxide anion which causes endothelial dysfunction in cardiovascular and cerebrovascular diseases.

However, the apocynum venetum tea is only used for protecting vascular endothelial cells, so that the problem of weak effect and strong side effect exists. In view of the above, through a large number of preliminary studies and experiments, the invention discovers that the apocynum venetum tea can actually inhibit Nox within a certain concentration range, so as to reduce the ROS concentration of vascular endothelial cells, but has the problems of reduction of cell viability and cell proliferation capacity and the like, and the effect and side effect of the apocynum venetum tea are reflected at the cell level. Relevant analysis and experimental verification show that intracellular ROS can be distinguished into a second signaling ROS with physiological concentration and an injury ROS with concentration exceeding physiological concentration, while Nox easily generates excessive ROS under external stimulation, and the injury ROS is formed with concentration exceeding physiological concentration, so that the intracellular ROS is not a good ROS source. In addition, the apocynum venetum tea inhibits Nox to reduce the intracellular ROS level, reduces the excessive generation of ROS serving as a damage reason, but also influences the second messenger effect of the ROS, and on the basis of the invention, the activity of NO pathway is improved to replace the NOx to produce the ROS required by the second messenger, and because NO is originally a protective signal pathway of vascular endothelial cells, the improved activity can generate a proper amount of ROS and play a good role in assisting the generation of the ROS serving as the second messenger.

As a functional beverage, the invention takes excellent safety as the first premise, and the improvement of the NO pathway activity is realized by supplementing nutrient substances required by the metabolic pathway. 5-hydroxytryptophan is supplemented to serve as a nutrient substance required by synthesis of BH4, so that the level of BH4 is up-regulated, eNOS coupling can be effectively promoted to improve the growth and proliferation capacity of vascular endothelial cells, and the proliferation capacity of the vascular endothelial cells after vascular injury is enhanced. In addition, pantothenic acid required by coenzyme A synthesis is supplemented, and coenzyme A is promoted to form SNO-CoA through combination with nitric oxide, so that the SNO-CoA can be used as an endogenous source of SNO, and NO can better play a protective messenger role.

In addition, the enzyme activity of the Nox is inhibited, NADPH is not easily over utilized by the Nox to generate ROS damage, the reducing power and free energy of the NADPH can be used for reducing substances such as oxidized glutathione and the like, and the dihydrolipoic acid serving as a polar and nonpolar amphiphilic ROS remover in the formula can eliminate lipid peroxidation damage excessively generated by ROS in an organism or a cell region, so that the effect of comprehensively protecting vascular endothelium is achieved.

On the basis of the analysis and experiments, the inventor refers to a theoretical framework that is established systematically in the earlier working research stage and is used for leading Nox to inappropriately activate a channel generating ROS damage, influencing NO and the like to generate vascular endothelial cell damage, which is a core mechanism causing cardiovascular and cerebrovascular diseases, and the theoretical framework is screened pertinently under the guidance of the theoretical framework, and after long-term cell experiments and random double-blind control experiments of human volunteers are improved and verified, a compound formula which is most suitable for preventing and treating the ROS-induced vascular endothelial cell damage is obtained, wherein the formula is based on auxiliary ingredients of apocynum tea (the original liquid amount is calculated according to the final concentration of total flavonoids of 3-100 mg/L), and the auxiliary ingredients of 5-hydroxytryptophan (4-200 mg/L), dihydrolipoic acid (4-100 mg/L), pantothenic acid (1-10 mg/L) and nicotinic acid (2-20 mg/L) are added subsequently.

Compared with the prior art, the invention has the following advantages.

1. The invention has strong innovation, the inventor gradually establishes a theoretical framework of 'ROS damage generated by improper activation of Nox, influencing pathways such as NO and the like, generating vascular endothelial cell damage, which is a core mechanism causing cardiovascular and cerebrovascular diseases' on the basis of long-term research on ROS damage vascular endothelial cell mechanism, aims at screening out a Nox low-activity technical scheme taking apocynum venetum tea as a main component under the guidance of the theoretical framework, regulates NO pathways by subsequently adding various nutrient components of metabolic pathways, avoids local ROS damage, improves the action effect of the apocynum venetum tea, inhibits the side effect to a certain extent, and can well relieve ROS-induced vascular endothelial cell damage.

2. The components of the formula of the invention have good interaction, and can generate synergistic synergism.

2.1. The formula components have good synergistic effect: only under the condition that the apocynum venetum tea inhibits Nox, more NADPH generated by nicotinic acid is supplemented, so that the NADPH is not metabolized into superoxide anions by the Nox, and ROS damage is further generated; the high-activity cardiovascular and cerebrovascular endothelial effect protection of the NO channel is based on inhibiting ROS and dihydrolipoic acid from Nox sources to properly eliminate the ROS, otherwise NO is easily combined with the ROS quickly to form active nitrogen such as peroxynitrite and the like, inflammatory load is increased by inducing nitrification stress, and vascular endothelial cells are seriously damaged. In addition, the high-activity NO induced by the combination of the 5-hydroxytryptophan and the pantothenic acid in the formula can supplement the proliferation and activity reduction of vascular endothelial cells caused by the low ROS level induced by the apocynum venetum tea and the dihydrolipoic acid.

2.2. The components of the formula maintain stability mutually: the dihydrolipoic acid is a ROS scavenger with amphiphilic polar phase and nonpolar phase, and can protect effective components such as flavone in herba Apocyni Veneti tea from oxidation and inactivation. 5-hydroxytryptophan, pantothenic acid and nicotinic acid are all compounds which are not easy to oxidize and have high thermal stability, and can maintain the stability of dihydrolipoic acid in the formula.

3. The cell experiment data show that the formula of the invention has obvious effect.

For details, see examples 1 and 2, the composite formulation has significant physiological effects on standardized high-throughput cellular experiments.

4. From the trial data of volunteers, the formula of the invention is obviously stronger than the mainstream scheme in the market.

In terms of action and effect, the formula is found to have better effect than certain marketed formulas through statistical analysis on the basis of acquiring a large amount of data by comprehensively utilizing a high-flux standardized cell experiment and a multicenter large-sample random double-blind control volunteer experiment (see example 3 in detail).

5. In terms of safety, the auxiliary components of the formula are from accepted low-toxicity metabolism regulation nutrient substances, and the cytotoxicity of the formula is specially checked in experiments, so that the use dosage is ensured not to exceed 5% of a toxicity threshold value; the effect is increased mainly by means of a small dose of the composite formulation. In addition, the early work of the invention accurately proves the action range and the toxicity threshold of each component of the formula through a standardized high-flux cell experiment, thereby ensuring that the use dosage of each component has certain tolerance and being beneficial to the development and marketing of products.

Detailed description of the preferred embodiments.

The technical embodiments of the present invention will be described in further detail with reference to specific examples.