TWI454269B - Compounds isolated from xanthoceras sorbifolia, methods for preparing same and uses thereof - Google Patents

Description

Compound isolated from XANTHOCERAS SORBIFOLIA, preparation method thereof and use thereof

This patent claims the priority of the International Patent Application (N. PCT/US04/33359, filed on Oct. 8, 2004), which is hereby incorporated herein by reference. Submit the rights required in USSerial N0s.60/532,101, filed on December 23, 2003. The content of these pending patent applications should therefore be fully incorporated into this patent application.

The contents of various publications are widely cited in this patent application in order to better clarify the rights to be obtained in this patent application.

Field of invention

The present invention discloses a plant extract, an extract of a plant called Xanthoceras sorbifolia Bunge, its composition, function and application, and their method of preparation.

Background of the invention

In recent years, the upsurge of exploring new drugs or health products from plant extracts has been a great success in countries around the world. In the treatment of encephalopathy and brain health, extracts such as ginkgo leaves are used to expand microvessels to improve Brain function; extract of small hypericum for the treatment of depression and ginseng extract can be used for wisdom and so on. Traditional Chinese medicine and folk herbal medicine are treasure trove of new medicines and health products. The three plants mentioned above are commonly used traditional Chinese medicines. The plant genus of the present invention is also a folk herb.

Xanthoceras is a plant of the genus Xanthoceras of the Sapindaceae family. The Latin name Xanthoceras sorbifolia Bunge, also known as the sacred flower, Wenguang fruit, Wenguan tree, cultural official fruit, cliff papaya and Syracuse Waiting, the English name is called "Goldenhorn, Yellowhorn". Deciduous shrubs or small trees up to 8 m high; odd-pinnate compound leaves; racemes, flowers white, heterozygous; capsules initially green, thick crust, woody, mature dark brown, ovoid, mostly native to China It is mainly distributed in northern China, wild or cultivated. The seeds contain more than 50% oil, edible, stems and leaves can be used as medicine, rheumatism, and kernels are used in the treatment of enuresis. Seeds, flowers and leaves are edible and can be used to save the wild.

The seeds of Xanthoceras are used in the folk to cure enuresis, and the stems and leaves are used to cure rheumatism in Mongolian medicine. It has a long history. However, in-depth development and research on the medicinal aspects of Xanthoceras is a matter of recent years.

Journal of Shenyang Pharmaceutical University (2001, 18(1): 53-56) reported the n-butanol extract of the wood of Xanthoceras sorbifolia and its anti-inflammatory effect.

The Chinese patents (CN 1092992A) and (CN 1092991A) disclose the powder of the crown of the fruit, which can effectively treat enuresis and improve brain function and intelligence. The Chinese patent (CN 1052636C) discloses an ethanol extract of the powder of the genus Pleurotus ostreatus, which can effectively treat enuresis and improve brain function and intelligence.

The U.S. Patent Application Publication (No. 20030096030) provides a substance for anti-aging and improving brain function, treating nocturia, urinary frequency, urinary incontinence, mental retardation, dementia and enhancing the body's ability to resist diabetes. The extract contains four saponins (Bunkankasaponin ABCD) and two sterol compounds. U.S. Patent Application Publication (No. 20030082293) discloses four saponins which are isolated and purified from the fruit husk of the genus Corolla. (Bunkankasaponin A.B.C.D), crude fat, crude protein, and sugars.

U.S. Patent (No. 6,616,943, issued September 9, 2003) provides a total extract of the genus Corolla, and its use and preparation methods. The preparation method comprises the steps of: harvesting the crown fruit, clearing the shell and/or the fruit stalk; drying and pulverizing the fruit husk and/or the fruit stalk; and extracting the crown with an organic solvent such as an alcohol (such as ethanol). Fruit shell and/or fruit stalk powder; solvent is recovered, concentrated to obtain a stream extract; dried, sterilized to obtain an organic solvent extract such as alcohol. The total extract of the husk is an organic solvent extract such as the fruit of the fruit of the genus Corolla, which mainly contains saponins, sugars, proteins and other substances. The total extract of the husk can be used to manufacture medicines or health foods for anti-aging, improve memory, and improve brain function, treatment of nocturia, frequent urination, urinary incontinence, mental retardation, dementia, senile dementia, autism, brain trauma, Jinsen disease and other conditions caused by impaired brain function or disorders. The total extract of the fruit of the canopy can also be combined with vitamin B, D, K, antioxidant, Cordyceps sinensis or its extract, Ginkgo biloba or its extract, Polygera or its extract, Shisong A, folic acid, Amino acids, creatine, fiber additives, etc. are made into various medicines or health foods.

Chen Yingjie Tadahiro Takeda, Yukio Ogihara et al. published four articles on the study of Physalis citrine in 1984 and 1985 in the Journal of Medicinal Chemistry (Chem. Pharm. Bull.) (1984, 32 (9): 3378-3383; 1985, 33 (1): 127-134; 1985, 1985 33 (3): 1043-1048; 1985, 33 (4): 1387-1394) reported four new saponins (Bunkankasaponin) isolated from the seeds of Xanthoceras ABCD), their chemical structure is as follows: (A) 22-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D -glucopyranosyl-(1→2)-β-D-glucuronopyranosyl] protoaecigenin, (B) 22-O-acetyl-21-O-(3,4-di-O-angeloyl)-β-D-fucopyranosyl-3- O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin, (C)28-O-acetyl-21-O-(4-O-acetyl-3-O-angeloyl)- β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin, (D)28-O-acetyl-21-O-(3,4-di -O-angeloyl)-β-D-fucopyranosyl-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]protoaecigenin. They also proposed the structure of prosapogenin: 16-O-acetyl-21-O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)protoaecigenin, 22-O-acetyl-21- O-(3,4-di-O-angeloyl-β-D-fucopyranosyl)protoaecigenin 3-O-β-D-glucuronopyranoside,21-O-(3,4-di-O-angeloyl)-β-D- Fucopyranosyltheasapogenol B,21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyltheasapogenol B,21-O-(4-O-acetyl-3-O-angeloyl)-β-D-fucopyranosyl -22-O-acetylprotoaescigenin.

Cancer is one of the important causes of modern human death. The causes of carcinogenesis are various. Cancer can be caused by different types of cell aberrations, and thus there are various cancers. Each cancer has its own unique characteristics that reflect its particular cause. Even the spread of cancer is beyond control and the cause can be identified. Therefore, it is important that the drugs developed must be targeted.

Ovarian cancer is the fifth leading cause of death in women. An important cause of death in gynecological diseases. In the United States, 1.4-2.5% of women may have ovarian cancer, and older women are at higher risk of developing ovarian cancer. More than 50% of women between the ages of 55 and 74 die of ovarian cancer. More than 25% of women between the ages of 35 and 54 die of ovarian cancer (see http://www.nlm.nih.gov/medlineplus/ency/article/000889.htm ).

The reason why ovarian cancer causes extremely high mortality in women is that, first of all, the symptoms are not obvious and are easily misdiagnosed. When diagnosed, the cancer cells have metastasized. At the same time, ovarian cancer often transfers malignant cells to the uterus, bladder, intestines and intestines, and before they are discovered, they have begun to form new tumors. In addition, there is no system for free monitoring of ovarian cancer, and 50% of women with ovarian cancer cannot be diagnosed at the beginning of cancer.

The compound (or synthetic) or a combination thereof isolated from the extract of the genus Corolla is provided by this patent, and has great potential for anti-ovarian cancer.

Summary of invention

The following is a summary of the patents of the present invention. In order to make the key terms of the invention obvious, the contents of other clauses may be simplified or even omitted. However, this does not mean that the invention patents are limited to those mentioned in the abstract.

The patent application of the present invention relates to the composition, function and application of the extract of Xanthoceras sorbifolia, and a preparation method thereof.

These extracts can be made into health care products or drugs to treat nocturia, enuresis, incontinence, and frequent urination. The extract of Xanthoceras sorbifolia is not only related to its ability to improve bladder function, but also to improve brain function in humans. It can improve the function of the brain's central nervous system and the transmission of urinary system information, so that the signal transmitted to the brain is increasing. Strong, the brain sends a signal to wake up the sleeping person to pee.

Avoid the occurrence of enuresis. Xanthoceras extract helps the growth of the bladder and sphincter, increases urine storage and reduces the number of urination, thus avoiding the occurrence of enuresis. The extract of Xanthoceras sorbifolia provided by the invention helps to eliminate mental stress, delay aging, improve bladder function, avoid detrusor instability, hyperactivity, hyperreflexia, thereby preventing urgency and frequent urination. The extract of Xanthoceras sorbifolia inhibits the synthesis of acetylcholine acetylase (AchE), mediates the release, absorption and decomposition of acetylcholine (Ach), thereby improving the transmission of information in the central nervous system and urinary system and avoiding the occurrence of enuresis. The extract of Xanthoceras sorbifolia provided by the invention can mediate the release of urinary hormone (ADH), thereby controlling the bladder to produce excessive urine volume and avoiding the occurrence of enuresis. Therefore, the extract of Xanthoceras sorbifolia can also be used to prevent brain aging and cerebrovascular disease, improve memory and develop intelligence, and treat dementia mentally retarded and other diseases caused by impaired brain function or disorder. At the same time, the extract also has an anti-inflammatory effect.

The patent application of the present invention relates to an extract of Xanthoceras sorbifolia which is an organic solvent extract of a fruit husk and/or a stalk, a seed, a seed shell, a branch, a bark and a root alcohol, a crude saponin extract, and a flavonoid. Extract, alkaloid extract, coumarin extract, aromatic organic acid extract, polysaccharide extract and tannin extract.

The patent application of the present invention relates to flavonoid extracts of Xanthoceras sorbifolia containing flavanols, flavonols, flavanones and the like and other compounds such as phenolic compounds, organic acids and the like. The patent application of the present invention relates to an organic solvent extract such as alcohol of Xanthoceras, which mainly contains saponins and other compounds such as sugar, crude protein and the like. This hair The patent application of the patent application relates to the crude saponin extract of Xanthoceras mainly containing the crude saponin compound. The patent application of the present invention relates to the alkaloid extract of Xanthoceras mainly containing alkaloids, coumarins, sugars and other compounds such as crude protein. The patent of the present application relates to the coumarin extract of Xanthoceras mainly containing coumarin, coumarin glycoside and other compounds such as sugar, crude protein and the like. The patent application of the present invention relates to an aromatic organic acid extract of Xanthoceras mainly containing aromatic organic acids, organic fatty acids, terpenoid organic acids and other compounds such as crude protein. The patent application of the present invention relates to a polysaccharide extract of Xanthoceras sorbifolia. The patent of the present invention relates to a tannin extract of Xanthoceras mainly containing tannin and other compounds such as organic acids, sugars, crude proteins and the like.

The patent application of the present invention provides a process for preparing extracts from the fruit husks and/or stalks, seeds, seed hulls, branches, bark and roots.

At the same time, the invention also discloses the function and application of these extracts. The extract of Xanthoceras sorbifolia provided by the present invention contributes to the growth and development of the bladder and sphincter; the extract of Xanthoceras sorbifolia can improve the sleep alert system and help prevent the occurrence of deep sleep; the extract of Xanthoceras sorbifolia can regulate the adrenal cortex hormone ( The role of ACTH) and its receptors, inhibiting the absorption of serotonin, thereby breaking deep sleep, allowing people to wake up when the bladder is full, avoiding bedwetting; extracts of the canopy can mediate urinary hormones (ADH) Release, thereby controlling the bladder to produce excessive urine volume and avoiding the occurrence of enuresis; the extract of Xanthoceras can mediate the action of norepinephrine (NE) and its receptors to prevent the occurrence of sleep paralysis; Xanthoceras extract can also regulate the action of neuropeptides and their receptors. The extract of the extract of Corundum provided by the present invention shows great potential for treating cancer and can be used as an anticancer drug such as ovarian cancer. Anti-bladder cancer, also anti-cervix Cancer, ovarian cancer, prostate cancer, lung cancer, breast cancer, colon cancer, liver cancer and brain cancer, as well as other cancers. The extract of Xanthoceras sorbifolia has a significant effect on the function of the lungs and bladder. The saponin (glycoside) = saponin = saponin expressed herein.

The invention also discloses a compound which is purified and separated from the saponin extract of Xanthoceras sorbifolia, the sapogenin thereof is a pentacyclic triterpenoid compound having one or more sugar chains, and has one side chain or one to the carbon 21 and 22 positions. A plurality of angelica amides form a biologically active compound.

The invention also discloses a compound having the following chemical structure:

Name of the compound: 3-0-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucopyranosyl -21,22-O-dipodoxime-3β,15α,16α,21β,22α,28-hexahydroxy-12-ene pentacyclic triterpenoid saponin.

The present invention also discloses compositions from the salts of the above compounds, and compositions formed from the above compounds and suitable carriers.

The invention also discloses a method for preparing an extract from Xanthoceras, the process of which is as follows: 冠 收 冠 冠 冠 冠 , , , , 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠, solvent extraction solution; ̇ collecting the extract, and then hot reflux (the appropriate number of times); ̇ collecting the combined extract; ̇ recovering the organic solvent to obtain a stream extract; ̇ drying and sterilizing the stream extract, powder Xanthoceras extract.

The present invention also discloses a triterpenoid saponin comprising three sugars and one or more dimeric sulfhydryl groups; containing two sugars and one or more angelica sulfhydryl groups; containing four sugars and one or more dijonins Base, as well as containing three sugars and one angelica thiol.

The invention also discloses a method for separating and purifying a compound from the extract of Xanthoceras sorbifolia. The process is as follows: ̇ 收 收 冠 冠 冠 , , , , , , , , , , , , 文 文 文 文 文 文 文 文 文 文 文 文 文Powder; immersing the extract powder of the sacred fruit with an appropriate amount of one or several organic solvents (the appropriate number of times and time, the solvent extract; the leaching solution, and then reflowing (the appropriate number of times); Collecting the combined extract; ̇ recovering the organic solvent to obtain a flow extract; ̇ drying and sterilizing the flow extract to obtain a powdered osmanthus extract; ̇ separating the powdered osmanthus extract into one or more components; a biologically active component of the above ingredients; The above biologically active components are separated and purified by fast-acting liquid chromatography (FPLC); the biologically active compounds are separated by high performance liquid chromatography (HPLC).

The present invention also discloses a method for determining the chemical structure of the above-mentioned biologically active compound by nuclear magnetic resonance. The chemical shifts of the two-dimensional HMQC NMR spectra of the compounds are shown in Table 10-2 (HMQC-peak shift) and Table 10.3 (HMBC-peak shift). The name of the compound: 3-0-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucopyran Base-21,22-O-dipodoxime-3β,15α,16α,21β,22α,28-hexahydroxy-12-ene pentacyclic triterpenoid saponin. Molecular formula: C ₅₇ H ₈₈ O ₂₃ . This compound and its derivatives have a strong anticancer effect. This compound or composition thereof modulates the metabolic activity of G protein cells or their receptors, Fas protein cells or their receptors, lysine kinases or their receptors, mitogens or their receptors and the like. These compounds can be isolated and purified from extracts of C. elegans, artificially synthesized, or raised from other organisms.

The extracts and compounds of the extracts provided by the present invention can inhibit the growth of cancer cells and can be used as an anticancer drug such as ovarian cancer. It is resistant to bladder cancer and also to cervical cancer, ovarian cancer, prostate cancer, lung cancer, breast cancer, colon cancer, liver cancer and brain cancer, as well as other cancers.

The daughter cells produced by normal stem cells have limited proliferative capacity. When the daughter cells divide to produce non-dividing cells, cell division stops and enters the final differentiation phase. If stem cell division does not produce non-stem cells, it will proliferate to form tumors. At the same time, if the daughter cells cannot proliferate normally, these fine The cells will proliferate to form tumors.

When normal cells mature, they enter specialized differentiation stages to form specialized cells and stop dividing. Once the cell population multiplies several times, it will stop adding value and enter a recession. If cell division cannot be terminated and does not enter the stage of decline and differentiation, cancer will develop. These cells are genetically unstable and ignore the signals that regulate cell proliferation both internally and externally. They can invade other tissues and add value to cause more tumors.

Many biological pathways are not functioning properly, causing cell division to fail, leading to cancer. For example, transforming growth factor alpha (TGF-alpha) and transforming growth factor beta (TGF-beta), transforming growth factor beta signaling pathway and SMAD-encoding regulatory genes, Ras-MAP kinase chain signaling pathway, gene regulatory protein Myc and other biochemical pathways The link or its receptor is inappropriately activated or overactive, causing the formation and proliferation of cancer cells. The extracts and novel saponin compounds provided by the present invention can regulate the biochemical link of these biological pathway activities or their receptors to prevent canceration. The signal that kills cells due to genetically modified cancer cells is blocked. As a result, cancer cells continue to divide, leading to cancer. The extract of the extract of the crown of the present invention and the novel saponin compound can clear the pathway of blocking the "suicide" signal transmission of the cancer cells, and cause the cancer cells to "suicide."

Simple illustration

Fig. 1 (a) and (b). Improvement of learning and memory in mice after 9 days of administration (water maze method).

Fig. 2 (a) and (b). Effect of extracts of Xanthoceras sorbifolia X and Y on memory impairment in mice given pentobarbital for 3 days (water maze method).

Figure 3. Effect of X and Y administration of Xanthoceras sorbifolia L. extract for 10 days on urine output in mice.

Figure 4. A typical sleep cycle for people.

Figure 5. High performance liquid chromatogram of the extract of the fruit of the corolla.

Figure 6. Comparison of the inhibitory effect of Xanthoceras sorbifolia extract on breast cancer (IC50 65 μg/ml) and leukemia cancer cells (IC50 35 μg/ml).

Figure 7. Canopy extract promotes bladder cell growth at low concentrations, and bladder cell growth is increased by 25% at a concentration of 10 μg/ml. The extract of Xanthoceras sorbifolia inhibits the growth of bladder cancer cells at high concentrations (IC50 is 45-60 μg/ml). At the same time, the extract of Xanthoceras sorbifolia inhibited the growth of ovarian cancer cells (IC50 was 15 μg/ml).

Figure 8. Extract of Campanula sinensis has no or slightly stimulating effects on brain cell growth at concentrations below 40 μg/ml. The extract of Xanthoceras sorbifolia has an inhibitory effect on the growth of the prostate (IC50 of 70 μg/ml) and brain cancer cells (IC50 is 40 μg/ml).

Figure 9. The growth of lung cells can be grown at a concentration of 10 μg/ml, and the growth is increased by 20% at 50 μg/ml. Bladder cells were grown 5% in RPMI-1640 medium containing 10 μg/ml of extract of C. elegans. However, the extract of Xanthoceras sorbifolia inhibited the growth of bladder cancer cells at a high concentration with an IC50 of 45-60 μg/ml. The extract of Xanthoceras sorbifolia inhibited the growth of liver cancer cells with an IC50 of 68 μg/ml.

Figure 10. The effect of the extract of C. elegans on the growth of ovarian cancer and cervical cancer cells is different. The therapeutic effect of extract of Xanthoceras sorbifolia on ovarian cancer is considerable, and the inhibition on the growth of cervical cancer cells is weak.

Figure 11. Osteoblasts grow 20% in low concentrations (eg 10 μg/ml) in RPMI-1640 medium containing extracts of Xanthoceras sorbifolia, inhibiting bone at high concentrations The growth of cancer cells has an IC50 of 40 μg/ml).

Figure 12. Separation of the extract of the extract of Xanthoceras on the μbondapak C18 column by high performance liquid chromatography (HPLC).

21A-B are representative elution profiles of fast liquid chromatography. Figure 12C-K is an HPLC chromatogram of the components obtained from fast liquid chromatography. Figure 12L: Original material; Figure 21M: Thin layer chromatography (TLC). The fractions selected from the fast liquid chromatography were stained with carbohydrates and analyzed on TLC.

Figure 13. Absorption spectra of extracts of Xanthoceras sorbifolia.

Abscissa: wavelength nm

Vertical coordinate: light intensity.

The extract of Xanthoceras sorbifolia has three absorption peaks at 207 nm, 278 nm and 500 nm.

Figures 14A-14E. MTT assay for the effect of extracts of C. elegans on cell growth. The sensitivity of various cells to extracts of Xanthoceras sorbifolia is as follows: Figure 14A is extremely sensitive: ovarian cells; Figure 14B is sensitive: bladder and bone cells; Figure 14C moderately sensitive: white blood cells and hepatocytes; Figure 14D slightly sensitive: prostate, breast and Brain cells; Figure 14E Less sensitive: colon, cervix and lung cells.

Figure 15. Gradient (10-80%) elution profile of Xanthoceras sorbifolia extract on fast liquid chromatography (FPLC).

Vertical coordinate: light intensity (245nm)

Abscissa: eluate volume (ml).

Figure 16A. Selected components of the extract of C. elegans obtained from fast liquid chromatography (FPLC).

Figure 16B. Effect of components of the extract of C. elegans obtained from fast liquid chromatography (FPLC) on bladder cell growth (according to MTT assay): component 5962 (Y) inhibits cell growth; components 610 and 1116 are slightly Stimulate cell growth.

Abscissa: concentration (ul/ml)

Vertical coordinate: cell growth (%).

Figure 17. A 64% Ethyl isocratic elution profile of the component 5962 of the extract of Coleoptera. The two main components X and Y are separated on the FPLC.

Abscissa: light intensity (254nm)

Vertical coordinate: component number (1 ml / component).

Figure 18. Effect of fractions X (2021) and Y (2728) of the extract of Campanula sinensis on bladder cell growth: only Y inhibits bladder cell growth.

Figure 19. High performance liquid chromatography (HPLC) 35% Ethyl isocratic gradient elution profile of the component Y of the extract of Xanthoceras sorbifolia L., showing that the Y(2728) component contains 4-5 compounds: Y0, Y1, Y2, Y3 and Y4.

Figure 20. High-performance liquid chromatography (HPLC) 45% Ethyl isocratic gradient elution of the component Y of the extract of Xanthoceras sorbifolia, showing that the peaks of Y3 and Y4 are superimposed, and Y1 and Y2 are clearly separated from Y3 and Y4. Open.

Figure 21. Equivalent gradient elution profile of high performance liquid chromatography (HPLC) 45% Ethyl acetate extract on the C18 column (Delta Pak C18). Y1 and Y2 are clearly separated.

Figure 22. Proton NMR of the up-sampling (upper half of the curve) and the lower half of the lower curve of the Y3 and Y4Y peaks of the extract of the extract of the extract of the genus Spectrum. Show them no difference.

Figure 23. Shows the purity of component compound Y (collected from a 45% ethyl acetate gradient elution profile on a high performance liquid chromatography Delta Pak C18 column).

Figure 24A. Shows the potential point scale of purified compound Y in inhibiting ovarian cancer (OCAR-3).

Figure 24B. shows the linear scale of the purified compound Y in inhibiting ovarian cancer (OCAR-3).

Abscissa: concentration (μg/ml), ordinate: cell growth (%) (detected according to MTT), IC50 about 1 μg/ml.

Figure 25. Comparison of the effect of compound Y on the growth of ovarian and cervical cancer cells: ovarian cancer cells are sensitive to compound Y, whereas cervical cancer cells are not sensitive. The IC50 of ovarian cancer cells is about 1 μg/ml, indicating that ovarian cancer cells are sensitive to purified compound Y.

Figure 26. Chemical shift map of proton nuclear magnetic resonance (NMR) of compound Y.

Figure 27. Two-dimensional NMR (HMQC) pattern of compound Y (see also chemical shift data in Table 10.2).

Figure 28A. HMBC Atlas Level 1 for Compound Y (see also Chemical Displacement Data in Table 10.3).

Figure 28B. HMBC Atlas Level 2 for Compound Y (see also Chemical Displacement Data in Table 10.3).

Figure 29A-1. One of the mass spectra of Compound Y: Y+Matrix (CHCA) + Angiotensin 1 "two point calibration".

Figure 29A-2. Mass spectrum of compound Y: Y+Matrix (CHCA) + Angiotensin "one point calibration".

Figure 29A-3. Mass spectrum of Compound Y: Y+Matrix (CHCA).

Figure 29A-4. Mass spectrum of Compound Y: Matrix (CHCA) only.

Figure 29A-5. Mass spectrum of Compound Y: Angiotensin 1+Matrix (CHCA).

Figure 29B. Mass spectrum of compound Y (ESI-MS).

Figure 30. Chemical structure and name of compound Y: 3-0-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3-β-D - glucopyranosyl-21,22-O-dipodoxime-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyoleolin-12-ene pentacyclic triterpenoid saponin.

Figure 31. Proton nuclear magnetic resonance (NMR) spectra of compound Y1. The chemical shift data is shown in Table 11.2.

Figure 32. Compound Y1 two-dimensional nuclear magnetic resonance (HMQC) map. The chemical shift data are shown in Tables 11.5 and 11.4.

Figure 33. Proton nuclear magnetic resonance spectroscopy of compound Y2. The chemical shift data is shown in Table 26.

Figure 34. Two-dimensional nuclear magnetic resonance (HMQC) pattern of compound Y2. The chemical shift data is shown in Table 27.

Figure 35. Proton nuclear magnetic resonance spectroscopy of compound Y5. The chemical shift data is shown in Table 28.

Figure 36. Compound Y5 two-dimensional nuclear magnetic resonance (HMQC) map. The chemical shift data is shown in Table 28.

Figure 37. Proton COSY-NMR spectrum of compound Y1. The chemical shift data is shown in Table 11.6.

Figure 38. Four possible chemical structures of Compound Y1: Figure 38A: Chemical Structure Y1-1; Figure 38B: Chemical Structure Y1-2; Figure 38C: Chemical Structure Y1-3; Figure 38D: Chemical Structure Y1-4.

Figure 39. Chemical structure of compound Y1.

Figure 40. A map of compound Y1 two-dimensional nuclear magnetic resonance (HMBC) level 1.

Figure 41. A map of compound Y1 two-dimensional nuclear magnetic resonance (HMBC) level 2.

Figure 42. A map of compound Y1 two-dimensional nuclear magnetic resonance (HMQC) level 2.

Figure 43. Comparison of the effects of compounds Y1 and Y2 on the growth of ovarian cancer cells (abscissa: extract concentration μg/ml, ordinate: % cancer cell growth).

Figure 44. Effect of extract of Xanthoceras on the urine output of mice in Table 5A-1 (25 days after the test).

Figure 45. Effect of extract of Xanthoceras on urination rate in mice in Table 5A-2 (25 days after the test).

Figure 46. Table 5A-3, Effect of extract of Xanthoceras on the specific gravity and pH of urine in mice (after 25 days with the test).

Figure 47. Effect of extract of Xanthoceras on the Na+, K+ and Cl- concentrations in mouse urine in Table 5A-4 (25 days after the test).

Figure 48. Effect of extract of Xanthoceras on urine output (25 days of administration).

Figure 49. Effect of extract of Cappari FS(X) on urination rate (25 days of administration).

Figure 50. Ethyl isocratic elution profile of the extract R of the extract of the extract of Xanthoceras sorbifolia L. by fast liquid chromatography (IS0-30)

Figure 51. Components #9, #10 and #11 were analyzed by high performance liquid chromatography.

Figure 52. Purification of compound R by high performance liquid chromatography (Delta-Pak C18 column).

Figure 53. Compound R proton nuclear magnetic resonance spectroscopy.

Figure 54. Proton nuclear magnetic resonance spectroscopy of compound R1.

Figure 55. Proton-NMR-expand-1 of the compound R1 proton nuclear magnetic resonance spectrum.

Figure 56. Proton-NMR-expand-2 of the compound R1 proton nuclear magnetic resonance spectrum.

Figure 57. Compound R1 proton-NMR-expand.

Figure 58. Two-dimensional HMQC nuclear magnetic resonance spectrum of compound R1.

Figure 59. Two-dimensional HMQC NMR level 1 spectrum of compound R1.

Figure 60. Two-dimensional HMQC NMR level 2 spectrum of compound R1.

Figure 61. Two-dimensional HMBC NMR level 1 spectrum of compound R1.

Figure 62. Two-dimensional HMBC NMR level 2 spectrum of compound R1.

Figure 63. Two-dimensional COSY nuclear magnetic resonance spectrum of compound R1.

Figure 64. Nuclear Magnetic Resonance Spectrum of Compound R1 Carbon 13.

Figure 65. Chemical structure of compound R1.

Figure 66. Compound compound Y-a structural formula.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C

R4=B or C

Figure 67. Compound compound Y-b structural formula.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C

R4=B or C

Figure 68. Compound Y-c structural formula.

Figure 69. Structure of compound Y1-a.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C; R3=A or B or C

R4=B or C

Figure 70. Structure of compound Y1-b.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C; R3=A or B or C

R4=B or C

Figure 71. Structure of compound Y1-c.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C; R2=A or B or C

R4=B or C

Figure 72. Component O is a gradient elution (iso-20) component plot of 20% Ethylene with FPLC and HPLC.

Figure 73. Secondary liquid chromatogram of component 28 (from iso-20).

Figure 74. Secondary liquid chromatogram of component 34 (from iso-20).

Figure 75. Secondary liquid chromatogram of component 54 (from iso-20).

Detailed description of the preferred embodiment

When a person sleeps, he or she enters a sleep cycle that includes 4-5 stages that alternate with each other, some stages are deep sleep stages, and some stages are mild sleep stages (Figure 4). Moreover, the alternation of these stages is not in a certain order. Sleeping people are more likely to be woken up when entering a mild sleep stage. However, when entering the deep sleep phase, it is more difficult to wake up. When a healthy person is sleeping, when the bladder is full, if you are sleeping lightly, it is easier to wake up to pee; if you are in deep sleep, it is not easy to wake up to pee, and enuresis may occur. Although enuresis, frequent urination and urgency are not dead, the pain and inconvenience caused to patients is hard to say. Moreover, children, adults, men and women can suffer from such conditions. Recent research has shown that enuresis, frequent urination and urgency are not only related to the development of the bladder, but also directly related to the function of the brain and nervous system.

At the same time, when the bladder is full of urine, the smooth muscle of the bladder will expand. When the smooth muscle of the bladder expands, it sends signals to the cerebral cortex and cerebellum through the pelvic and sacral spinal nerves. After the cerebral cortex and cerebellum receive signals, the bladder continues to contract and the sphincter relaxes. At this time, the urine will be discharged. When a person enters the bladder during sleep, the detrusor stretches to enlarge the bladder. As the urine accumulates in the bladder, it stimulates the bladder's stretch receptor to continuously signal the brain. The strength of the signal depends on how much urine is accumulated in the bladder. When the amount of urine in the bladder accumulates enough, the signals transmitted to the brain continue to increase, causing the brain to signal the sleeping person to pee. but, Enuresis occurs if the signal is transmitted or is not strong enough due to a defect in the function of the brain or bladder, resulting in a person who cannot wake up to sleep. The extract of the crown of the apple provided by the invention helps to improve the function of the brain or the bladder, makes the transmission signal smooth or strengthen, promptly wakes up the sleeping person, and prevents enuresis from occurring.

Bladder smooth muscle has two functions. When it relaxes, the bladder stores urine; when it contracts, the bladder urinates. There is a telescopic sensor receiver in the bladder wall that senses the amount of urine in the bladder and transmits this information to the spinal cord system through the spinal thalamus bundle. When the detrusor is needed to relax, the brain sends a suppression signal. When the detrusor contraction is needed, the brain emits an excitatory signal. Acetylcholine (AchE) inhibits the synthesis of acetylcholine (Ach), and the deficiency of acetylcholine affects the transmission of the above information, causing enuresis. The extract of Xanthoceras sorbifolia provided by the invention can inhibit the synthesis of acetylcholine acetylase (AchE), thereby contributing to the formation of acetylcholine and preventing enuresis.

There is a urinary hormone (ADH) in the posterior pituitary gland that regulates the state of water in the body through the kidneys. This affects how much kidneys produce urine. The release of urinary hormones is controlled by osmotic receptors and pressure receptors. Osmotic receptors are specialized cells of the hypothalamus that are sensitive to the concentration of particles in the blood. When the concentration of particles in the blood is high, the pituitary will release more urinary hormones, and conversely, the urinary hormone released by the pituitary will decrease. The pressure receptors are located very close to the right atrium, the large veins and the cervical sinus, and are sensitive to blood flow and blood pressure. When blood pressure is low, the heart sends a signal to the hypothalamus and pituitary, releasing more urinary hormones. Conversely, the released urinary hormones are reduced. The extract of Xanthoceras sorbifolia provided by the invention can regulate the synthesis of urinary hormone (ADH) to reduce the amount of urine production and prevent enuresis.

At the same time, other factors, such as various stresses, mental stress, or the aging of the body due to age, the bladder will shrink and become smaller, but the ability to store urine and detrusor instability, hyperactivity, hyperreflexia, resulting in bladder storage When not full, it is forced to contract in advance, eager to urinate, urgency and frequent urination.

The extract of Xanthoceras sorbifolia provided by the invention can inhibit the absorption of serotonin. The presence of 5-hydroxytryptamine maintains and enhances deep sleep in humans. The extract of Xanthoceras sinensis inhibits the absorption of 5-hydroxytryptamine, thereby breaking deep sleep, allowing people to wake up when the bladder is full and avoid bedwetting. The extract of the extract of Corundum provided by the present invention can improve the sleep alert system and help prevent the occurrence of sleep paralysis. The extract of Xanthoceras sorbifolia provided by the invention inhibits the synthesis of acetylcholine acetylase (AchE), mediates the release, absorption and decomposition of acetylcholine (Ach), thereby improving the transmission process of brain central nervous system and urinary system information, and avoiding The occurrence of enuresis. The extract of the extract of the present invention provides the growth and development of the bladder and the sphincter, increases the urine storage and reduces the number of urination, thereby avoiding the occurrence of enuresis. The extract of Xanthoceras sorbifolia provided by the invention helps to eliminate mental stress, delay aging, improve bladder function, avoid detrusor instability, hyperactivity, hyperreflexia, thereby preventing urgency and frequent urination. Therefore, the drug is more effective in treating enuresis, urgency and urinary frequency than any of the current similar drugs, and is currently the most effective drug of choice.

Xanthoceras is a plant of the genus Xanthoceras in the Sapindaceae family. The Latin name Xanthoceras sorbifolia Bunge, also known as the sacred flower, Wenguang fruit, Wenguan tree, cultural official fruit, cliff papaya and Syracuse Waiting, the English name is called "Goldenhorn, Yellowhorn". Deciduous shrubs or small trees up to 8 m high; odd-pinnate compound leaves; racemes, flowers white, heterozygous; capsules initially green, shell thick, woody, mature dark brown, ovoid, mostly native to China, It is mainly distributed in northern China, wild or cultivated. The seeds contain more than 50% oil, edible, stems and leaves can be used as medicine, rheumatism, and kernels are used in the treatment of enuresis. Seeds, flowers and leaves are edible and can be used to save the wild.

The invention discloses a crude extract of flavonoids which is a fruit husk and/or a stalk of the genus. The preparation procedure is as follows: ̇ harvesting the crown fruit, clearing the husk and/or the stalk, drying; smashing the husk of the genus and/or the stalk; extracting the husk and/or the stalk with ethanol Powder 3-4 times, get ethanol extract; ̇ recover ethanol to obtain a stream extract; ̇ dry, sterilized stream extract to obtain a yellow powdery flavonoid extract.

The flavonoid extract contains a flavanol, a flavonol, a flavanone, and the like, and other compounds such as a phenol compound, an organic acid, and the like.

The invention discloses a crude flavonoid extract of the leaves of the genus Coleoptera, and the crude flavonoid extract of the leaves of the genus Corydalis extends the water-soluble and water-insoluble flavonoids. The preparation procedure is as follows: ̇ 收 文 冠 冠 , , , , , , , , , , , , , , , , , , 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠̇ concentrating the ethanol extract; immersing the concentrated ethanol extract with hot water to obtain a water-soluble extract and a water-insoluble extract; The resulting water-soluble and water-insoluble flavonoid extract is dried and sterilized to obtain a water-soluble and water-insoluble yellow powdery flavonoid extract.

The two flavonoid extracts contain flavanols, flavonols, flavanones and the like and other compounds such as phenolic compounds, organic acids and the like.

The invention discloses a crude flavonoid extract of the stem of the crown. The preparation method comprises the following steps: picking and collecting the crown of the crown, clearing and drying; smashing the dried fruit of the crown of the crown and extracting the dried fruit of the crown; and extracting the dried fruit of the dried fruit of the crown with ethanol for 4 times to obtain an ethanol extract; ̇Recovering ethanol to obtain a stream extract; drying and sterilizing to obtain a yellow powdery flavonoid extract.

The flavonoid extract contains a flavanol, a flavonol, a flavanone, and the like, and other compounds such as a phenol compound, an organic acid, and the like.

Another disclosed in the present invention is a crude flavonoid extract of the genus Corydalis. The preparation method comprises the following steps: ̇ collecting the crown fruit, selecting the seed, drying; licking the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; drying the smashing seed cake, obtaining the seed kernel Powder; 提取 extract the seed powder with n-hexane to further remove oil; ̇ recover n-hexane; ̇ dry, grind to get the oil seed cream; ̇ extract the seed cream with ethanol 3-4 times; ̇ Recover ethanol to obtain a liquid extract; ̇ dry, sterilize the flow extract to obtain a yellow powdery flavonoid extract.

The flavonoid extract contains a flavanol, a flavonol, a flavanone, and the like, and other compounds such as a phenol compound, an organic acid, and the like.

Another disclosed in the present invention is a crude extract of flavonoids from the root of the crown. The preparation method comprises the following steps: ̇ harvesting the crown of the crown, clearing and drying; smashing the root of the crown of the genus canopy root powder; ̇ extracting the crown root powder with ethanol for 3-4 times to obtain ethanol Leaching solution; ̇Recovering ethanol to obtain a stream extract; ̇drying, sterilizing the stream extract to obtain a yellow powdery flavonoid extract.

The flavonoid extract contains a flavanol, a flavonol, a flavanone, and the like, and other compounds such as a phenol compound, an organic acid, and the like.

Another disclosed in the present invention is a crude flavonoid extract of the crown bark. The preparation method comprises the following steps: ̇ harvesting the crown bark, clearing and drying; ̇ crushing the crown tree bark to obtain the crown fruit bark powder; ̇ extracting the crown tree bark powder with ethanol for 3-4 times to obtain ethanol Leaching solution; ̇Recovering ethanol to obtain a stream extract; ̇drying, sterilizing the stream extract to obtain a yellow powdery flavonoid extract.

The flavonoid extract contains a flavanol, a flavonol, a flavanone, and the like, and other compounds such as a phenol compound, an organic acid, and the like.

The invention discloses a crude flavonoid extract of the genus Corolla. The method of preparation is as follows: 种 收 收 的 的 的 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 文 文 文 文 文 文 文 文 文 文 文 文 文 文Extracting liquid; ̇ recovering ethanol to obtain a liquid extract; ̇ drying, sterilizing the flow extract to obtain a yellow powdery flavonoid extract.

The flavonoid extract contains a flavanol, a flavonol, a flavanone, and the like, and other compounds such as a phenol compound, an organic acid, and the like.

The invention discloses an organic flux extract such as an alcohol of a fruit shell and/or a stalk of a scutellaria, mainly containing saponins and other substances, and the specific content is as follows:

The production is carried out according to the following steps: ̇ harvesting the crown fruit, clearing the husk and/or the stalk, drying; smashing the fruit husk and/or the stalk of the sorghum; extracting the sacred fruit from an organic solvent such as sterols Shell and/or fruit stalk powder; ̇ recovering solvent, concentrated to obtain a liquid extract; ̇ drying, sterilizing to obtain an organic solvent extract such as alcohol; and an organic solvent extract such as an alcohol of leaves of the leaves of the genus It mainly contains saponins and other substances. The procedure of the preparation method is as follows: ̇ ̇ 文 冠 冠 , , , , , , , , , , , , , , , , , , , , 冠 冠 冠 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文, the solvent extract; ̇Recovering the organic solvent to obtain a flow extract; ̇ extracting the flow extract with ethanol and water to obtain a secondary flow extract; immersing the secondary flow extract with n-butanol to obtain an n-butanol extract; The n-butanol was recovered to obtain a three-stream extract; the mash was dried and sterilized to obtain three leaching leaching to obtain a leaf saponin extract.

The leaf saponin extract mainly contains saponins and other compounds such as sugar, crude protein and the like.

The invention discloses an organic solvent extract such as an alcohol of the crown of the crown, which mainly contains saponins and other substances.

The procedure of the preparation method is as follows: ̇ harvesting the crown of the crown, clearing, drying; smashing the dried fruit of the crown, obtaining the dry powder; 浸 extracting the dried fruit of the crown with an organic solvent (ethanol, methanol, etc.) to obtain an organic solvent Leaching solution; ̇Recovering organic solvent to obtain a flow extract; ̇drying, sterilizing the flow extract to obtain a saponin extract.

The saponin extract mainly contains saponins and other compounds such as sugars, crude proteins and the like.

The invention discloses an organic solvent extract such as an alcohol of the genus Corydalis, which mainly contains saponins and other substances. The preparation process procedure is as follows: ̇ harvesting the crown fruit, clearing the seed, drying; licking the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ drying, crushing the seed cake to obtain the seed powder; ̇ Extract the seed powder with n-hexane to further remove the oil; ̇ recover n-hexane; ̇ dry, grind, get the oil seed cream; 浸 extract the seed cream with organic solvent (ethanol, methanol, etc.), get organic solvent dip Extract liquid; ̇Recover organic solvent to obtain extract; ̇ dry, sterilize flow extract to obtain saponin extract.

The saponin extract mainly contains saponins and other compounds such as sugars, crude proteins and the like.

Another organic solvent extract such as an alcohol of the crown of the present invention mainly contains saponins and other substances. The preparation process procedure is as follows: ̇ harvesting the crown root, clearing, drying; ̇ crushing the crown of the fruit root, the root powder; ̇ extracting the crown root powder with an organic solvent (ethanol, methanol, etc.), organic Solvent extract; ̇Recover organic solvent to obtain a flow extract; ̇ dry, sterilize the flow extract to obtain a saponin extract.

The saponin extract mainly contains saponins and other compounds such as sugars, crude proteins and the like.

Another organic solvent extract such as an alcohol of the crown bark of the present invention mainly contains saponins and other substances. The procedure of the preparation method is as follows: ̇ Harvesting the crown bark, clearing and drying; ̇ crushing the crown bark of the crown, obtaining the bark powder; 浸Immerse the crown bark powder with an organic solvent (ethanol, methanol, etc.) to obtain an organic solvent extract; ̇ recover the organic solvent to obtain a stream extract; ̇ dry, sterilize the stream extract to obtain a saponin extract.

The saponin extract mainly contains saponins and other compounds such as sugars, crude proteins and the like.

Another organic solvent extract, such as an alcohol of the genus Corolla, is mainly composed of saponins and other substances. The procedure of the preparation method is as follows: ̇ 收 冠 冠 冠 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 冠 , , , 冠 冠 冠 , 冠 冠 冠 , , Powder, organic solvent extract; ̇ recovery of organic solvent to obtain a stream extract; ̇ dry, sterilized stream extract, to obtain saponin extract.

The saponin extract mainly contains saponins and other compounds such as sugars, crude proteins and the like.

The crude saponin extract of the husk (or stalk) of the crown of the present invention is prepared as follows: a) harvesting the crown fruit, clearing the husk, drying; b) smashing the crown fruit Shell; c) alcohol and other organic solvent extraction of the crown fruit shell powder, according to the ratio of organic solvent: shell powder 2:1, at room temperature, leaching 4-5 times, each time 20-35 hours; d) collecting the extract, and then refluxing (80 ° C) for 2-3 times; e) collecting the combined extract; f) removing the solvent to obtain a flow extract; g) drying and sterilizing to obtain an ethanol extract; h) extracting the extract and then dissolving in water, n-butanol extraction; i) n-butanol extract The crude saponin is obtained by chromatography.

The crude saponin extract mainly contains saponin.

The invention discloses a crude saponin extract which is a leaf of the genus Coleoptera, and mainly contains crude saponin. The procedure of the preparation method is as follows: ̇ harvesting the crown of the fruit, clearing, drying; ̇ ̇ 文 文 文 文 文 得 文 文 文 文 文 ; ; 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文2:1) 4-5 times, each time 20-35 hours, get the solvent extract; ̇ collect the extract, then reflux and heat (80 ° C) 2-3 times; ̇ collect the combined extract; ̇ recovery The organic solvent is obtained as a liquid extract; the hydrazine is dissolved in the water to obtain an aqueous extract; the immersion solution is extracted with n-butanol to obtain an n-butanol extract; and the n-butanol leaching is separated by chromatography. The extract gives a crude saponin extract.

The crude saponin extract mainly contains saponin.

The invention discloses a crude saponin extract which is a dried saponin of the crown of the genus. The procedure of the preparation method is as follows: ̇ 收 文 冠 冠 冠 , , , , , , , , , 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 文 文 文 文 文 文:1) 4-5 times, each time 20-35 hours, get the solvent extract; ̇ collect the extract, then reflow hot (80 ° C) 2-3 times; ̇ collect the combined extract; ̇ recover the organic solvent flow The extract is dissolved in water to obtain an aqueous extract; the aqueous extract is extracted with n-butanol to obtain an n-butanol extract; and the n-butanol extract is separated by chromatography. Crude saponin extract.

The crude saponin extract mainly contains saponin.

The invention discloses a crude saponin extract which is mainly a crude saponin. The preparation process procedure is as follows: ̇ harvesting the crown fruit, clearing the seed, drying; licking the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ drying, crushing the seed cake to obtain the seed powder; ̇ Extract the seed powder with n-hexane to further remove the oil; ̇ recover n-hexane; ̇ dry, grind to remove the oil seed cream; 浸 extract the seed cream with organic solvent (ethanol, methanol, etc.), (2:1) 4-5 times, each time 20-35 hours, get the organic solvent extract; ̇ collect the extract, then reflux and heat (80 ° C) 2-3 times; ̇ collect the combined extract; ̇ recover the organic solvent flow The extract is immersed in the water to obtain a water leaching solution; the hydrazine extract is extracted with n-butanol to obtain an n-butanol extract; The n-butanol extract was separated by chromatography to obtain a crude saponin extract.

The crude saponin extract mainly contains saponin.

Another disclosed in the present invention is a crude saponin extract of the root of the crown, which mainly contains saponins and other substances. The preparation process procedure is as follows: ̇ 收 文 冠 冠 , , , , , , , , , , , , , 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠(2:1) 4-5 times, each time 20-35 hours, get the solvent extract; ̇ collect the extract, then reflow hot (80 ° C) 2-3 times; ̇ collect the combined extract; ̇ Recovering the organic solvent to obtain a flow extract; ̇ dissolving the flow extract in water to obtain an aqueous extract; immersing the aqueous extract with n-butanol to obtain n-butanol extract; and separating n-butanol by chromatography The extract is obtained as a crude saponin extract.

The crude saponin extract mainly contains saponin.

Another disclosed in the present invention is a crude saponin extract of the crown bark of the crown, which mainly contains saponins and other substances. The procedure of the preparation method is as follows: ̇ Harvesting the crown bark, clearing and drying; ̇ crushing the crown bark of the crown, obtaining the crown bark powder; 浸 extracting the crown bark powder with an organic solvent (ethanol, methanol, etc.) (2:1) 4-5 times, each time 20-35 hours, get the solvent extract; ̇ collect the extract, then reflow hot (80 ° C) 2-3 times; ̇ collect the combined extract; ̇ Recycling organic solvent to obtain a flow extract; ̇ Dissolve the flow extract in water to obtain an aqueous extract; 浸 extract the aqueous extract with n-butanol to obtain n-butanol extract; 分离 separate the n-butanol extract by chromatography to obtain crude saponin extract Object.

The crude saponin extract mainly contains saponin.

Another disclosed in the present invention is a crude saponin extract of the genus Corolla, which mainly contains saponins and other substances. The preparation process procedure is as follows: ̇ 收 文 冠 冠 冠 , , , , , , , , , , , , , , , , , , , , , , , , , 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠Fruit shell powder (2:1) 4-5 times, each time 20-35 hours, get the solvent extract; ̇ collect the extract, then reflow hot (80 ° C) 2-3 times; Extracting liquid; ̇recovering organic solvent to obtain a liquid extract; ̇ dissolving the flow extract in water to obtain an aqueous extract; ̇ extracting the aqueous extract with n-butanol to obtain an n-butanol extract; The n-butanol extract was separated to obtain a crude saponin extract.

The crude saponin extract mainly contains saponin.

A coumarin extract of the genus Corolla and/or the stalk of the present invention is disclosed. The preparation method comprises the following steps: ̇ harvesting the crown fruit, clearing the husk and/or the stalk, drying; smashing the husk and/or the stalk to obtain the husk and/or the stalk powder; Extracting the husk and/or the stalk powder with NaOH solution to obtain the water extract; immersing the husk and/or the stalk powder water extract with diethyl ether to obtain ether leaching The extract is neutralized with HCl, and the extract is concentrated, and the ether extract is acidified to obtain a coumarin extract.

The coumarin extract mainly contains coumarin, coumarin glycoside and other compounds such as sugar, crude protein and the like.

One of the coumarin extracts disclosed in the present invention is a coumarin extract. The preparation method comprises the following steps: ̇ harvesting the crown of the fruit, clearing, drying; smashing the fruit of the fruit of the genus, and extracting the fruit of the genus of the genus; and extracting the genus of the genus of the genus with 0.5% NaOH solution, Water extract; ̇ extract the water extract of the leaves of the genus Phyllanthus with diethyl ether to obtain an ether extract; 中 neutralize the ether extract with HCl; concentrate the hydrazine, acidify the ether extract to obtain the coumarin extract .

The coumarin extract mainly contains coumarin, coumarin glycoside and other compounds such as sugar, crude protein and the like.

A coumarin extract which is a dried fruit of the crown of the present invention. The preparation method comprises the following steps: picking and collecting the crown of the crown, clearing and drying; smashing the dried fruit of the crown of the fruit, obtaining the dried fruit of the crown of the fruit; licking the dried fruit of the crown with 0.5% NaOH solution to obtain the water immersion The extract was extracted with diethyl ether and extracted with water to obtain an ether extract; the extract was neutralized with HCl, and the extract was concentrated to acidify the ether extract to obtain a coumarin extract.

The coumarin extract mainly contains coumarin, coumarin glycoside and others Compounds such as sugars, crude proteins, and the like.

The invention discloses a coumarin extract which is a seed cultivar. The preparation method comprises the following steps: ̇ collecting the crown fruit, selecting the seed, drying; ̇ peeling the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ drying, smashing the seed cake to obtain the seed powder ̇ extract the seed powder with n-hexane to further remove oil; ̇ recover n-hexane; ̇ dry, grind to obtain oil seed powder; 浸 extract 0.5 cm NaOH solution to extract the fruit seed powder, obtain water extract The extract was extracted with diethyl ether and extracted with diethyl ether extract to obtain an ether extract; the extract was neutralized with HCl, and the extract was concentrated to acidify the ether extract to obtain a coumarin extract.

The coumarin extract mainly contains coumarin, coumarin glycoside and other compounds such as sugar, crude protein and the like.

One of the coumarin extracts disclosed in the present invention is the coumarin extract. The preparation method comprises the following steps: ̇ harvesting the crown root, clearing and drying; smashing the root of the crown fruit, obtaining the root of the crown; ̇ extracting the crown root powder with 0.5% NaOH solution, Water extract; 浸 extract the water extract of the root of the crown of the genus with diethyl ether to obtain an ether extract; 中 neutralize the ether extract with HCl; The hydrazine is concentrated, and the ether extract is acidified to obtain a coumarin extract.

The coumarin extract mainly contains coumarin, coumarin glycoside and other compounds such as sugar, crude protein and the like.

A coumarin extract of the crown bark of the present invention is disclosed. The preparation method comprises the following steps: ̇ harvesting the crown bark, clearing and drying; smashing the fruit of the crown fruit fruit bark, obtaining the crown tree bark powder; immersing the crown tree bark powder with 0.5% NaOH solution, Water extract; ̇ extract the water extract of the crown bark with diethyl ether to obtain an ether extract; 中 neutralize the ether extract with HCl; concentrate and acidify the ether extract to obtain the coumarin extract .

The coumarin extract mainly contains coumarin, coumarin glycoside and other compounds such as sugar, crude protein and the like.

The invention discloses a coumarin extract which is a crustacean seed shell. The preparation method comprises the following steps: ̇ harvesting the crown and seed shell, clearing and drying; smashing the stalk of the fruit of the genus, and obtaining the cultivar powder; ̇ extracting the genus of the genus with 0.5% NaOH solution Powder, obtain water extract; ̇ extract the water extract of C. elegans with diethyl ether to obtain ether extract; 中 neutralize ether extract with HCl; concentrate ,, acidify ether extract, obtain fragrant Soy extract.

The coumarin extract mainly contains coumarin, coumarin glycoside and other compounds such as sugar, crude protein and the like.

The invention discloses a sugar which is a fruit husk and/or a stalk of the genus Take things. The preparation method comprises the following steps: ̇ harvesting the crown fruit, clearing the husk and/or the stalk, drying; smashing the husk and/or the stalk to obtain the husk and/or the stalk powder; The husk and/or the stalk powder is extracted under temperature for 24 hours to obtain a water extract; the hydrazine is further extracted at a temperature of 60-70 ° C for 1-2 hours to obtain a secondary water extract; The second water extract is obtained by extracting the extract.

The flow extract mainly contains polysaccharides, glycosides, other sugars, and other compounds such as saponins and tannins.

One disclosed in the present invention is a sugar extract of the leaves of the genus. The preparation method comprises the following steps: ̇ harvesting the crown of the fruit, clearing and drying; smashing the leaves of the genus of the genus, and extracting the leaves of the genus of the genus; the cockroach is immersed in the water for about 24 hours at room temperature, The water extract can be obtained by hot extraction at a temperature of 60-70 ° C for 1-2 hours to obtain a secondary water extract;

The flow extract mainly contains polysaccharide glycosides, other sugars, and other compounds such as saponins and tannins.

The invention discloses a sugar extract which is a dried branch of the crown. The preparation method comprises the following steps: ̇ harvesting the crown of the crown, clearing, drying; ̇ 文 文 文 文 文 , , 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文The second water extract is obtained; the ruthenium is filtered, and the second water extract is concentrated to obtain a flow extract.

The flow extract mainly contains polysaccharides, glycosides, other sugars and saponins, and other compounds such as tannins.

The invention discloses a sugar extract which is a seed of the genus Corolla. The preparation method comprises the following steps: ̇ collecting the crown fruit, selecting the seed, drying; ̇ peeling the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ drying, smashing the seed cake to obtain the seed powder ̇ extract the seed powder with n-hexane to further remove oil; ̇ recover n-hexane; ̇ dry, grind to remove oil seed powder; ̇ water to digest the crown fruit seed powder at room temperature for 24 hours, get water immersion The extract is further hot-extracted at a temperature of 60-70 ° C for 1-2 hours to obtain a secondary water extract; the mixture is filtered, and the secondary water extract is concentrated to obtain a flow extract.

The flow extract mainly contains polysaccharides, glycosides, other sugars, and other compounds such as saponins and tannins.

The invention discloses a sugar extract which is a root of the crown. The preparation method comprises the following steps: ̇ harvesting the crown of the crown, clearing and drying; smashing the leaves of the genus of the genus, and obtaining the root powder of the genus; the cockroach root is immersed in the water for 24 hours at room temperature, The water extract can be obtained by hot extraction at a temperature of 60-70 ° C for 1-2 hours to obtain a secondary water extract;

The flow extract mainly contains polysaccharides, glycosides, other sugars, and other compounds such as saponins and tannins.

A saccharide extract of the crown bark of the present invention is disclosed. The preparation method comprises the following steps: ̇ harvesting the crown bark, clearing and drying; smashing the crown bark of the crown, obtaining the crown bark powder; immersing the crown bark powder at room temperature for 24 hours with water, The water extract can be obtained by hot extraction at a temperature of 60-70 ° C for 1-2 hours to obtain a secondary water extract;

The flow extract mainly contains polysaccharides, glycosides, other sugars, and other compounds such as saponins and tannins.

The invention discloses a sugar extract which is a seed coat of the genus Corolla. The preparation method comprises the following steps: ̇ harvesting the crown and fruit seed shell, clearing and drying; ̇ ̇ 文 文 文 文 , , , 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文After 1-2 hours, a second water extract is obtained; ̇ is filtered, and the secondary water extract is concentrated to obtain a flow extract.

The extract preferably contains polysaccharides, glycosides, other sugars, and other compounds such as saponins and tannins.

An alkaloid extract of the genus Corolla and/or the stalk of the present invention is disclosed. The preparation method comprises the following steps: cultivating the crown fruit, clearing the husk and/or the stalk, drying; smashing the husk and/or the stalk to obtain the husk and/or the stalk powder; Fruit shell and / or fruit stalk powder (6:1) 3-4 times, each time 10-15 hours, get water extract; ̇ collect combined water extract; 碱 alkalized combined water extract with NaOH To obtain a pH of 10-12, to obtain an alkalized extract; to extract the alkalized extract with toluene to obtain a toluene extract; and to pass the toluene extract to a 2% dicarboxylic acid having a pH of 5-7 The dicarboxylic acid extract is obtained; the dicarboxylic acid extract is concentrated under reduced pressure to obtain a crude alkaloid extract.

The crude alkaloid extract mainly contains alkaloids, coumarins, sugars and other compounds such as crude protein.

One disclosed in the present invention is an alkaloid extract of the leaves of the crown. preparation The method is as follows: ̇ 收 文 冠 冠 , , , , , , , , , , , , , , , , 冠 冠 冠 冠 冠 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文4 times, each time 10-15 hours, to obtain water extract; ̇ collecting combined water extract; 碱 alkalizing the combined water extract with NaOH to make the pH reach 10-12, to obtain alkalized extract; The alkalized extract is extracted with toluene to obtain a toluene extract; the toluene extract is passed through a 2% dicarboxylic acid having a pH of 5-7 to obtain a dicarboxylic acid extract; The acid extract is extracted to obtain a crude alkaloid extract.

The crude alkaloid extract mainly contains alkaloids, coumarins, sugars and other compounds such as crude protein.

The invention discloses an alkaloid extract which is a dried branch of the crown. The preparation method comprises the following steps: picking and collecting the crown of the crown, clearing and drying; smashing the dried fruit of the crown, obtaining the dried fruit of the crown of the fruit; immersing the dried fruit of the crown with water (6:1) 3-4 times , each time 10-15 hours, the water extract can be obtained; ̇ collecting the combined water extract; 碱 alkalizing the combined water extract with NaOH to make the pH reach 10-12, to obtain the alkalized extract; Extracting the alkalized extract with toluene to obtain a toluene extract; The toluene extract is passed through a 2% dicarboxylic acid having a pH of 5-7 to obtain a dicarboxylic acid extract; the dicarboxylic acid extract is concentrated under reduced pressure to obtain a crude alkaloid extract.

The crude alkaloid extract mainly contains alkaloids, coumarins, sugars and other compounds such as crude protein.

The invention discloses an alkaloid extract which is a seed of the genus Corolla. The preparation method comprises the following steps: ̇ collecting the crown fruit, selecting the seed, drying; ̇ peeling the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ drying, smashing the seed cake to obtain the seed powder ̇ Extract the seed powder with n-hexane to further remove the oil; ̇ recover n-hexane; ̇ dry, grind, get the oil seed powder; ̇ water extracting the fruit seed powder (6:1) 3-4 times , each time 10-15 hours, the water extract can be obtained; ̇ collecting the combined water extract; 碱 alkalizing the combined water extract with NaOH to make the pH reach 10-12, to obtain the alkalized extract; The alkalized extract is extracted with toluene to obtain a toluene extract; the toluene extract is passed through a 2% dicarboxylic acid having a pH of 5-7 to obtain a dicarboxylic acid extract; The liquid is extracted to obtain a crude alkaloid extract.

The crude alkaloid extract mainly contains alkaloids, coumarin, sugar and its His compounds are such as crude protein.

One of the disclosed inventions is an extract of the root of astronomical alkaloids. The preparation method comprises the following steps: ̇ harvesting the crown root, clearing and drying; ̇ crushing the crown of the fruit of the crown, obtaining the root of the crown; ̇ extracting the root of the crown of the genus with water (6:1) 3 -4 times, 10-15 hours each time, obtaining water extract; ̇ collecting combined water extract; 碱 alkalizing the combined water extract with NaOH to bring the pH to 10-12, to obtain alkalized extract The alkalized extract is extracted with toluene to obtain a toluene extract; the toluene extract is passed through a 2% dicarboxylic acid having a pH of 5-7 to obtain a dicarboxylic acid extract; A carboxylic acid extract is obtained to obtain a crude alkaloid extract.

The crude alkaloid extract mainly contains alkaloids, coumarins, sugars and other compounds such as crude protein.

An alkaloid extract of the crown bark of the present invention is disclosed. The preparation method comprises the following steps: ̇ harvesting the crown bark, clearing and drying; smashing the crown fruit bark, obtaining the crown fruit bark powder; ̇ extracting the crown fruit bark powder with water (6:1) 3 -4 times, each time 10-15 hours, the water extract is obtained; the cockroach collects the combined water extract; 碱 alkalized the combined water extract with NaOH to make the pH reach 10-12, The alkalized extract; the alkalized extract is extracted with toluene to obtain a toluene extract; the toluene extract is passed through a 2% dicarboxylic acid having a pH of 5-7 to obtain a dicarboxylic acid extract; The dicarboxylic acid extract was concentrated under reduced pressure to obtain a crude alkaloid extract.

The crude alkaloid extract mainly contains alkaloids, coumarins, sugars and other compounds such as crude protein.

The invention discloses an alkaloid extract which is a seed coat of the genus Corolla. The preparation method comprises the following steps: ̇ harvesting the crown and seed shell, clearing and drying; smashing the cultivar of the fruit of the genus, and extracting the shell seed powder; ̇ extracting the genus of the genus of the genus of the genus (6) :1) 3-4 times, each time 10-15 hours, obtain water extract; ̇ collect and combine the water extract; 碱 alkalized the combined water extract with NaOH to make the pH reach 10-12, alkali Extracting the leaching extract with toluene to obtain a toluene extract; ̇ passing the toluene extract through a 2% dicarboxylic acid having a pH of 5-7 to obtain a dicarboxylic acid extract; The dicarboxylic acid extract was concentrated under reduced pressure to obtain a crude alkaloid extract.

The crude alkaloid extract mainly contains alkaloids, coumarins, sugars and other compounds such as crude protein.

An organic acid extract of the present invention is a fruit husk and/or a stalk. The preparation method comprises the following steps: ̇ harvesting the crown fruit, clearing the husk and/or the stalk, drying; smashing the husk and/or the stalk to obtain the husk and/or the stalk powder; The hydrochloric acid is soaked in the husk and/or the stalk powder to obtain an acid leaching solution; the leaching solution is extracted with an organic solvent (ether or benzene) to obtain an organic solvent extract; ̇ 5-10% HaHCO ₃ immersion The organic solvent extract is extracted to obtain HaHCO ₃ extract; the HaHCO ₃ extract is extracted, filtered, precipitated, and the precipitate is extracted with an organic solvent to obtain a secondary organic solvent; the solvent of the recovery machine is used to obtain crude organic acid extraction. Things.

The crude organic acid extract mainly contains aromatic organic acids, organic fatty acids, terpenoid organic acids and other compounds such as crude protein.

One of the disclosed inventions is an organic acid extract of the leaves of the genus. The preparation method comprises the following steps: ̇ harvesting the crown of the fruit, clearing and drying; smashing the leaves of the genus of the genus, and extracting the leaves of the genus, and extracting the powder of the genus of the genus with 10% hydrochloric acid to obtain acid The extract is extracted with an organic solvent (diethyl ether or benzene) to obtain an organic solvent extract; the organic solvent extract is extracted with 5-10% HaHCO ₃ to obtain a HaHCO ₃ extract; The HaHCO ₃ extract is acidified, filtered, precipitated, and the precipitate is extracted with an organic solvent to obtain a secondary organic solvent; the solvent is recovered in a recovery solvent to obtain a crude organic acid extract.

The crude organic acid extract mainly contains aromatic organic acids, organic fatty acids, terpenoid organic acids and other compounds such as crude protein.

The invention discloses an organic acid extract which is a dried branch of the crown. The preparation method comprises the following steps: picking and collecting the crown of the crown, clearing and drying; smashing the dried fruit of the crown, obtaining the dried fruit of the crown, and extracting the dried fruit of the dried fruit of the crown with 10% hydrochloric acid, obtaining acid pickling The extract is extracted with an organic solvent (diethyl ether or benzene) to obtain an organic solvent extract; the organic solvent extract is extracted with 5-10% HaHCO ₃ to obtain a HaHCO ₃ extract; The HaHCO ₃ extract is filtered, precipitated, and the precipitate is extracted with an organic solvent to obtain a secondary organic solvent; the solvent of the recovery machine is used to obtain a crude organic acid extract.

The crude organic acid extract mainly contains aromatic organic acids, organic fatty acids, terpenoid organic acids and other compounds such as crude protein.

The invention discloses an organic acid extract which is a seed of the genus Corolla. The preparation method comprises the following steps: ̇ collecting the crown fruit, selecting the seed, drying; ̇ peeling the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ drying, smashing the seed cake to obtain the seed powder ̇ extract the seed powder with n-hexane to further remove the oil; ̇ recover n-hexane; ̇ dry, grind to remove the oil seed powder; 浸 leaching the extract of the fruit of the fruit with 10% hydrochloric acid to obtain the acid extract The organic acid extract is extracted with an organic solvent (diethyl ether or benzene) to obtain an organic solvent extract; the organic solvent extract is extracted with 5-10% HaHCO ₃ to obtain a HaHCO ₃ extract; the tannic acid HaHCO ₃ extract, filter, precipitate, and then extract the precipitate with an organic solvent to obtain a secondary organic solvent; ̇ recover the solvent of the machine to obtain a crude organic acid extract.

The crude organic acid extract mainly contains aromatic organic acids, organic fatty acids, terpenoid organic acids and other compounds such as crude protein.

The invention discloses an organic acid extract which is a root of the crown. The preparation method comprises the following steps: ̇ harvesting the crown of the crown, clearing and drying; smashing the root of the crown, obtaining the root powder of the crown; ̇ extracting the root powder of the crown with 10% hydrochloric acid, obtaining acid The extract is extracted with an organic solvent (diethyl ether or benzene) to obtain an organic solvent extract; the organic solvent extract is extracted with 5-10% HaHCO ₃ to obtain a HaHCO ₃ extract; The HaHCO ₃ extract is acidified, filtered, precipitated, and the precipitate is extracted with an organic solvent to obtain a secondary organic solvent; the solvent is recovered in a recovery solvent to obtain a crude organic acid extract.

The crude organic acid extract mainly contains aromatic organic acid, organic fat Acids, terpenoid organic acids and other compounds such as crude protein.

An organic acid extract of the crown bark of the present invention is disclosed. The preparation method comprises the following steps: ̇ harvesting the crown bark, clearing and drying; smashing the crown bark of the crown, obtaining the crown bark powder; immersing the crown bark powder with 10% hydrochloric acid to obtain acid The extract is extracted with an organic solvent (diethyl ether or benzene) to obtain an organic solvent extract; the organic solvent extract is extracted with 5-10% HaHCO ₃ to obtain a HaHCO ₃ extract; The HaHCO ₃ extract is acidified, filtered, precipitated, and the precipitate is extracted with an organic solvent to obtain a secondary organic solvent; the solvent is recovered in a recovery solvent to obtain a crude organic acid extract.

The crude organic acid extract mainly contains aromatic organic acids, organic fatty acids, terpenoid organic acids and other compounds such as crude protein.

The invention discloses an aromatic organic acid extract which is a seed coat of the genus Corolla. The preparation method comprises the following steps: ̇ harvesting the crown and seed shell, clearing and drying; smashing the cultivar of the genus of the genus, obtaining the husk of the genus of the genus; cultivating the cultivar with the 10% hydrochloric acid The powder is obtained by extracting the acid extract; the acid extract is extracted with an organic solvent (diethyl ether or benzene) to obtain an organic solvent extract; and the organic solvent extract is extracted with 5-10% HaHCO ₃ to obtain HaHCO ₃ Extracting; leaching HaHCO ₃ extract, filtering, precipitating, and extracting the precipitate with an organic solvent to obtain a secondary organic solvent; ̇ recovering the solvent to obtain a crude organic acid extract.

The crude organic acid extract mainly contains aromatic organic acids, organic fatty acids, terpenoid organic acids and other compounds such as crude protein.

The invention discloses a tannin extract which is a fruit husk and/or a fruit stalk of the genus Corolla, and has two preparation methods.

The method of the first method is as follows: ̇ harvesting the crown fruit, clearing the husk and/or the stalk, drying; smashing the husk and/or the stalk to obtain the husk and or the stalk powder; Extracting the husk and/or the stalk powder with ethanol to obtain an ethanol extract; concentrating the ethanol extract under reduced pressure to obtain a tannin extract;

The second method is as follows: ̇ harvesting the crown fruit, clearing the husk and or the stalk, drying; smashing the husk and/or the stalk to obtain the husk and or the stalk powder; 1:1) extracting the husk and/or stalk powder for 2-7 days to obtain an aqueous extract of acetone; ̇ recovering acetone at 50 ° C to obtain a concentrated extract; ̇ filtering the concentrated extract; ̇ The filtered extract was extracted with diethyl ether, and an aqueous extract of diethyl ether was recovered; the aqueous extract was extracted with ethyl acetate; and ethyl acetate was recovered to obtain a tannin extract.

The tannin extract mainly contains tannins and other compounds such as organic acids, sugars, crude proteins and the like.

The invention discloses a tannin extract which is a crown of the fruit of the genus, and has two preparation methods.

The method of the first method is as follows: ̇ ̇ 文 冠 冠 , , , , , , , , , , , , , , , , 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠The extract is concentrated; the ethanol extract is concentrated under reduced pressure to obtain a tannin extract;

The method of the second method is as follows: ̇ ̇ 文 冠 冠 , , , , , , , , , , , , , , , , , , , 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠 冠Leaf powder for 2-7 days, obtaining aqueous extract of acetone; 丙酮 recovering acetone at 50 ° C to obtain concentrated extract; ̇ filtering concentrated concentrated extract; extracting filtered extract with diethyl ether, recovering ether An aqueous solution extract; the aqueous extract is extracted with ethyl acetate; the ethyl acetate is recovered to obtain a tannin extract.

The tannin extract mainly contains tannins and other compounds such as organic acids, sugars, crude proteins and the like.

The invention discloses a kind of extract of the dried fruit tannin of the crown of the genus, and has two preparation methods.

The method of the first method is as follows: ̇ harvesting the crown of the crown, clearing, drying; smashing the dried fruit of the crown, obtaining the dried fruit of the crown of the fruit; ̇ extracting the dried fruit of the crown with 95% ethanol, and extracting with ethanol Liquid; 乙醇 concentrated ethanol extract under reduced pressure to obtain tannin extract.

The second method of the method is as follows: ̇ picking the crown of the crown, clearing, drying; smashing the dried fruit of the crown, obtaining the dried fruit of the crown; ̇ extracting the dried fruit of the crown with an aqueous solution of acetone (1:1) 2 -7 days, an aqueous extract of acetone is obtained; the acetone is recovered at 50 ° C to obtain a concentrated extract; the concentrated extract is filtered by hydrazine; the filtered extract is extracted with diethyl ether, and the aqueous solution is extracted with diethyl ether. Extracting the aqueous extract with ethyl acetate; extracting ethyl acetate to obtain a tannin extract.

The tannin extract mainly contains tannins and other compounds such as organic acids, sugars, crude proteins and the like.

Another type disclosed in the present invention is a candidum seed tannin extract, which has two preparation methods.

The method of the first method is as follows: ̇ collecting the crown fruit, selecting the seed, drying; licking the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ dry, crush the seed cake to obtain seed powder; ̇ extract the seed powder with n-hexane to further remove oil; ̇ recover n-hexane; ̇ dry, grind to remove oil seed powder; ̇ use 95% ethanol extract The crown fruit seed powder is obtained from an ethanol extract; the ethanol extract is concentrated under reduced pressure to obtain a tannin extract.

The second method of the method is as follows: ̇ harvesting the crown fruit, clearing the seed, drying; licking the shell to obtain the seed kernel; rolling the seed kernel to the oil to obtain the seed cake; ̇ drying, smashing the seed cake to obtain the seed powder ̇ Extracting the seed powder with n-hexane to further remove oil; ̇ recovering n-hexane; ̇ drying, grinding to obtain oil seed powder; ̇ extracting wengan fruit seed powder with aqueous solution of acetone (1:1) 2- 7 days, an aqueous extract of acetone was obtained; 丙酮 was recovered at 50 ° C to obtain a concentrated extract; 浓缩 filtered concentrated extract; 过滤 extracted with diethyl ether to extract the extract, and the ether was extracted to obtain an aqueous extract The aqueous extract was extracted with ethyl acetate; ethyl acetate was recovered to obtain a tannin extract.

The tannin extract mainly contains tannins and other compounds such as organic acids, sugars, crude proteins and the like.

Another disclosed in the present invention is a canopy root tannin extract, which has two preparation methods.

The method of the first method is as follows: ̇ picking the root of the crown, clearing and drying; smashing the root of the crown, obtaining the root powder of the crown; ̇ extracting the root of the crown with 95% ethanol to obtain ethanol The extract is concentrated; the ethanol extract is concentrated under reduced pressure to obtain a tannin extract.

The method of the second method is as follows: ̇ harvesting the crown of the crown, clearing and drying; smashing the root of the crown, obtaining the root powder of the crown; immersing the root of the crown with an aqueous solution of acetone (1:1) Powder 2-7 days, obtaining aqueous extract of acetone; 丙酮 recovering acetone at 50 ° C to obtain concentrated extract; ̇ filtering concentrated concentrated extract; extracting filtered extract with diethyl ether, recovering ether to obtain aqueous solution The extract was extracted with an aqueous solution of ethyl acetate; the ethyl acetate was recovered to obtain a tannin extract.

The tannin extract mainly contains tannins and other compounds such as organic acids, sugars, crude proteins and the like.

The invention discloses a kind of extract of the root of the crown of the fruit of the corolla, and the preparation method has two.

The method of the first method is as follows: ̇ Harvesting the crown bark, clearing and drying; ̇Crushing the crown bark of the crown, obtaining the crown bark powder; 浸 extracting the crown bark powder with 95% ethanol to obtain the ethanol extract; concentrating the ethanol extract under reduced pressure to obtain the tannin extract.

The method of the second method is as follows: ̇ Harvesting the crown bark of the crown, clearing and drying; smashing the bark of the crown fruit, obtaining the bark powder of the crown fruit; immersing the bark of the crown fruit with an aqueous solution of acetone (1:1) Powder 2-7 days, obtained aqueous solution of acetone; ̇ recover acetone at 50 ° C to obtain concentrated extract; ̇ filter concentrated concentrated extract; 萃取 extract the filtered extract with ether, recover ether to obtain aqueous solution Extract; extract the aqueous extract with ethyl acetate; extract ethyl acetate to obtain tannin extract.

The tannin extract mainly contains tannins and other compounds such as organic acids, sugars, crude proteins and the like.

The invention discloses a kind of tannin extract of the genus Corolla, and there are two preparation methods.

The method of the first method is as follows: ̇ harvesting the crown and seed shells, clearing and drying; ̇ extracting the crown fruit seed shell powder with 95% ethanol to obtain an ethanol extract; , get tannin extract.

The second method of the method is as follows: ̇ Harvesting the crown and seed shell, clearing and drying; ̇ ̇ 文 文 文 , , , , 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文 文The acetone is recovered at °C to obtain a concentrated extract; the concentrated extract is filtered by hydrazine; the filtered extract is extracted with diethyl ether, and the aqueous extract of diethyl ether is recovered; the aqueous extract is extracted with ethyl acetate; Ethyl acetate gave a tannin extract.

The tannin extract mainly contains tannins and other compounds such as organic acids, sugars, crude proteins and the like.

Organic solvent extracts such as scutellaria can be used to mediate and improve the function of the cranial nervous system in various dosage forms. The effect of the drug on enuresis is always more effective than any of the current similar drugs. An effective drug of choice. At the same time, it can also prevent brain aging and cerebrovascular disease, improve memory and develop intelligence, treat nocturia, enuresis, incontinence, urinary frequency and mental retardation, senile dementia, Parkinson's disease and other diseases caused by impaired brain function or disorder. And can prevent rheumatism, arthritis, vascular sclerosis, blood circulation disease, Raynaud's syndrome and other symptoms have significant effects.

Traditional Chinese medicine theory believes that enuresis, frequent urination, and urinary incontinence are caused by kidney deficiency, often using kidney-tonifying drugs, such as Cordyceps sinensis, ginseng, Morinda, Eucommia, Cistanche, etc., and using solid sputum such as Schisandra, Gallic, are to strengthen kidney function, Through the "kidney meridian" to regulate the circulation of water in the body, to achieve effective medical results. The experiment of the present patent application proves that the extract of the canopy of the canopy can promote the growth of bladder cells. Strengthen the bladder storage and discharge function, its role is to regulate the circulation and discharge function of water in the body through the "bladder meridian", so if the application of the patent application for the canopy shell extract is compatible with ginseng or extract, or Cordyceps sinensis or its Traditional Chinese medicine for tonifying kidney and its extracts, such as extracts, can treat enuresis, urinary frequency and urinary incontinence through the "bladder meridian" and "kidney meridian" to achieve better medical results.

These extracts of Coleoptera can also be combined with vitamin B, D, K, grape seed extract or other antioxidants, Cordyceps sinensis or its extract, Ginkgo biloba or its extract, ginseng and American ginseng or its extract, polygera or Its extract, small hypericum or its extract, puerarin or its extract, gastrodia elata or its extract, honey fungus or its extract, salvia miltiorrhiza or its extract, notoginseng or its extract, red peony, jaundice or Extract thereof, Rehmannia or its extract, Angelica sinensis or its extract, Polygala or its extract, Ganoderma lucidum or its extract, Lycium barbarum or its extract, Licorice or its extract, Stone Pine A, Trichlorfon, Lacithin, brainwashing, nocetile, folic acid, amino acids, creatine kinase and fiber additives make a variety of new and health foods. For example, the husk and/or stalk extract of the genus Corolla can be combined with ginseng or extract, or Cordyceps sinensis or its extract to make a new treatment for enuresis, frequent urination, and urinary incontinence.

Extracts of the fruit husks and/or fruit stalks, seeds, seed coats, branches, bark and roots can be used to mediate and improve the function of the brain's nervous system and health foods. The extract of Xanthoceras sorbifolia provided by the present invention can inhibit the absorption of 5-hydroxytryptamine. The presence of 5-hydroxytryptamine maintains and enhances deep sleep in humans. The extract of Xanthoceras sorbifolia inhibits the absorption of serotonin, which breaks deep sleep and allows people to wake up when the bladder is full and avoid bedwetting. The invention provides The extract of the dried fruit can improve the sleep alert system and help prevent sleep paralysis. The extract of Xanthoceras sorbifolia provided by the invention inhibits the synthesis of acetylcholine acetylase (AchE), mediates the release, absorption and decomposition of acetylcholine (Ach), thereby improving the transmission process of brain central nervous system and urinary system information, and avoiding The occurrence of enuresis. The extract of the extract of Corundum provided by the present invention contributes to the growth and development of the bladder and the sphincter, thereby avoiding the occurrence of enuresis. The extract of Xanthoceras sorbifolia provided by the invention helps to eliminate mental stress, delay aging, improve bladder function, avoid detrusor instability, hyperactivity and hyperactivity, thereby preventing urgency and frequent urination. Therefore, the drug is more effective in treating enuresis, urgency and urinary frequency than any of the current similar drugs, and is currently the most effective drug of choice.

The extract of Xanthoceras sorbifolia provided by the present invention, which is detected by MTT, shows a strong activity for inhibiting the growth of cancer cells, especially ovarian cancer cells. Thus, these extracts, at appropriate concentrations and their appropriate carriers, can be used as anticancer agents, such as ovarian cancer, at appropriate dosages. It is resistant to bladder cancer and also to cervical cancer, ovarian cancer, prostate cancer, lung cancer, breast cancer, colon cancer, liver cancer and brain cancer, as well as other cancers.

The thirteen new saponin compounds disclosed in the present invention, the saponin is a triterpenoid, and the carbon 21 and 22 have an angelica thiol or other side chain, and even a plurality of sugar chains.

One of them is the saponin compound Y, and its chemical structure is as follows:

Name of saponin compound Y: 3-0-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucopyran Mercapto-21,22-O-dipodoxime-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene pentacyclic triterpenoid saponin. Molecular formula: C ₅₇ H ₈₈ 0 ₂₃ . That is Xanifolia-Y.

The chemical structure of the other 12 compounds is as follows: chemical structure of compound Y1:

Name of saponin compound Y1: 3-O-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucopyran Mercapto-21-O-(3,4-dihydrocarbyl)-α-L-rhamnosepyranoyl-22-O-acetamido-3β,16α,21β,22α,28-pentahydroxy Oleanol-12-ene pentacyclic triterpenoid saponin. Molecular formula: C ₆₅ H ₁₀₀ 0 ₂₇ . That is Xanifolia-Y1.

Chemical structure of compound Y1-1: (see Figure 38A)

Chemical structure of compound Y1-2: (see Figure 38B)

Chemical structure of compound Y1-3: (see Figure 38C)

Chemical structure of compound Y1-4: (see Figure 38D)

Chemical structure of compound Ya: (see Figure 66)

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound Yb: (see Figure 67)

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound Yc: (see Figure 68)

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound Y1-a: (see Figure 69)

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

R3=A or B or C

Chemical structure of compound Y1-b: (see Figure 70)

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

R3=A or B or C

Chemical structure of compound Y1-c: (see Figure 71)

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

R3=A or B or C

Chemical structure of compound R1: (see Figure 65)

Name of saponin compound R1: 3-O-[Danggui thiol (1→3)-β-D-glucopyranosyl-(1-6)]-β-D-glucopyraninyl-28-O -[ α -L-rhamnose pyranyl-(1-2)-β-D-glucopyranosyl-(1-6)-β-D-glucopyranin-3β,21β, 22α, 28-tetrahydroxy olean-12-ene pentacyclic triterpenoid saponin. Molecular formula: C ₆₅ H ₁₀₉ O ₂₉ . That is Xanifolia-R1.

The present invention also provides salts formed by the above novel compounds.

The present invention also provides a medicament made of the above novel compound or a salt thereof, characterized in that the medicament consists of an appropriate concentration of the above novel compound or a salt thereof and a suitable carrier.

The present invention also provides the above novel compounds, salts formed therefrom and synthetic methods for preparing the medicaments therefrom.

The method for detecting the cell-specific toxicity of the extract of the extract of the crown of the apple by the MTT disclosed in the present patent is described below.

Methods and materials

Cells from the American Model Culture Collection (American Type Culture Collection) obtained: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 (breast), k562 (white blood cells), HCT116 (colon), HepG2 (liver), U20S (bone), T98G (brain), and OVCAR-3 (ovary).

Culture: HeLa-S3 (cervix), DU145 (prostate), MCF-7 (breast), HepG2 (liver) and T98G (brain) cells were cultured on MEN (Earle salt) medium. HTB-9 (bladder), H460 (lung), 562 (leukocytes) and OVCAR-3 (ovary) were cultured on RPMI-1640 medium, and other cells were on McCoy-5A medium.

These media are supplemented with 10% fetal bovine serum, glutamine and antibiotics. Incubate in an incubator with a CO ₂ concentration of 5%.

MTT detection

The test method is basically in accordance with the method of Carmicheal et al. 1987, and only a few places have been changed. These cells were cultured in small wells with 96 small wells for 24 hours; 10,000 HTB-9 (bladder) per hole, HeLa-S3 (cervix), H460 (lung), HCT116 (colon), T98G (brain), And OVCAR-3 (ovary) cells; 15,000 DU145 (prostate), MCF-7 (breast), HepG2 (liver) and U20S (bone) cells per well; 40,000 k562 per well (white blood cells). Then, a sample of the extract of the dried fruit is placed in a hole, and cultured for another 48 hours (liver and bone cancer cells) for 72 hours, and breast cancer cells for 96 hours. Then, MTT (0.5 mg/ml) was added to each well and cultured for 1 hour. The resulting formazan was dissolved in DMSO and its O.D. value (TD) was measured using an ELISA reader (Dynatech, Model R700). The MTTO.D. value (TO) before the addition of the sample is also measured. The percentage of each cell growth (%G) can be determined by the following formula: %G=(TD-TO/TC-TO)×100

(TC is the O.D. value of the control cell group)

When TO < TD, the cell-specific toxicity (LC) value is: % LC = (TD-TO / TO) × 100

Cell-specific toxicity of Xanthoceras sorbifolia extract by MTT, compound Y, Y1, R1, Y1-1, Y1-2, Y1-3, Y-4, Ya, Yb, Yc, Y1-a, Y1- Both b and Y1-c inhibit the growth of cancer cells. The above novel compounds, which can be prepared from the method of artificial extraction or artificial synthesis.

Cancer is one of the important causes of modern human death. The causes of carcinogenesis are various. For example, the Wnt signaling pathway regulates embryonic development and cell proliferation, death and morphogenesis, and plays an important role in the development of cancer cells. Some cancer occurrences were found to be associated with inappropriate activation of the Wnt signaling system. The invention provides the extract of the crown of the fruit and the novel saponin compound or the composition thereof, can regulate the biochemical link or the receptor thereof in the Wnt signaling pathway, avoid inappropriate activation of the Wnt signaling system, thereby preventing the formation of cancer cells and proliferation.

Cell mitosis is involved in the Ras-MAP kinase chain signaling pathway. If the activity of the Ras-MAP kinase chain is too active, it will lead to the production of the gene regulatory protein Myc. Myc will accelerate the replication of some genes. If this activity is excessively active, it will lead to cancer. Cell production and proliferation. The invention provides the extract of the crown and the new saponin compound or the composition thereof, can regulate the activity of the Ras-MAP kinase chain, and the activity of the Ras-MAP kinase pathway can not be excessively active, thereby causing no generation and proliferation of cancer cells. .

Sometimes the amino acid in the Ras signaling pathway is mutated, causing this to occur. The amino acid protein is permanently overactive, resulting in a subordinate Ras signaling pathway that is too active in the absence of mitotic stimulation. Similarly, mutations in amino acids can also cause excessive activity of the Myc signaling pathway. This can lead to abnormal cell growth and the formation of cancer cells. The extract of the genus Angustifolia and the novel saponin compound or the combination thereof provided by the invention can regulate the Ras signaling pathway and the Myc signaling pathway of the mutation of the amino acid, so that they are not excessively active, thereby causing abnormal growth of the cells and causing canceration.

There is a checkpoint mechanism within the cell that monitors malformations of mitosis and causes abnormally overactive cells to die. The monitoring point mechanism of cancer cells cannot be activated due to mutations in the genes that compile the monitoring point mechanism, resulting in unrestricted growth and proliferation of cancer cells. The extract of the crown of the genus and the novel saponin compound or the combination thereof provided by the invention can initiate an intracellular monitoring point mechanism to timely prevent abnormal mitotic activity and stop the growth and proliferation of cancer cells.

The extracellular growth factor that stimulates cell growth initiates an intracellular signaling pathway through receptors on the cell surface, initiating PI3 kinase to promote protein synthesis, at least by activating the kinases eif4e and s6 to increase mRNA amplification, thereby stimulating cell growth. The extract of the genus Angustifolia and the novel saponin compound or the combination thereof provided by the invention can regulate the link of the extracellular growth signal pathway or its receptor, thereby affecting the signal pathway of intracellular growth, inhibiting excessive proliferation of cells and preventing the production of cancer cells. .

Cell division is also controlled by the Myc protein in the nucleus. Excessive Myc protein causes excessive proliferation of cells and the formation of cancer cells. The invention provides the extract of the crown of the fruit and the novel saponin compound or the composition thereof, and controls the cell Excessive production of Myc protein, thereby preventing excessive proliferation of cells, leading to tumor formation.

Transforming growth factor alpha (TGF-alpha) induces long-term proliferation of immature pre-hematopoietic cells (eg, BFU-E) in mice and chickens, and also induces the conversion of BFU-EDE cells into red blood cells. Transforming growth factor alpha can also stimulate proliferation of cultured endothelial cells. It plays an important role in the vascularization of tumor tissue. The transforming growth factor α is produced by keratimcyte, macrophages, hepatocytes and platelets, and viral infection can stimulate the synthesis of the transformation growth factor α. The extract of the crown of the genus and the novel saponin compound or the combination thereof provided by the invention can regulate the link of the transforming growth factor alpha signal pathway or its receptor, thereby inhibiting the growth of ovarian and bladder cancer cells.

Transforming growth factor beta (TGF-beta) regulates cell growth and prevents the growth of many cells. It has two receptors: receptor 1 receptor 2. They are serine-threonine kinases that regulate signaling family signaling via SMAD. Transforming growth factor beta signaling pathways are involved in mutations and carcinogenesis of SMAD-encoding regulatory genes. The extract of the crown of the genus and the novel saponin compound or the combination thereof provided by the invention can regulate the link of the transforming growth factor beta signal pathway or its receptor, thereby inhibiting the growth of ovarian and bladder cancer cells.

DAN tumor virus can cause cancer by interfering with cell loop control protein E6 and protein p53. Mutation of the protein p53 gene causes cancer cells to continue to survive and grow in the event of DAN damage. The papillomavirus uses proteins E6 and E7 to sequence the proteins p53 and Rb, respectively, which initiates the mutated cells, allowing them to survive, divide and grow. The growth of damaged cells can lead to cancer. The invention provides the extract of osmanthus and the novel saponin compound or the composition thereof, which is adjustable The proteins E6 and E7 are released and the proteins p53 and Rb are released, thereby inhibiting the divisional growth of the deformed cells. At the same time, it can also control or react with these proteins to cause cancer cells to die.

Protein p53 can help multicellular organisms safely self-replicate in DAN damage and other adverse conditions, and stop cell division and growth in dangerous situations. Cancer cells often contain a large number of mutated proteins, p53, suggesting that genetic mutations or growth repression can trigger the protein p53 under adverse conditions. Thus, loss of protein p53 activity is dangerous because it allows the mutated cells to continue to complete its growth cycle and avoid death. Therefore, if DAN is damaged, some cells will die, and those cells that survive without growth damage and can continue to grow may carry a corrupt genome, which leads to the loss of genes that inhibit cancer during gene amplification. Start the oncogene. At the same time, gene amplification may also cause cells to develop resistance. The extract of the crown of the genus and the novel saponin compound or the combination thereof provided by the invention can regulate the link of the protein 53 signaling pathway or its receptor, thereby inhibiting the proliferation of cancer cells.

All cells have a variety of dormant procaspases in the nucleus, waiting for commands to kill cells. Procaspase is a dormant zymogen formed by a suicide protease. It can be initiated by another caspase enzyme group by cleavage of the protease. The two lysed fragments combine to form the active part of the caspase. An active enzyme is considered to be a tetrad of one of these two parts. Each activated caspase molecule can be cleaved into multiple procaspase molecules, which in turn initiate more caspase molecules. Through this chain reaction, an explosive activity of a large number of procaspase molecules is caused. Some startup The procaspase is cleaved into multiple key proteins, including cytosolic proteins and nuclear-lamins, which control cell death. The initiation of extracellular death receptors also initiates procaspase. However, the signal that kills cells due to genetically modified cancer cells is blocked. As a result, cancer cells continue to divide, leading to cancer. The extract of the crown of the genus and the novel saponin compound or the combination thereof provided by the invention can clear the pathway for blocking the "suicide" signal transmission of the cancer cells and cause the cancer cells to "suicide".

A method for inhibiting the growth of cancer cells according to the present application, characterized in that the method comprises contacting a certain amount of a compound having the following chemical structure and a suitable drug carrier to contact the cancer cells, and adjusting the amount thereof. And drug carrier:

Wherein R1, R2, R3, R4 are short aliphatic side chains, R1 = R2 = R3 = R4 = CH3, and R5 = OH. Contains at least one sugar, sapogenin, a triterpenoid, attached to a triterpenoid, at C21 or C22 There is at least one side chain or the Angelica Group.

Another method for inhibiting the growth of cancer cells of the present application is characterized in that the method comprises contacting a certain amount of a compound having the following chemical structure and a suitable drug carrier to the cancer cells, and adjusting the same Dosage and drug carrier:

Compound Ya chemical structure:

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound Yb:

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound Yc:

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Saponin compound Y1 chemical structure:

Name of saponin compound Y1: 3-O-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucopyran Mercapto-21-O-(3,4-dihydrocarbyl)-α-L-rhamnosepyranoyl-22-O-acetamido-3β,16α,21β,22α,28-pentahydroxy Oleanol-12-ene pentacyclic triterpenoid saponin. Chemical structure of compound Y1-a:

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound Y1-b:

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound Y1-c:

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

Where R5=B or C or S1

R1=A or B or C

R2=A or B or C

R4=B or C

Chemical structure of compound R1: (see Figure 65)

Name of saponin compound R1: 3- 0 -[Danggui thiol (1→3)-β-D-glucopyranosyl-(1-6)]-β-D-glucopyraninyl-28-O -[ α -L-rhamnose pyranyl-(1-2)-β-D-glucopyranosyl-(1-6)-β-D-glucopyranin-3β,21β, 22α, 28-tetrahydroxy olean-12-ene pentacyclic triterpenoid saponin.

Detailed description (1)

The promoting effect of the extract of the crown of the present invention disclosed in the patent on mice is further illustrated by the following example (1).

Effect of Saponin Extract X and Y on Memory Function of Aged Mice (Water Maze Method): Experimental Male Old Mice, 16 Months Mouse, Weight 35-55g, Cage-raised, Full-price Pellet Feed Free drinking water, room temperature 20±2°C, humidity 55-66%; water maze automatic test, water height 11cm, water temperature 25±1°C, water maze from starting point, four blind spots, end steps and corresponding passages, etc. Structure group to make. Before the experiment, the mice were trained to calculate the time required to reach the end point and the number of errors made by each training mouse. A total of three trainings were performed, and mice that reached the end point in 2 minutes were selected as the animals of the test. The eligible mice were then randomly divided into 9 groups, 1 in each group: 1) blank control group, equal amount of NC; 2) positive group, Xienkai 0.9 g/kg; 3) X drug group I: 100 mg/kg; X drug group II: 200mg/kg; 5) X drug group III: 400mg/kg; 6) Y drug group I: 125mg/kg; 7) Y drug group II: 250mg/kg; 8) Y drug group III: 500mg/kg. The above groups of animals were intragastrically administered with a dose of 20 ml/kg, and water maze training was performed for 3 days, 6 days, and 9 days, respectively, to calculate the improvement effect of learning and memory on each training mouse after administration. Data were analyzed by t test.

result

The results of the experiment showed that after 3 days of administration, the dose of 500 mg/kg of Y medicine could significantly shorten the time of arrival of the mice, which was significantly different from that of the saline group (P<0.05). The remaining dose groups can reduce the number of errors and shorten the time to reach the end point (see Table 1.1).

After 6 days of administration, the number of errors was reduced in each dose group of X and Y drugs, and there was a significant difference compared with the saline group (P<0.05, P<0.01). The dose of Y medicine in the 500 mg/kg group significantly shortened the time to reach the end point of the mice (P<0.05). The remaining administration groups also reduced the time to reach the end point (see Table 1.2).

After 9 days of administration, the number of errors was reduced in each dose group of X and Y drugs, and there was a significant difference compared with the saline group (P<0.05). The X drug 400mg/kg dose group and the Y drug 250mg/kg and 500mg/kg dose groups significantly shortened the time to reach the end point of the mice, with significant difference (P<0.05, P<0.01) (see Table 1.3).

The above experimental results show that X and Y extracts of Xanthoceras sorbifolia have a significant improvement on the memory function of aged mice, and the improvement of learning and memory in mice is more obvious with the prolonged administration time.

The following is an example (2) to further explain the effects of Xantholic extracts X and Y on the memory dysfunction of aged mice induced by pentobarbital (water maze method): each of the details (1, 1) The mice in the group were injected with pentobarbital 15 mg/kg on the tenth day after administration, and a water maze test was performed 30 minutes later, and the time required to reach the end point of each training mouse and the number of errors made in 2 minutes were calculated. Continuous injection of pentobarbital for 3 days was performed to observe the effect of each administration group on memory dysfunction of aged mice induced by pentobarbital.

result

The results of the experiment showed that the saponin extract X and Y contained the dysfunction of memory in aged mice given pentobarbital, 1 day, X drug 100mg/kg dose group, Y drug three dose groups Significantly shorten the time the mouse reaches the end point, There was a significant difference (P<0.05, P<0.01). The Y drug 500 mg/kg dose group can reduce the number of errors, and there is a significant difference (P<0.05) compared with the saline group (see Table 2.1).

After 2 days of injection, the X-drug and Y-drug groups significantly shortened the time to reach the end point of the mice, and both reduced the number of errors, with a significant difference (P < 0.05) (see Table 2.2).

After 3 days of injection, the doses of X and Y drugs significantly shortened the time of arrival of the mice (P<0.05), and the doses of 250 mg/kg and 500 mg/kg of Y drugs all reduced the number of errors, compared with the saline group. Significant differences (P < 0.05) (Figure 2 (a) and (b)) (see Table 2.3).

The above experiments showed that Xanthocera extracts X and Y have a significant improvement in the recovery of memory dysfunction induced by pentobarbital in mice.

The following is an example (3) to further explain the effect of extracts X and Y of Xanthoceras sorbifolia L. on memory dysfunction in aged mice caused by Dongpu (Skip method): ICR mice were used for platform training. The mice were lightly placed on a platform and the latency (SDL) of the mice staying on the platform was recorded. The mice jumped from the platform and immediately contacted the copper with a 36-volt electrical stimulation to record the latency (EL) of the mouse to escape to the platform. Mice of SDL and EL at 2-60 seconds were selected as the animals of the experiment. The mice were randomly divided into 9 groups, 10 in each group, half male and half female, weighing 16-20 g. The doses of the groups were the same as those in the example (1), and the administration volume was 20 ml/kg. After the administration, the platform training was performed once on the 3rd, 6th, and 9th days, and the SDL, EL, and the number of times the electric shock was jumped from the jumping platform within 5 minutes (the number of errors) was recorded. After the 10th day of administration, the abdominal cavity was injected into the sputum 3 ml/kg. After 30 minutes, the platform training was conducted. Repeated on the 11th day to observe the effects of extracts X and Y on the memory dysfunction of aged mice induced by Dongpu.

result

1) The X and Y doses of the extracts of Xanthoceran extract can shorten the mouse EL and prolong the SDL after 3 days of administration; after 6 and 9 days, the number of errors can be reduced, but it is not statistically significant (see Table 3.1 for drug administration). Data for Days 9 and 6) Data for Days 3 and 6 are not listed.

2) After the administration on the 10th day, the abdominal cavity was injected into the sputum to cause memory impairment in the mice, and the platform training was carried out. The experimental results showed that the extract of Xanthoceras sorbifolia X400mg/kg, the group of Y 250mg/kg and 500mg/kg The number of errors was significantly reduced, and there was a significant difference compared with the saline group (P<0.05, P<0.01) (see Table 3.2).

3), after the 11th day of administration, the abdominal cavity was injected into Dongpu to cause memory impairment in mice, and the platform training was carried out. The results showed that the X and Y doses of extracts of Xanthoceras sorbifolia could significantly reduce the number of errors (P<0.05). The Y 250 mg/kg dose group prolonged the SDL time and was significantly different from the saline group (P < 0.05) (see Table 3.3).

The above-mentioned mouse platform test showed that the extracts of Xanthoceras sorbifolia X and Y had a significant improvement on the recovery of memory dysfunction caused by Dongpu.

The following is an example (4) to further explain the effect of Xanthocera extract X and Y on memory dysfunction induced by sodium nitrite in mice (water maze method): experimental male ICR mice, weighing 16-19g, caged , full-price pellet feed, free drinking water, room temperature 20±2°C, humidity 55-66%; water maze from The instrument test, the water height is 11 cm, the water temperature is 25±1 °C, and the water maze consists of the starting point, four blind spots, the end step and the corresponding passage. Before the experiment, the mice were trained to calculate the time required to reach the end point and the number of errors made by each training mouse. A total of two trainings were performed, and mice that reached the end point in 2 minutes were selected as the animals of the test. The eligible mice were then randomly divided into 9 groups, 11 in each group: 1) blank control group, equal amount of NC; 2) positive group, Xienkai 0.9 g/kg; 3) X drug group I: 100 mg/kg; X drug group II: 200mg/kg; 5) X drug group III: 400mg/kg; 6) Y drug group I: 125mg/kg; 7) Y drug group II: 250mg/kg; 8) Y drug group III: 500mg/kg. The above groups of animals were intragastrically administered, and water maze training was performed at 3, 6, and 9 days, respectively, to calculate the improvement effect of learning and memory on each training mouse after administration. On the 10th day, each group of animals was injected subcutaneously with sodium nitrite 120 mg/kg. After 24 hours, water maze test was performed to observe the effect of each administration group on memory dysfunction caused by sodium hyponitrate-induced hypoxia in mice. Data were analyzed by t test.

result:

1) The results of the learning and memory test after the administration of the water maze method showed that after 3 days of administration, each dose group of X and Y drugs can reduce the number of errors and shorten the time to reach the end point (see Table 4.1).

After 6 days of administration, the X400mg/kg and Y drug 500mg/kg dose groups were able to reduce the number of errors, which was significantly different from the saline group (P<0.01) (see Table 4.2).

After 9 days of administration, the dose of 250 mg/kg of X medicine and the dose of 250 mg/kg and 500 mg/kg of Y medicine significantly shortened the time to reach the end point of the mice, with significant difference (P<0.05) (see Table 4.3 and Figure). 1 (a) and (b)).

After 10 days of administration, the 250 mg/kg and 500 mg/kg dose groups of Y drugs significantly reduced the number of errors. The Y mg 500 mg/kg dose group shortened the time to reach the end point of the mice, with a significant difference (P<0.05, P<0.01) (see Table 4.4).

2), the memory dysfunction caused by hypoxia in the brain of mice to mice showed that X drug 100mg / kg, 200mg / kg dose group, Y drug three dose groups can significantly reduce the number of errors in mice Compared with the model group, there was a significant difference (P<0.05) (see Table 4.5).

The above water maze test showed that Xanthocera extract X and Y have a significant improvement effect on the recovery of memory dysfunction caused by sodium nitrite in mice. Combining all the above experiments on learning and memory of mice showed that X and Y extracts of Xanthoceras sorbifolia had obvious promoting effects.

Detailed description (2)

The effect of the extract of the extract of the crown of the present invention on the urine volume of the mouse is further illustrated by the following example (5). Xanthoceras extract X = Xanthoceras X component extract = Xanthoceras extract FS (X), Xanthoceras extract Y = Xanthoceras Y component extract = Crested fruit extract FS (Y), (see Figure 12 for the X and Y groups).

1, the effect of extracts of Xanthocera sorbifolia X and Y on urine volume in mice (3 days of administration):

Experimental male ICR mice 112, weighing 18-22g, caged, full-price pellet feed, free drinking water, room temperature 20 ± 2 ° C, humidity 55-66%. The mice were randomly divided into 9 groups, 14 in each group: 1) blank control group, equal amount of NC; 2) hydrochlorothiazide group, 400 mg/kg; 3) X drug group I: 100 mg/kg; 4) X drug group II : 200 mg / kg; 5) X drug group III: 400 mg / kg; 6) Y drug group I: 125 mg / kg; 7) Y drug group II: 250 mg / kg; 8) Y drug group III: 500 mg / kg. The above groups of animals were intragastrically administered, and the administration volume was 20 ml/kg, which was administered for 3 days and 3 times a day. After the last administration, each group of mice was placed in a 500 ml beaker in which filter paper was previously placed. The filter paper was taken out at 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and 360 minutes, and weighed by an electronic analytical balance. The urine volume was calculated by weight loss method, and the extraction of the crown fruit was compared in each time period. The effect of X and Y on the urine volume of mice. According to the t test.

The experimental results showed that the urine volume was significantly reduced after 30 minutes of administration of the X drug 400 mg/kg dose group. After 60 minutes of administration of the Y drug 500 mg/kg dose group, the urine volume was significantly reduced, and there was a significant difference compared with the saline group (P<0.01). After 180 minutes of administration of the X drug 200 mg/kg dose group and the Y drug 125 mg/kg and 500 mg/kg dose groups, the urine volume increased, and there was a significant difference compared with the saline group (P<0.01) (see Table 5.1).

2, the effect of extracts of Xanthocera sorbifolia X and Y on the urine volume of mice (administered for 5 days):

Experimental male ICR mice 112, weighing 18-22g, caged, full-price pellet feed, free drinking water, room temperature 20 ± 2 ° C, humidity 55-66%. The mice were randomly divided into 9 groups, 14 in each group: 1) blank control group, equal amount of NC; 2) hydrochlorothiazide group, 400 mg/kg; 3) X drug group I: 100 mg/kg; 4) X drug group II : 200 mg / kg; 5) X drug group III: 400 mg / kg; 6) Y drug group I: 125 mg / kg; 7) Y drug group II: 250 mg / kg; 8) Y drug group III: 500 mg / kg. The above groups of animals were intragastrically administered, and the administration volume was 20 ml/kg, respectively, once a day, once a day. After the last administration, each group of mice was placed in a 500 ml beaker in which filter paper was previously placed. The filter paper was taken out at 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and 360 minutes, and weighed by an electronic analytical balance. The urine volume was calculated by weight loss method, and the extraction of the crown fruit was compared in each time period. The effect of X and Y on the urine volume of mice. Data were analyzed by t test.

The experimental results showed that the urine volume increased significantly after 240 minutes of administration of the X drug 400 mg/kg dose group and the Y drug dose group, and there was a significant difference compared with the saline group (P<0.01, P<0.05). X medicine 400mg/kg dose group, Y medicine After 30 minutes of administration in the 500 mg/kg dose group, the amount of urine decreased (see Table 5.2).

3. Effect of extracts of Xanthocera sorbifolia X and Y on urine volume in mice (administration for 7 days):

Experimental male ICR mice 112, weighing 18-22g, caged, full-price pellet feed, free drinking water, room temperature 20 ± 2 ° C, humidity 55-66%. The mice were randomly divided into 9 groups, 14 in each group: 1) blank control group, equal amount of NC; 2) hydrochlorothiazide group, 400 mg/kg; 3) X drug group I: 100 mg/kg; 4) X drug group II : 200 mg / kg; 5) X drug group III: 400 mg / kg; 6) Y drug group I: 125 mg / kg; 7) Y drug group II: 250 mg / kg; 8) Y drug group III: 500 mg / kg. The above groups of animals were intragastrically administered, and the administration volume was 20 ml/kg, which was administered for 7 days and once a day. After the last administration, each group of mice was placed in a 500 ml beaker in which filter paper was previously placed. The filter paper was taken out at 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and 360 minutes, and weighed by an electronic analytical balance. The urine volume was calculated by weight loss method, and the extraction of the crown fruit was compared in each time period. The effect of X and Y on the urine volume of mice. According to the t test.

The results of the experiment showed that after taking the X drug 200mg/kg, 400mg/kg dose group and the Y drug 250mg/kg, the 500mg/kg dose group for 120 minutes, the urine volume was significantly reduced, and there was a significant difference compared with the saline group (P< 0.05); After 300 minutes, the amount of urine increased significantly (P < 0.05) (see Table 5.3).

4, the effect of extracts of Xanthocera sorbifolia X and Y on the urine volume of mice (administered for 10 days)

Experimental male ICR mice 112, weighing 18-22g, caged, full-price pellet feed, free drinking water, room temperature 20 ± 2 ° C, humidity 55-66%. The mice were randomly divided into 9 groups, 14 in each group: 1) blank control group, equal amount of NC; 2) hydrochlorothiazide group, 400 mg/kg; 3) X drug group I: 100 mg/kg; 4) X drug group II : 200 mg / kg; 5) X drug group III: 400 mg / kg; 6) Y drug group I: 125 mg / kg; 7) Y drug group II: 250 mg / kg; 8) Y drug group III: 500 mg / kg. The above groups of animals were intragastrically administered, and the administration volume was 20 ml/kg, which was administered for 10 days and once a day. After the last administration, each group of mice was placed in a 500 ml beaker in which filter paper was previously placed. The filter paper was taken out at 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and 360 minutes, and weighed by an electronic analytical balance. The urine volume was calculated by weight loss method, and the extraction of the crown fruit was compared in each time period. The effect of X and Y on the urine volume of mice. According to the t test.

The results of the experiment showed that after taking the X drug 200mg/kg, 400mg/kg dose group and the Y drug 250mg/kg, the 500mg/kg dose group for 120 minutes, the urine volume was significantly reduced, and there was a significant difference compared with the saline group (P< 0.05) (see Table 5.4 and Figure 3).

5, the effect of extracts of Xanthocera sorbifolia X and Y on urine volume in mice (administered for 20 days)

Experimental male ICR mice 84, weighing 18-22g, caged, full-price pellet feed, free drinking water, room temperature 20 ± 2 ° C, humidity 55-66%. The mice were randomly divided into 6 groups, 14 in each group: 1) blank control group, equal amount of NC; 2) hydrochlorothiazide group, 33.4 mg/kg; 3) X drug group I: 100 mg/kg; 4) X drug II Group: 200 mg/kg; 5) Y drug group I: 125 mg/kg; 6) Y drug group II: 250 mg/kg. All the above groups of animals were intragastrically administered, and the administration volume was 20 ml/kg, which was administered for 20 days and once a day. After the last administration, each group of mice was placed in a 500 ml beaker in which filter paper was previously placed. The filter paper was taken out at 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, and 360 minutes, and weighed by an electronic analytical balance. The urine volume was calculated by weight loss method, and the extraction of the crown fruit was compared in each time period. The effect of X and Y on urine output in mice. According to the t test.

The results of the experiment showed that the amount of urine was significantly reduced after administration of X drug 100 mg/kg, 200 mg/kg dose group and Y drug 125 mg/kg, and 250 mg/kg dose group for 30 minutes. After 60 minutes of administration of the X drug 200 mg/kg and the Y drug 250 mg/kg dose group, the urine volume was significantly reduced, and there was a significant difference compared with the saline group (P<0.05, P < 0.01).

In summary, all the experimental results show that X and Y extracts of Xanthoceras sorbifolia have a significant regulatory effect on urine output.

Detailed description (3)

The urinary effect of the extracts of Xanthocera sorbifolia X and Y on mice (standard metabolic cage method) is further illustrated by the following example (5A):

OBJECTIVE: To study the urinary effect of extract of Cappari FS(X) on mice by standard metabolic cage method

Drug: Xanthoceras extracts FS (X) and FS (Y).

Experimental animals: 100 male SD mice were used for experimentation, weighing 150-200 g, and caged at 200 (20×30×45 cm), 5 per cage. The bottom of the cage is polished with a polished wooden board, which is changed every two days on average and dried for use. Squirrel cages should be disinfected before use. Full price pellet feed (animal experiment center), free drinking water, room temperature 23 ± 2 ° C, humidity 40-70%, air conditioning, automatic exhaust and ventilation, natural light source, 12 hours light, 12 hours dark.

Mouse training: The mice were placed in cages for adaptive training for two days, 6-10 hours per day. Before entering the cage, squeeze the lower part of the abdomen of the mouse to remove the urine. Distilled water was then injected into the stomach (38 ° C), 25 ml / kg. After the injection, the urine in the cage was collected every two hours, and the lower part of the abdomen of the mouse was carefully squeezed to collect the residual urine. Mice with a urine volume exceeding 40% of the injected water will be used as an experiment.

Experimental equipment: standard metabolic cage, automatic urine analyzer (Miditron Junior II), urine pressure tester, urine ion analyzer (EL-ISE, Beckman).

method:

The mice were randomly divided into 6 groups, 10 in each group: 1) blank control group, irrigation Gastric administration, sodium carboxymethyl cellulose (CMC-Na) 0.5% suspension, 10 ml/kg per day; 2) positive control group, the mice were placed in the abdomen cavity of the mouse after the pre-cage injection. 0.25u/kg; 3) FS (X) drug group I: 100mg/kg; 4) FS (X) drug group II: 200mg/kg; 5) FS (X) drug group III: 400mg/kg; 6) FS (Y) Drug group: 400 mg/kg; administered for 25 days, once a day, 30 minutes after the cage was given to the test.

Urine volume was collected and observed: urine samples were collected 18 hours after the cage was placed. Squeeze the lower part of the abdomen of the mouse to exclude urine. Distilled water was then injected into the stomach (38 ° C), 50 ml / kg. Mice were placed in cages after injection and urine volume was collected at 0.25 hours, 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours. After collecting urine volume for the sixth hour, the lower part of the abdomen of the mouse was squeezed to collect urine in the bladder.

Routine indicators for detecting urine: pH, red blood cells, white blood cells, proteins, etc.; determination of Na+, K+, Cl- ion concentration and urine osmotic pressure.

Data processing: The urinary rate, relative urine volume, Na+, K+, and Cl- ion concentrations in the reagent group were compared with the control group by t test.

result

1), the effect on urine volume: the urinary effect of FS (X) in the reagent group was related to the dose, the drug group II: 200mg/kg; the FS (X) drug group III: 400mg/kg had obvious urinary action (0.25 hours to 6 hours) (Table 5A-1, see Figure 44; Figure 48). The FS (Y) and FS (X) 400 mg/kg dose groups had the most significant effect on urination rate (Table 5A-2, see Figure 45; Figure 49), which allowed urine to be administrated for 2 hours. The dose of FS (X) 400 mg / kg dose can significantly reduce urine output, but the effect on total urine volume has nothing to do with the dose. The positive control group did not reduce the total urine volume, however, It can be used for 2 hours.

2) Effect on Na+, K+, Cl- ions in urine: Because the reagent can reduce the amount of urine, the concentration of ions in the urine is increased to varying degrees, but it is independent of the dose. It has almost no effect on the concentration of Na+ and Cl- ions, and has the effect of promoting K+ excretion. All the concentrations and discharges of urinary ions in the positive control group were significantly promoted (Table 5A-4, see Figure 47).

3) Effects on pH and urine osmotic pressure: The reagents FS(X) and FS(Y) have no significant effect on pH and urine osmotic pressure, and have a slight effect on urine specific gravity (Table 5A-3, see figure 46).

Detailed description (4)

The preparation method of the crown fruit bioactive substance disclosed in the patent is further illustrated by the following example (6). Summarized as follows: ̇ 收 收 冠 冠 冠 冠 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : Get the sample powder (the seed kernel should be pre-oiled); 浸 Extract the dried fruit sample powder (2:1) with organic solvent, 4-5 times, each time 20-35 hours, get the organic solvent extract; The extract is reheated and refluxed (80 ° C) for 2-3 times; the combined extract is collected by hydrazine; the organic solvent is recovered by hydrazine to obtain a flow extract; ̇ dried, sterilized, and the extract of sempervivin is obtained.

Detailed description (5)

The following is an example (7) to further illustrate the crown fruit organism disclosed in this patent. Method for separation, identification and purification of active substances.

1, separation and purification of extracts of Xanthoceras sorbifolia by high pressure liquid chromatography (HPLC)

Methods and materials:

Column: A C18 reverse phase μbondapak column (Water P/N 27324), acetonitrile (10%), trifluoroacetic acid (TFA, 0.005%) was used as the mobile phase.

Injection: The sample was dissolved in 10% acetonitrile-TFA (0.005%) at a concentration of 1 mg/ml; injection: 20 μg.

Elution: Gradient elution with acetonitrile, the concentration was increased from 10% to 80% in 70 minutes; flow rate: 0.5 ml/min. Then, it was kept at a concentration of 80% for another 10 minutes (70-80 minutes). The acetonitrile concentration was then reduced to 10% (80-85 minutes) and held for 25 minutes (85-110 minutes).

The absorption wavelength was measured: 207 nm.

Recording paper speed: 0.25 cm/min.

Optical density (OD): full range, 0.128.

Instruments used: Waters Model 510 solvent delivery system; Waters 484 absorbance adjustable detector; Waters 745/745B data model.

Spectral Absorption Analysis: The extract of Xanthoceras sorbifolia was detected using a spectrophotometer (Spectronic Ins. Model Gene Sys2). The extract of Xanthoceras sorbifolia is dissolved in 10% acetonitrile-TFA with a detection wavelength of 200-700 nm.

result:

The HPLC spectrum showed 60-70 peaks. Four of them are the main peaks, 10 are medium peaks, and the rest are small peaks (see Figure 5). 27 of them are major The peaks are numbered a-z in increasing order of concentration of the acetonitrile eluate (see Figure 12).

The three largest absorption wavelengths detected were: 207 nm, 278 nm, and 500 nm (see Figure 13).

The method for detecting the cell-specific toxicity of the extract of the extract of Xanthocergia by MTT is further illustrated by the following example (8).

Methods and materials

Cells: The following human cancer cells were obtained from the American Type Culture Collection: HTB-9 (bladder), HeLa-S3 (cervix), DU145 (prostate), H460 (lung), MCF-7 ( Mammary gland), k562 (leukocyte), HCT116 (colon), HepG2 (liver), U20S (bone), T98G (brain), and OVCAR-3 (ovary).

Culture: HeLa-S3 (cervix), DU145 (prostate), MCF-7 (breast), HepG2 (liver) and T98G (brain) cells were cultured on MEN (Earle salt) medium. HTB-9 (bladder), H460 (lung), 562 (leukocytes) and OVCAR-3 (ovary) were cultured on RPMI-1640 medium, and other cells were on McCoy-5A medium.

These media are supplemented with 10% fetal bovine serum, glutamine and antibiotics. Incubate in an incubator with a CO ₂ concentration of 5%.

MTT detection

The test method is basically in accordance with the method of Carmicheal et al. 1987, and only a few places have been changed. These cells were cultured in small wells with 96 small wells for 24 hours; 10,000 HTB-9 (bladder) per hole, HeLa-S3 (cervix), H460 (lung), HCT116 (colon), T98G (brain), And OVCAR-3 (ovarian) cells; 15,000 DU145 (prostate) per well, MCF-7 (breast), HepG2 (liver) And U20S (bone) cells; 40,000 k562 per hole (white blood cells). Then, a sample of the extract of the dried fruit is placed in a hole, and cultured for another 48 hours (liver and bone cancer cells) for 72 hours, and breast cancer cells for 96 hours. Then, MTT (0.5 mg/ml) was added to each well and cultured for 1 hour. The resulting formazan was dissolved in DMSO and its O.D. value (TD) was measured using an ELISA reader (Dynatech, Model R700). The MTTO.D. value (TO) before the addition of the sample is also measured. The percentage of growth of each cell (%G) can be obtained by the following formula: %G=(TD-TO/TC-TO)×100

(TC is the O.D. value of the control cell group)

When TO < TD, the cell-specific toxicity (LC) value is: % LC = (TD-TO / TO) × 100

result:

Among the 10 kinds of cancer cells, they can be divided into 4 groups according to their sensitivity to extracts of Xanthoceras sorbifolia: the most sensitive are ovarian cancer cells; the second one is bladder, bone, prostate and leukemia cancer cells; It is liver, breast and brain cancer cells; less sensitive are colon, cervix and lung cancer cells (Figures 6, 10, 11 and 14A-E). See their LC50 values (see Table 8-1).

It was also found that low concentrations of extracts of the extract of the genus Canopy stimulate the growth of bladder, bone, and lung cells (Figures 7, 9 and 11). The ability of the pure C. sinensis extract Y component to inhibit cancer cells increased with increasing concentration (compare Figures 14A and 16B, 18). It was initially found that the C. elegans bioactive substance was in components 610 and 1116 of the FPLC (Fig. 16B).

The extraction of the extract of Xanthoceras by fast liquid chromatography (FPLC) is further illustrated by the following example (9).

1, method:

Column: An octadecyl activated silica gel column, 2 cm x 28 cm, with acetonitrile (10%)-TFA (0.005%) as the mobile phase.

Injection: The sample was dissolved in 10% acetonitrile-TFA (0.005%) at a concentration of 100 mg/ml; injection volume: 1-2 ml.

Gradient elution: elute with a gradient of 10-80% acetonitrile (500 ml total).

The absorption wavelength was measured: 254 nm.

Separation of fractions: 90 fractions were obtained from 10% to 72% acetonitrile eluate, 5 ml per component.

Instrument used: AKTA-FPLC; pump: P920; detector: UPC-900; Frac-900.

result:

The FPLC elution profile shows that there are 4-5 large components (see Figure 15). These components were further analyzed by high performance liquid chromatography (HPLC), some of which were localized on the FPLC component map by a particular small peak (component) numbered a-z in Figure 21L.

The fractions isolated by FPLC were divided into 7 groups and used for MTT assay of bladder cell growth. The results showed that at least one group (#5962) inhibited bladder cell growth (Fig. 16B).

2. Further separation of Xanthoceras bioactive substance component #5962 by fast liquid chromatography (FPLC).

method:

Column: octadecyl activated silicone, 50 ml, 2 cm x 28 cm, using acetonitrile (64%)-TFA (0.005%) as the mobile phase.

Injection: The sample is dissolved in 65% acetonitrile-TFA at a concentration of 1-2 mg/ml; Injection volume: 1-2ml.

Elution: Elution with a gradient of 64% acetonitrile.

The absorption wavelength was measured: 254 nm.

Collecting separated components: collecting the first 90 components from the eluent, each component 1ml.

Instrument used: AKTA-FPLC; pump: P920; detector: UPC-900; Frac-900.

result:

Fraction #5692 was further separated and purified by elution with an open ODS-C18 splitter column and 64% isocratic acetonitrile to give two major components, X and Y (Figure 17). However, the MTT assay showed that only component Y cancer cells had an inhibitory response (Fig. 18).

3, high pressure liquid chromatography (HPLC) separation of the fruit of the fruit of the fruit of the fruit of the crown of the Y method:

Column: Waters μbondapak C18 (3.9 mm x 300 cm).

Elution: 35% or 45% isocratic elution.

Flow rate: 0.5 ml/min, 207 nm; dilution: 0.128; detection: O.D. scale 0.128; recording paper speed: 0.25 cm/min.

result:

Elution with a gradient of 35%, component Y separated 4-5 components (Y0, Y1, Y2, Y3, Y4). Among them, Y3 and Y4 are main components. Y1 and Y2 can be a group, and Y3 and Y4 are a group (Fig. 19).

Elution with a 45% isocratic gradient, the components Y1 and Y2 were polymerized into one peak, and Y3 and Y4 were polymerized into one peak (Fig. 20). There is also a component Y5 after the peaks Y3 and Y4.

4. Separating and purifying the biologically active components in component Y by HPLC:

Column: Waters Delta Pak C18-300A.

Elution: 45% isocratic elution.

Flow rate: 1.0 ml/min.

The absorption wavelength was measured: 207 nm.

The peaked fractions were collected and lyophilized in vacuo.

result:

Y1 and Y2 are well separated and collected separately. However, Y3, Y4 and Y5 are not separated (Fig. 21).

Further analysis of the ascending and descending fractions of the components in which Y3 and Y4 were polymerized into one peak by proton NMR spectroscopy revealed that Y3 and Y4 were the same component (Fig. 22). This component is referred to as "Compound Y". The collected compound Y was reanalyzed by HPLC on a C18 column reversed-phase chromatography, and the result was also a peak (Fig. 23).

The finally purified compound Y was in the form of an amorphous white powder. Soluble in hydroalcohol (methanol and ethanol), 50% acetonitrile and 100% pyridine.

The compound Y-MTT assay showed that it has a strong inhibitory activity on the growth of ovarian cancer cells (OSCAR-3) with an IC50 value of only 1 μg/ml, which is 10-15 times higher than its crude extract activity (Fig. 24a and b). Moreover, it selectively inhibits the performance of cancer cells, such as inhibiting ovarian cancer cells and not inhibiting cervical cancer cells (HeLa) (Fig. 25).

Detailed description (6)

The method for determining the chemical structure of the Guanluo bioactive substance disclosed in the present patent is further illustrated by the following example (10).

Determination of the chemical structure of the bioactive substance compound Y of Astragalus membranaceus: A), nuclear magnetic resonance (NMR)

Pure Xanthocera extract Compound Y sample was dissolved in pyridine-d5 (pydine-D5) containing 0.05% TMS using Bruker Avance 600MHz NMR with QXI probe ( ¹ H/ ¹³ C/ ¹⁵ N/ ³¹ P) Resonance meter, 298k NMR spectra of all samples were obtained. The number of scans (16-128) of the one-dimensional ¹ H nuclear magnetic resonance spectrum depends on the concentration of the sample. The two-dimensional HMQC NMR spectrum has a spectral width of 6000×24000 Hz, and the data points of the t1 and t2 dimensions are 2024×256, and the number of scans is 4-128. The two-dimensional HMBC NMR spectrum has a spectral width of 6000×30000 Hz, and the data points of the t1 and t2 dimensions are 2024×256, respectively, and the number of scans is 64. These two-dimensional data were subjected to Fourier transform by mathematical calculus and XWIN-NMR software. The size of the matrix of the two-dimensional HMQC and HMBC NMR spectra obtained was 2048×256 and 2048×512 (data points, F2×F1), respectively.

B), mass spectrometry

The quality of the extract of Xanthoceras sorbifolia L. was determined by MALDI-TOF and ESL-MS mass spectrometry.

1) ALDI-TOF assay

The sample was first dissolved in acetonitrile and mixed with the CHCA mother liquor (α-cyano-4-hydroxycarnitonic acid 10 mg/ml in 50:50 water/acetonitrile and 0.1% TFA). The molecular weight is determined by a high resolution mass spectrometer.

2) SI-MS assay

The samples were measured by a mass spectrometer manufactured by LCQ DECA XP and Thermo Finningan. The sample was electrospray ionized (ESI) and the solvent was acetonitrile.

result

The proton nuclear magnetic resonance spectrum of component Y is shown in Figure 26. The chemical shift data of the protons of compound Y are shown in Table 10-1.

The data of the two-dimensional HMQC nuclear magnetic resonance spectrum of component Y is shown in Fig. 27 and Figs. 28A and B. The chemical shift data are shown in Table 10-2 (HMQC-peak shift) and Table 10.3 (HMBC-peak shift).

The chemical functional group determined according to the data of the chemical shifts of ¹³ C and ¹ H of the composition component (Compound) Y is shown in Table 10-4.

Figure 29 is a MALDI-TOF and ESL-MS mass spectrometric map of the composition of the crown of the fruit (compound) Y.

Based on these data and analysis, the composition of the canopy component (compound) Y is C ₅₇ H ₈₈ O ₂₃ .

The chemical structure of the composition component (Compound) Y is shown in the following figure:

The chemical name of compound Y is: 3-0-[β-D-galactopyranosyl (1→2)]- α -L-arabinofuranosyl (1→3)-β-D-glucopyran Mercapto-21,22-O-dipodoxime-3β,15α,16α,21β,22α,28-hexahydroxyolean-12-ene pentacyclic triterpenoid saponin. It belongs to triterpenoid saponins, which are pentacyclic triterpenoids with one sugar chain and two dimeric guanidine groups.

The method for determining the chemical structure of the Guanluo bioactive substance disclosed in the present patent is further illustrated by the following example (11).

1, Determination of the chemical structure of the extract of Y. glabra L.

5 mg of Xanthoceras extract Y was dissolved in 3 ml of methanol, and then treated with 3 ml of 3N HCl acid, and refluxed for 4 hours to carry out hydrolysis. After hydrolysis, it was neutralized with 5% Na ₂ CO ₃ and then extracted three times with EtOAc to obtain a water-soluble portion, a sugar-containing portion, and a fat-soluble portion containing an aglycon. The aglycone (aglycon) was further purified by gel column chromatography (CHCl ₃ : MeOH, 1:9) or C18 ODS column high pressure liquid chromatography (HPLC). Approximately 2 mg of the new compound Yc was obtained (see figure below). (See Method: Essentials of Carbohydrate Chemistry. By John F. Robyt, Springer, 1998).

Oligosaccharides are cleaved by incomplete acid hydrolysis and special enzymatic hydrolysis. For example, the arabinofuranosonic acid 1→4 bond can be cleaved by an alpha-amylase. Other enzymes such as β- Amylase, isozyme, glucose oxidase, mannanase and maltase can also be used to cleave oligosaccharides in saponins.

The guanidine amino group in the saponin can be cleaved by hydrazine hydrolysis. For example, compound Y is dissolved in 1 M NaOH, stirred at room temperature for 2-3 hours, acidified or neutralized with 2M HCl, and the hydrolyzed saponin is extracted with ethyl acetate. It was then further purified by HPLC on a C18 column.

By enzymatic hydrolysis, compound Y can be converted to the following compounds: and compounds

2, Determination of the chemical structure of the Y1 component of the biological active substance method: A), nuclear magnetic resonance analysis

A sample of the compound of the extract of the extract of the compound Y1 or other components was dissolved in pyridine d5 containing 05% TMS (v/v). A 298 k NMR spectrum of all samples was obtained using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe ( ¹ H/ ¹³ C/ ¹⁵ N/ ³¹ P). The number of scans (16-128) of the one-dimensional ¹ H nuclear magnetic resonance spectrum depends on the concentration of the sample. The two-dimensional HMQC NMR spectrum has a spectral width of 6000 x 24000 Hz, and the data points of the t1 and t2 dimensions are 2024 x 256 and the number of scans is 4-128. The two-dimensional HMBC NMR spectrum has a spectral width of 6000 x 30000 Hz, and the data points of the t1 and t2 dimensions are 2024 x 256, respectively, and the number of scans is 64. These two-dimensional data were subjected to Fourier transform by mathematical calculation and XWIN-NMR software. The matrix of the two-dimensional HMQC and HMBC NMR spectra obtained were 2048 x 256 and 2048 x 512 (data points, F2 x F1), respectively.

B), mass spectrometry

The quality of the extract of Xanthoceras sorbifolia L. was determined by MALDI-TOF and ESL-MS mass spectrometry.

1) ALDI-TOF assay: The sample is first dissolved in acetonitrile and then mixed with the CHCA mother liquor (α-cyano-4-hydroxycarnitonic acid 10 mg/ml in 50:50 water/acetonitrile and 0.1% TFA) . The molecular weight is determined by a high resolution mass spectrometer.

2) SI-MS assay: Samples were determined by mass spectrometers manufactured by LCQ DECA XP and Thermo Finningan. The sample was electrospray ionized (ESI) and the solvent was acetonitrile.

result

1) The chemical functional group determined according to the nuclear magnetic resonance data of ¹³ C and ¹ H of the crown fruit compound Y1 is shown in Table 11.1.

2) The two-dimensional HMBC nuclear magnetic resonance spectrum of Compound Y1 is shown in Figures 40 and 41. The data of chemical shifts of HMBC NMR spectra are shown in Table 11.2 and Table 11.3.

3) The two-dimensional HMQC nuclear magnetic resonance spectrum of the Xanthoceras compound Y1 is shown in Fig. 42. The NMR data for two-dimensional HMQC are shown in Tables 11.4 and 11.5.

4) The COSY nuclear magnetic resonance spectrum of the Wolverine component (compound) Y1 is shown in Table 11.6.

According to these data and analysis, the extract component (compound) Y1 is a triterpenoid saponin containing 2 sugar chains, 4 sugars, 2 angelica sulfhydryl groups, and linked to a sugar chain containing 1 sugar. The chemical structure of the Xanthium fruit component (compound) Y1 is shown in the following figure:

3-0-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3)-β-D-glucopyranyl-21-O -(3,4-dihydrocarbyl)-α-L-rhamnosepyranoyl-22-0-acetamido-3β,16α,21β,22α,28-pentahydroxy olean-12- Triterpenoid saponin. Xanthoceras component (compound) Y1 Molecular formula: C ₆₅ H ₁₀₀ O ₂₇

The MTT assay of the Y1 component showed that it had a strong inhibitory activity on the growth of ovarian cancer cells (OSCAR-3) with an IC50 value of only 1 μg/ml (Fig. 43).

The method for determining the chemical structure of the Guanluo bioactive substance disclosed in the present patent is further illustrated by the following example (12).

Purification of compound R1 from extract of Xanthoceras A) Separation of components of the extract of Xanthoceras sorbifolia by fast liquid chromatography (FPLC) (gradient elution) method:

Column: octadecyl activated silicone, 50ml, 2cm X 28cm, with acetonitrile

(10%)-TFA (0.005%) is the mobile phase.

Injection: The sample was dissolved in 10% acetonitrile-TFA (0.005%) at a concentration of 1-2 mg/ml; injection volume: 1-2 ml.

Elution: Elution with a gradient of 10-80% acetonitrile (500 ml total).

The absorption wavelength was measured: 254 nm.

Separate fractions were collected: The first 90 fractions, 5 ml each, were collected from a 10-72% acetonitrile eluate.

Instrument used: AKTA-FPLC; pump: P920; detector: UPC-900; Frac-900.

result:

The FPLC elution profile shows that there are 4-5 large components (see Figure 15). These components were further analyzed by high performance liquid chromatography (HPLC), and some of the specific components numbered a-z were identified on the component map of the fast liquid chromatography (FPLC).

B) Separation of component R by fast liquid chromatography (FPLC) (30% acetonitrile gradient elution) method:

Column: octadccyl activated silicone, 50ml, 2cm X 28cm, with acetonitrile

(30%)-TFA (0.005%) is the mobile phase.

Injection: The sample is dissolved in 65% acetonitrile-TFA (0.005%) at a concentration of 1-2 mg/ml; injection volume: 0.2 ml.

Elution: Elution with a gradient of 30% acetonitrile.

The absorption wavelength was measured: 254 nm.

Separation of fractions was collected: the first 90 fractions were collected from the acetonitrile eluate, 5 ml per fraction.

Instrument used: AKTA-FPLC; pump: P920; detector: UPC-900; Frac-900.

result:

Fractions 39-41 were collected together and further separated and purified by elution with an open ODS-C18 splitter column and 30% acetonitrile to yield two groups of six identifiable components (Figure 50). Components 6-13 were further separated and purified by high performance liquid chromatography (HPLC).

C) Purification of component R1 by HPLC method:

Column: Waters Delta μbonda Pak C18 column and Waters Delta Pak C18 column, 7.8 mm X 30 cm.

Elution: 10-80% acetonitrile gradient elution and 30% acetonitrile gradient elution.

Flow rate: 0.5 ml/min, dilution: 0.128; recording paper speed: 0.25 cm/min.

The absorption wavelength was measured: 207 nm.

result:

Elution with a gradient of 10-80% acetonitrile, component 9-11 contains 1 major Divided into several small components (Figure 51). These fractions were further eluted on a Delta Pak C18 column using a Delta Pak C18 column to give 4-5 components, the main component at R1 (Fig. 52, left side). R1 is separately separated and collected from the column (Fig. 52, right side).

The final purified R1 has an amorphous white powder appearance. Soluble in hydroalcohol (methanol and ethanol), 50% acetonitrile and 100% pyridine.

Determination of chemical structure of R1 method 1), nuclear magnetic resonance analysis

A sample of the extract of R. ostreatus R1 and other components was dissolved in pyridine d5 containing 05% TMS (v/v). A 298 k NMR spectrum of all samples was obtained using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe ( ¹ H/ ¹³ C/ ¹⁵ N/ ³¹ P). The number of scans (16-128) of the one-dimensional ¹ H nuclear magnetic resonance spectrum depends on the concentration of the sample. The two-dimensional HMQC NMR spectrum has a spectral width of 6000 x 24000 Hz. The data points of the t1 and t2 dimensions are 2024 x 256 and the number of scans is 4-128. The two-dimensional HMBC NMR spectrum has a spectral width of 6000 x 30000 Hz, and the data points of the t1 and t2 dimensions are 2024 x 256, respectively, and the number of scans is 64. These two-dimensional data were subjected to Fourier transform by mathematical calculation and XWIN-NMR software. The matrix of the two-dimensional HMQC and HMBC NMR spectra obtained were 2048 x 256 and 2048 x 512 (data points, F2 x F1), respectively.

2), mass spectrometry

The quality of the extract of Xanthoceras sorbifolia L. was determined by MALDI-TOF and ESL-MS mass spectrometry.

A) ALDI-TOF assay: The sample is first dissolved in acetonitrile and then mixed with the CHCA mother liquor ( α -cyano-4-hydroxycarnitonic acid 10 mg/ml in 50:50 water/acetonitrile and 0.1% TFA) . The molecular weight is determined by a high resolution mass spectrometer.

B) SI-MS assay: Samples were determined by mass spectrometers manufactured by LCQ DECA XP and Thermo Finningan. The sample was electrospray ionized (ESI) and the solvent was acetonitrile.

result

1), the proton nuclear magnetic resonance spectrum of component R is shown in Fig. 53. Nuclear magnetic resonance spectrum of protons of component R1 pure. The proton nuclear magnetic resonance spectrum of component R1 is enlarged as shown in Figures 55-57. The two-dimensional HMQC nuclear magnetic resonance spectrum of the component R1 is shown in Figures 58-60. The two-dimensional HMBC nuclear magnetic resonance spectrum of the component R1 is shown in Figures 61-62. The two-dimensional COSY nuclear magnetic resonance spectrum of the component R1 is shown in Fig. 63. The carbon 13 nuclear magnetic resonance spectrum is shown in Fig. 64.

2) For all NMR chemical shifts, see Table 12.1; 12.2; 12.3; 12.4; 12.5; 12.6.

3) The chemical functional group of component R1 determined according to all the above materials is shown in Table 12.7.

4), component R1 formula: C ₆₅ H ₁₀₆ O ₂₉ .

Based on these data and analysis, the extract component (compound) R1 of the extract is a triterpenoid saponin containing five sugar chains of five sugars, one angelica thiol group, and a sugar chain containing two sugars.

The chemical name of the compound R1 is: 3-0-[Dangdangyl-(1→3)-β-D-glucopyranosyl (1→6)]-β- β -D-glucopyraninyl-28 -O-[α-L-rhamnopyrano-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranin-3β,21β , 22α,28-tetrahydroxyolean-12-ene pentacyclic triterpenoid saponin (3-O-[angeloyl-(1→3)-β-D-glucopyranosyl-(1→6)]-β-D- Glucpyranosyl-(1→6)-β--28-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosy-1-3β, 21β, 22α, 28-tetrahydroxyolean-12-ene].

The chemical structural formula of the compound R1 is as follows:

The method for determining the chemical structure of the active material of the extract of the crown of the living organism of the present invention disclosed in the patent is further illustrated by the following example (13).

1, purification of compound O from the extract of Xanthoceras A) Separation of components of the extract of Xanthoceras sorbifolia by fast liquid chromatography (FPLC) (gradient elution) method:

Column: octadecyl activated silicone, 50 ml, 2 cm X 28 cm, using acetonitrile (10%)-TFA (0.005%) as the mobile phase.

Injection: 100 mg/ml of the sample was dissolved in 10% acetonitrile-TFA (0.005%) at a concentration of 1-2 mg/ml; injection volume: 1-2 ml.

Elution: Elution with a gradient of 10-80% acetonitrile (500 ml total).

The absorption wavelength was measured: 254 nm.

Separate fractions were collected: The first 90 fractions, 5 ml each, were collected from a 10-72% acetonitrile eluate.

Instrument: AKTA-FPLC; pump: P920; detector: UPC-900; Frac-900.

result:

The FPLC elution profile shows that there are 4-5 large components (see Figure 15). These components were further analyzed by high performance liquid chromatography (HPLC), and some of the specific components numbered a-z were identified on the component map of the fast liquid chromatography (FPLC).

B) Separation of component O by rapid liquid chromatography (FPLC) (20% acetonitrile gradient elution) method:

Column: HPLC column (Waters Delta Pak C18-300A); Injection: Fraction #28 (from FPLC gradient elution) combined for HPLC.

Injection speed: 1 ml / sec.

Elution: Elution with a gradient of 20% acetonitrile.

The absorption wavelength was measured: 207 nm.

Separate fractions were collected: fractions 28, 34 and 54 were collected from the acetonitrile eluate. These components were freeze-dried and analyzed again using the same chromatographic method.

result:

Sixteen components were resolved on the elution profile (Fig. 72). Under the same conditions, components 28, 34 and 54 were further analyzed by HPLC. It can be seen from Figures 82, 83 and 84 that components 28, 34 and 54 are single peaks indicating that they have all been purified.

The final purified O-23, and O-34 are in the form of an amorphous yellowish powder. Soluble in hydroalcohol (methanol and ethanol), 50% acetonitrile and 100% In pyridine. The final purified O-54 is in the form of an amorphous white powder. Soluble in hydroalcohol (methanol and ethanol), 50% acetonitrile and 100% pyridine.

2, Determination of the chemical structure of the extract of Ophiopogon japonica method 1), nuclear magnetic resonance analysis

A sample of the extract of O. glabra L. and other components was dissolved in pyridine d5 containing 05% TMS (v/v). A 298 k NMR spectrum of all samples was obtained using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe ( ¹ H/ ¹³ C/ ¹⁵ N/ ³¹ P). The number of scans (16-128) of the one-dimensional ¹ H nuclear magnetic resonance spectrum depends on the concentration of the sample. The two-dimensional HMQC NMR spectrum has a spectral width of 6000 x 24000 Hz. The data points of the t1 and t2 dimensions are 2024 x 256 and the number of scans is 4-128. The two-dimensional HMBC NMR spectrum has a spectral width of 6000 x 30000 Hz, and the data points of the t1 and t2 dimensions are 2024 x 256, respectively, and the number of scans is 64. These two-dimensional data were subjected to Fourier transform by mathematical calculation and XWIN-NMR software. The matrix of the two-dimensional HMQC and HMBC NMR spectra obtained were 2048 x 256 and 2048 x 512 (data points, F2 x F1), respectively.

2), mass spectrometry

The quality of the extract of Xanthoceras sorbifolia L. was determined by MALDI-TOF and ESL-MS mass spectrometry.

a. ALDI-TOF assay: first dissolve the sample in acetonitrile and then mix with the CHCA mother liquor ( α -cyano-4-hydroxycarnitonic acid 10 mg/ml in 50:50 water/acetonitrile and 0.1% TFA) . The molecular weight is determined by a high resolution mass spectrometer.

b. SI-MS assay: Samples were determined by mass spectrometers manufactured by LCQ DECA XP and Thermo Finningan. The sample was dissolved by electrospray ionization (ESI) The agent is acetonitrile.

references

1, Chen Qi editor of "Chinese medicine pharmacology research methodology" People's Health Publishing House 1995 first edition, P892

2. Huang Zhaosheng, Liu Mingping, Chen Changzhou, etc., Yang Shoudan's experimental study on improving learning and memory function, Chinese Journal of Integrated Traditional and Western Medicine 1997,9(17)9, P553

3. Zhang Yan, Zhang Heyun, Li Weiping, An Jifu's research on promoting intelligence, Chinese Journal of Pharmacology, 1995,7,11(3)P233

4. Yang Jun, Wang Jing, Feng Ping and other brain Kangtai capsules to improve learning and memory in mice, new Chinese medicine and clinical pharmacology 2000,1 (11) 1, P29

5. Yang Jun, Wang Jing, Zhang Jixun et al. The improvement effect of total glycosides of Radix Paeoniae Alba on learning and memory in mice. Chinese Pharmacological Bulletin 2000, 2(16)1, P46

6. Xia Weijun, Jin Miaowen, Zhang Li et al. Experimental study on the treatment of senile vascular dementia with Dangdang Decoction, Chinese Pharmacology and Clinical Practice 2000, 16(4)

7. Qi Huimin, Yu Jingzao, Gong Yuning et al. Effects of Tongmai Yizhi Capsule on memory function of mice, pharmacology and clinical practice of Chinese medicine 2000,16(5)P40

8. Wei Xiaolong, Zhang Yongxiang, Research progress in animal models of Alzheimer's disease, Chinese Pharmacological Bulletin 2000, 8, (16) 4, P372

9. Ministry of Health, Drug Administration, Guidelines for New Drug Research, Guiding Principles for Neuropharmacological Pharmacodynamics (Spirit) P45

10. Zhang Danshen, Zhang Juntian, Effects of total ginsenosides on memory impairment in mice induced by B-amyloid peptide, Chinese Journal of Pharmacology 2000,8,(16)4,P422

Figure 1 (a) and (b). Improvement of learning and memory in mice after 9 days of administration (water Maze method).

Fig. 2 (a) and (b). Effect of extracts of Xanthoceras sorbifolia X and Y on memory impairment in mice given pentobarbital for 3 days (water maze method).

Figure 3. Effect of X and Y administration of Xanthoceras sorbifolia L. extract for 10 days on urine output in mice.

Figure 4. A typical sleep cycle for people.

Figure 5. High performance liquid chromatogram of the extract of the fruit of the corolla.

Figure 6. Comparison of the inhibitory effect of Xanthoceras sorbifolia extract on breast cancer (IC50 65 μg/ml) and leukemia cancer cells (IC50 35 μg/ml).

Figure 7. Canopy extract promotes bladder cell growth at low concentrations, and bladder cell growth is increased by 25% at a concentration of 10 μg/ml. The extract of Xanthoceras sorbifolia inhibits the growth of bladder cancer cells at high concentrations (IC50 is 45-60 μg/ml). At the same time, the extract of Xanthoceras sorbifolia inhibited the growth of ovarian cancer cells (IC50 was 15 μg/ml).

Figure 8. Extract of Campanula sinensis has no or slightly stimulating effects on brain cell growth at concentrations below 40 μg/ml. The extract of Xanthoceras sorbifolia has an inhibitory effect on the growth of the prostate (IC50 of 70 μg/ml) and brain cancer cells (IC50 is 40 μg/ml).

Figure 9. The growth of lung cells can be grown at a concentration of 10 μg/ml, and the growth is increased by 20% at 50 μg/ml. Bladder cells were grown 5% in RPMI-1640 medium containing 10 μg/ml of extract of C. elegans. However, the extract of Xanthoceras sorbifolia inhibited the growth of bladder cancer cells at a high concentration with an IC50 of 45-60 μg/ml. The extract of Xanthoceras sorbifolia inhibited the growth of liver cancer cells with an IC50 of 68 μg/ml.

Figure 10. The effect of the extract of C. elegans on the growth of ovarian cancer and cervical cancer cells is different. The therapeutic effect of extract of Xanthoceras sorbifolia on ovarian cancer is considerable, and the inhibition on the growth of cervical cancer cells is weak.

Figure 11. Bone cells contain extracts of Xanthoceras sorbifolia at low concentrations (eg 10 μg/ml) Growth in RPMI-1640 medium increased by 20%, and at high concentrations, inhibited the growth of bone cancer cells with an IC50 of 40 μg/ml).

Figure 12. Separation of the extract of the extract of Xanthoceras on the μbondapak C18 column by high performance liquid chromatography (HPLC).

21A-B are representative elution profiles of fast liquid chromatography. 21C-K are HPLC chromatograms of the components obtained from fast liquid chromatography. Figure 21L: Original material; Figure 21M: Thin layer chromatography (TLC). The fractions selected from the fast liquid chromatography were stained with carbohydrates and analyzed on TLC.

Figure 13. Absorption spectra of extracts of Xanthoceras sorbifolia.

Abscissa: wavelength nm

Vertical coordinate: light intensity.

The extract of Xanthoceras sorbifolia has three absorption peaks at 207 nm, 278 nm and 500 nm.

Figures 14A-23E. MTT assay for the effect of extracts of C. elegans on cell growth. The sensitivity of various cells to extracts of Xanthoceras sorbifolia is as follows: Figure 14A is extremely sensitive: ovarian cells; Figure 14B is sensitive: bladder and bone cells; Figure 14C moderately sensitive: white blood cells and hepatocytes; Figure 14D slightly sensitive: prostate, breast and Brain cells; Figure 14E Less sensitive: colon, cervix and lung cells.

Figure 15. Gradient (10-80%) elution profile of Xanthoceras sorbifolia extract on fast liquid chromatography (FPLC).

Vertical coordinate: light intensity (245nm)

Abscissa: eluate volume (ml).

Figure 16A. Selected components of the extract of C. elegans obtained from fast liquid chromatography (FPLC).

Figure 16B. Effect of components of the extract of C. elegans obtained from fast liquid chromatography (FPLC) on bladder cell growth (according to MTT assay): component 5962 (Y) inhibits cell growth; components 610 and 1116 are slightly Stimulate cell growth.

Abscissa: concentration (ul/ml)

Vertical coordinate: cell growth (%).

Figure 17. A 64% Ethyl isocratic elution profile of the component 5962 of the extract of Coleoptera. The two main components X and Y are separated on the FPLC.

Abscissa: light intensity (254nm)

Vertical coordinate: component number (1 ml / component).

Figure 18. Effect of fractions X (2021) and Y (2728) of the extract of Campanula sinensis on bladder cell growth: only Y inhibits bladder cell growth.

Figure 19. High performance liquid chromatography (HPLC) 35% Ethyl isocratic gradient elution profile of the component Y of the extract of Xanthoceras sorbifolia L., showing that the Y(2728) component contains 4-5 compounds: Y0, Y1, Y2, Y3 and Y4.

Figure 20. High-performance liquid chromatography (HPLC) 45% Ethyl isocratic gradient elution of the component Y of the extract of Xanthoceras sorbifolia, showing that the peaks of Y3 and Y4 are superimposed, and Y1 and Y2 are clearly separated from Y3 and Y4. Open.

Figure 21. Equivalent gradient elution profile of high performance liquid chromatography (HPLC) 45% Ethyl acetate extract on the C18 column (Delta Pak C18). Y1 and Y2 are clearly separated.

Figure 22. Comparison of the proton nuclear magnetic resonance spectra of the ascending (top half of the curve) and the lower half of the descending curve of the Y3 and Y4Y peaks of the extract of the extract of the extract. Show them no difference.

Figure 23. Shows the purity of component Y (collected from a 45% ethyl acetate gradient elution profile on a high performance liquid chromatography Delta Pak C18 column).

Figure 24A. Shows the potential point scale of purified compound Y in inhibiting ovarian cancer (OCAR-3).

Figure 24B. shows the linear scale of the purified compound Y in inhibiting ovarian cancer (OCAR-3).

Abscissa: concentration (μg/ml), ordinate: cell growth (%) (detected according to MTT), IC50 about 1 μg/ml.

Figure 25. Comparison of the effect of compound Y on the growth of ovarian and cervical cancer cells: ovarian cancer cells are sensitive to compound Y, whereas cervical cancer cells are not sensitive. The IC50 of ovarian cancer cells is about 1 μg/ml, indicating that ovarian cancer cells are sensitive to purified compound Y.

Figure 26. Chemical shift map of proton nuclear magnetic resonance (NMR) of compound Y.

Figure 27. Two-dimensional NMR (HMQC) pattern of compound Y (see also chemical shift data in Table 10.2).

Figure 28A. HMBC Atlas Level 1 for Compound Y (see also Chemical Displacement Data in Table 10.3).

Figure 28B. HMBC Atlas Level 2 for Compound Y (see also Chemical Displacement Data in Table 10.3).

Figure 29A-1. One of the mass spectra of Compound Y: Y+Matrix (CHCA) + Angiotensin 1 "two point calibration".

Figure 29A-2. Mass spectrum of Compound Y: Y+Matrix (CHCA) + Angiotensin "one point calibration".

Figure 29A-3. Mass spectrum of Compound Y: Y+Matrix (CHCA).

Figure 29A-4. Mass spectrum of Compound Y: Matrix (CHCA) only.

Figure 29A-5. Mass spectrum of Compound Y: Angiotensin 1+Matrix (CHCA).

Figure 29B. Mass spectrum of compound Y (ESI-MS).

Figure 30. Chemical structure and name of compound Y: 3-0-[β-D-galactopyranosyl (1→2)]-α-L-arabinofuranosyl (1→3-β-D - glucopyranosyl-21,22-O-dipodoxime-3β, 15α, 16α, 21β, 22α, 28-hexahydroxyoleolin-12-ene pentacyclic triterpenoid saponin.

Figure 31. Proton nuclear magnetic resonance (NMR) spectra of compound Y1. The chemical shift data is shown in Table 11.2.

Figure 32. Compound Y1 two-dimensional nuclear magnetic resonance (HMQC) map. The chemical shift data are shown in Tables 11.5 and 11.4.

Figure 33. Proton nuclear magnetic resonance spectroscopy of compound Y2. The chemical shift data is shown in Table 26.

Figure 34. Two-dimensional nuclear magnetic resonance (HMQC) pattern of compound Y2. The chemical shift data is shown in Table 27.

Figure 35. Proton nuclear magnetic resonance spectroscopy of compound Y5. The chemical shift data is shown in Table 28.

Figure 36. Compound Y5 two-dimensional nuclear magnetic resonance (HMQC) map. The chemical shift data is shown in Table 28.

Figure 37. Proton COSY-NMR spectrum of compound Y1. See chemical shift data Table 11.6.

Figure 38. Four possible chemical structures of Compound Y1: Figure 38A: Chemical Structure Y1-1; Figure 38B: Chemical Structure Y1-2; Figure 38C: Chemical Structure Y1-3; Figure 38D: Chemical Structure Y1-4.

Figure 39. Chemical structure of compound Y1.

Figure 40. A map of compound Y1 two-dimensional nuclear magnetic resonance (HMBC) level 1.

Figure 41. A map of compound Y1 two-dimensional nuclear magnetic resonance (HMBC) level 2.

Figure 42. A map of compound Y1 two-dimensional nuclear magnetic resonance (HMQC) level 2.

Figure 43. Comparison of the effects of compounds Y1 and Y2 on the growth of ovarian cancer cells (abscissa: extract concentration μg/ml, ordinate: % cancer cell growth).

Figure 44. Effect of extract of Xanthoceras on the urine output of mice in Table 5A-1 (25 days after the test).

Figure 45. Effect of extract of Xanthoceras on urination rate in mice in Table 5A-2 (25 days after the test).

Figure 46. Table 5A-3, Effect of extract of Xanthoceras on the specific gravity and pH of urine in mice (after 25 days with the test).

Figure 47. Effect of extract of Xanthoceras on the Na+, K+ and Cl- concentrations in mouse urine in Table 5A-4 (25 days after the test).

Figure 48. Effect of extract of Xanthoceras on urine output (25 days of administration).

Figure 49. Effect of extract of Cappari FS(X) on urination rate (25 days of administration).

Figure 50. Ethyl isocratic elution profile of the extract R of the extract of the extract of Xanthoceras sorbifolia L. by fast liquid chromatography (IS0-30)

Figure 51. Components #9, #10 and #11 were analyzed by high performance liquid chromatography.

Figure 52. Purification of compound R by high performance liquid chromatography (Delta-Pak C18 column).

Figure 53. Compound R proton nuclear magnetic resonance spectroscopy.

Figure 54. Proton nuclear magnetic resonance spectroscopy of compound R1.

Figure 55. Proton-NMR-expand-1 of the compound R1 proton nuclear magnetic resonance spectrum.

Figure 56. Proton-NMR-expand-2 of the compound R1 proton nuclear magnetic resonance spectrum.

Figure 57. Compound R1 proton-NMR-expand.

Figure 58. Two-dimensional HMQC nuclear magnetic resonance spectrum of compound R1.

Figure 59. Two-dimensional HMQC NMR level 1 spectrum of compound R1.

Figure 60. Two-dimensional HMQC NMR level 2 spectrum of compound R1.

Figure 61. Two-dimensional HMBC NMR level 1 spectrum of compound R1.

Figure 62. Two-dimensional HMBC NMR level 2 spectrum of compound R1.

Figure 63. Two-dimensional COSY nuclear magnetic resonance spectrum of compound R1.

Figure 64. Nuclear Magnetic Resonance Spectrum of Compound R1 Carbon 13.

Figure 65. Chemical structure of compound R1.

Figure 66. Compound compound Y-a structural formula.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C

R4=B or C

Figure 67. Compound compound Y-b structural formula.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C

R4=B or C

Figure 68. Compound Y-c structural formula.

Figure 69. Structure of compound Y1-a.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C; R3=A or B or C

R4=B or C

Figure 70. Structure of compound Y1-b.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C; R3=A or B or C

R4=B or C

Figure 71. Structure of compound Y1-c.

A=Angelica Angeloyl

B=acetyl group (acetyl)

C=H

S1 = sugar chain containing one or more sugars or uronic acids such as D -glucose, D -galactose, L -rhamnose, L -arabinose, D -xylose, D -glucuronic acid, D- Galacturonic acid, and its derivatives.

When R5=B or C or S1

Then R1=A or B or C

R2=A or B or C; R3=A or B or C

R4=B or C

Figure 72. Component O is a gradient elution (iso-20) component plot of 20% Ethylene with FPLC and HPLC.

Figure 73. Secondary liquid chromatogram of component 28 (from iso-20).

Figure 74. Secondary liquid chromatogram of component 34 (from iso-20).

Figure 75. Secondary liquid chromatogram of component 54 (from iso-20).

Claims (40)

A compound or a salt thereof comprising the following structures: A compound according to claim 1 for use in the preparation of a health care product or a medicament for inhibiting the growth of tumor cells. The compound of claim 1, wherein the compound is a salt. A composition for inhibiting growth of an ovarian cancer cell, which comprises a compound as claimed in claim 1 and a suitable carrier. A pharmaceutical composition comprising an effective amount of a compound as claimed in claim 1 and a pharmaceutically acceptable carrier. An anti-ovarian cancer composition comprising the pharmaceutical composition according to item 5 of the patent application. A method for preparing a medicament for inhibiting growth of ovarian cancer cells, characterized by comprising an effective concentration of the fifth item of the patent application scope The pharmaceutical composition is used to prepare a drug. A method of preparing a medicament for treating or preventing ovarian cancer comprising administering an effective amount of a composition as in claim 4 of the patent application to prepare a medicament for treating or preventing ovarian cancer. A pharmaceutical composition comprising a compound as claimed in claim 1 for use as a cancer cell inhibitor. A pharmaceutical composition comprising a compound according to claim 1 or a salt thereof for treating cancer, wherein the cancer is breast cancer, blood cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, bone cancer, colon Cancer, cervical cancer, lung cancer or brain cancer. A pharmaceutical composition according to claim 10, which is for use in the treatment of bone cancer. A method for separating a compound according to claim 1 from Xanthoceras, which comprises the steps of: a. leaching an extract of a plant with an organic solvent (one or more) for an appropriate amount of time to obtain an organic extract b. collecting the leachate; c. reheating the organic leach solution to obtain a second leach solution; d. removing the organic solvent of the second leach solution; e. drying the second leach solution Take the crude extract powder of the fruit. f. Separating and purifying the crude extract powder by high pressure phase chromatography (HPLC) and fast liquid chromatography (FPLC) using tannin extract, C18 and other equivalent solid materials to obtain a component; g. detecting absorption wavelength: 207 nm or 254 nm ; h. identifying the biologically active component of the extract; i. purifying the biologically active component(s) of the extract powder with FPLC to obtain one or more portions of the biologically active component; and j. The compound was isolated by preparative HPLC. The method of claim 12, wherein the canopy powder is a shell, a shoot, a stem, a leaf, a nut, a root, a bark or a seed shell prepared from the herb of the genus Corolla. The method of claim 12, wherein the organic solvent is ethanol, methanol, diethyl ether, chloroform, alcohol or acetone. The method of claim 12, wherein the ratio of the crown fruit powder to the organic solvent is 1:2. The method of claim 12, wherein the leaching step (a) is carried out 4-5 times for 20-35 hours each time. The method of claim 12, wherein the reflux hot lifting step (c) is carried out 2-3 times. The method of claim 12, wherein the identifying step (g) is performed using an MTT assay. The method of claim 12, wherein the compound is biologically active. A composition for treating bladder cancer characterized by comprising a compound as in claim 1 of the patent application. A method for preparing a medicament for treating or preventing ovarian cancer, which is characterized in that a pharmaceutical composition according to claim 5 of the patent application is used to prepare a medicament for treating or preventing ovarian cancer. A compound comprising the structure as follows: A compound comprising the structure as follows: A compound comprising the structure as follows: A compound comprising the structure as follows: A compound comprising the structure as follows: A compound comprising the following compound name: 3-0-[ β -D-galactofuranyl (1→2)]- α -L-arabinofuranosyl (1→3) -β -D- Glucosylpyridinyl-21-O-(3,4-dihydrocarbyl) -α -L-rhamnose piperidinyl-22-0-acetamido-3β,16α,21β,22α,28- Pentahydroxy olean-12-ene. A compound comprising the structure as follows: A pharmaceutical composition comprising a compound as claimed in claim 26 for use as a cancer cell inhibitor. A pharmaceutical composition comprising a compound as claimed in claim 26 for the treatment of brain damage. A pharmaceutical composition comprising a compound according to claim 26 of the patent application for treating cancer, wherein the cancer is breast cancer, leukemia, liver cancer, ovarian cancer, bladder cancer, prostate cancer and/or brain cancer. A pharmaceutical composition comprising a compound as claimed in claim 26 for the treatment of bone cancer. A pharmaceutical composition against bladder cancer comprising a compound as in claim 26 of the patent application. A pharmaceutical composition comprising an effective amount of a compound of the structure: and a pharmaceutically acceptable carrier: Wherein R1, R2, R3, and R4 are short aliphatic chains and R5=OH. A pharmaceutical composition according to claim 34, wherein R1 = R2 = R3 = R4 = CH3. The pharmaceutical composition according to claim 34 of the patent application is used for preparing a medicament for inhibiting the growth of tumor cells and treating cancer, which comprises breast cancer and blood cancer. , liver cancer, ovarian cancer, bladder cancer, prostate cancer, brain cancer, bone cancer colon cancer, cervical cancer or lung cancer. The compound according to any one of claims 22-26 for use in the preparation of a medicament for inhibiting the growth of tumor cells and treating cancer, which comprises breast cancer, blood cancer, liver cancer, ovarian cancer, bladder cancer, prostate cancer, brain cancer, bone cancer , colon cancer, cervical cancer or lung cancer. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 22-26 and a pharmaceutically acceptable carrier. A method for preparing a medicament for inhibiting growth of a tumor cell, characterized in that a drug is prepared by inhibiting the growth of the tumor cell with an effective amount of a compound having the following structure and a pharmaceutically acceptable carrier: Wherein R1, R2, R3, and R4 are short aliphatic chains and R5=OH. The method of claim 39, wherein R1 = R2 = R3 = R4 = CH3.