CN109851655B

CN109851655B - Dammarane type saponin compound with anti-tumor activity, preparation method and application thereof

Info

Publication number: CN109851655B
Application number: CN201811397122.3A
Authority: CN
Inventors: 朴香兰; 崔伟业
Original assignee: Minzu University of China
Current assignee: Minzu University of China
Priority date: 2018-11-22
Filing date: 2018-11-22
Publication date: 2022-10-04
Anticipated expiration: 2038-11-22
Also published as: CN109851655A

Abstract

The invention discloses a gynostemma pentaphylla extract compound shown in formulas (I) and (II) with an anti-tumor effect, a preparation method thereof and application of the compound in medicines, and belongs to the field of phytochemistry. The process for the preparation of the compounds of formulae (I) and (II) according to the invention comprises the following steps: treating herba Gynostemmatis at high temperature and high pressure, extracting with ethanol under reflux, and concentrating; suspending the ethanol extract with water, and eluting with HP-20 macroporous resin, water, 50% ethanol, and 95% ethanol respectively; concentrating the 95% ethanol eluate under reduced pressure, mixing with silica gel 1: 1, separating with silica gel column chromatography, eluting with dichloromethane/methanol at volume ratio of 20: 1-1: 1, segmenting, eluting 8 th segment with preparative chromatographic column with acetonitrile/water at volume ratio of 50: 50 to obtain the final product. Pharmacodynamic tests show that the compounds shown in the formulas (I) and (II) have stronger activity of inhibiting cancer cell proliferation.

Description

Dammarane type saponin compound with anti-tumor activity, preparation method and application thereof

Technical Field

The invention relates to a saponin compound with anti-tumor activity, which is extracted from plant Gynostemma pentaphylla (Gynostemma pentaphylum); the invention also relates to a preparation method of the saponin compound and application of the saponin compound in medicines, belonging to the field of phytochemistry.

Background

Gynostemma pentaphylla is whole herb of Gynostemma pentaphylla (Gynostemma) Makino in Gynostemma of Cucurbitaceae, and has effects of clearing away heat and toxic materials, relieving cough and eliminating phlegm.

The main medicinal component in the gynostemma pentaphylla is saponin component. Research shows that the dalbergine B and the dalbergine A with double bonds of C20 (21) and C20 (22) have stronger cancer cell proliferation inhibiting effect than gypenoside L and gypenoside LI with hydroxyl connected to the C20 position of the side chain, and the content of the gypenoside L and the gypenoside LI in the gynostemma pentaphylla is very small. For these rare saponins, which have a strong activity, their source is limited and their content in the plant is low. If the separation is carried out by the traditional method, not only a large amount of medicinal resources are consumed, but also the yield is low. Therefore, reasonable component transformation and special method treatment of the compound become hot spots of modern pharmaceutical research, and can create greater economic, social and ecological benefits.

Disclosure of Invention

The present inventors isolated novel compounds having structural formulae (I) and (II) in the study of the active ingredients of a product of heat treatment of gynostemma pentaphyllum, and have not reported in the prior literature, and further conducted pharmacological experiments thereon, found that they have inhibitory effects on human gastric cancer cell SGC-7901, human lung cancer cell a549, human lung cancer cell H1299, human medulloblastoma cell strain SH-SY5Y, human bladder cancer cell T24, and human myeloid leukemia cell K562, and thus completed the present invention.

One object of the present invention is to provide a compound of the following formula (I) or (II):

wherein R is ₁ 、R ₂ And R ₃ Independently of one another, OH or a sugar radical;

preferably: r ₁ Or R ₃ Is OH; r ₂ Is a glycosyl;

particularly preferably, the structural formula of the compound is shown as the formula (III) or (IV):

it is a further object of the present invention to provide a process for the preparation of the above compounds of formula (I) or (II).

Another purpose of the invention is realized by the following technical scheme:

a process for preparing a compound of formula (I) or (II) above, comprising:

(1) Heat treating coarse powder of dried whole plant of Gynostemma pentaphyllum Makino at high temperature and high pressure, reflux extracting with ethanol, recovering solvent from the extractive solution under reduced pressure to obtain ethanol extract;

(2) Passing the ethanol extract through HP20 resin, eluting with 20% ethanol and 50% ethanol, eluting with 95% ethanol, and concentrating 95% ethanol eluate under reduced pressure to obtain 95% ethanol eluate; subjecting 95% ethanol eluate to silica gel column chromatography, eluting with dichloromethane/methanol at volume ratio of 20: 1-2: 1, collecting eluate in segments to obtain 13 components, subjecting 8 th component to semi-preparative chromatographic column (250 × 2.5mm,5 μm) with flow rate of 1mL/min, and eluting with acetonitrile/water at volume ratio of 50: 50.

In the preparation method, in the step (1), the coarse powder of dried whole plant of gynostemma pentaphyllum is preferably subjected to heat treatment for 3h-10h at 125 ℃ and under 0.24MPa, and is extracted for 3 times by 80% ethanol under reflux, wherein the reflux extraction time for 3 times is 2h, 2h and 2h in sequence; mixing extractive solutions, recovering solvent under reduced pressure to obtain ethanol extract;

eluting with 95% ethanol in step (2), and recovering total saponin extract of solvent.

The compound shown in the formula (III) or (IV) has stronger cancer cell proliferation inhibition activity, and particularly, the compound shown in the formula (III) or (IV) has obvious inhibition effects on human gastric cancer cells SGC-7901, human lung cancer cells A549, human lung cancer cells H1299, human bone marrow neuroblastoma cell strains SH-SY5Y, human bladder cancer cells T24 and human myeloid leukemia cells K562, and the half inhibition rate concentration of the compound is less than 100 mu g/mL.

Drawings

FIG. 1 LC-MS spectra of the compounds of the present invention, dalbergine E and dalbergine F

Detailed Description

EXAMPLE 1 preparation of the Compounds of the invention

The dried coarse powder of the whole plant of the gynostemma pentaphylla is thermally treated for 3 to 10 hours at the temperature of 125 ℃ and under the pressure of 0.24MPa, and is extracted for 3 times by refluxing with 80 percent ethanol for 2 hours, 2 hours and 2 hours respectively. Mixing the extractive solutions, and recovering solvent under reduced pressure to obtain 80% ethanol extract. Suspending 80% ethanol extract with distilled water, suspending the ethanol extract with distilled water, and eluting with HP-20 macroporous resin, water, 50% ethanol, and 95% ethanol respectively; recovering solvent under reduced pressure to obtain water extract and ethanol extract; concentrating 95% ethanol extract under reduced pressure, drying, mixing with silica gel 1: 1, separating with silica gel column chromatography, eluting with dichloromethane/methanol at volume ratio of 20: 1-1: 1, collecting eluate in stages to obtain 14 components, eluting 8 th component with preparative chromatographic column (250 × 2.5mm,5 μm) at flow rate of 1mL/min with acetonitrile/water at volume ratio of 50: 50, and rotary evaporating and freeze drying the two components to obtain 2 kinds of white powder (named as dalbergine E and dalbergine F); the structure identification spectra data for the resulting compounds (dalbergine E and dalbergine F) are given in table 1 below.

TABLE 1 isolation of the Compounds ¹³ C NMR data

Dahling F in ¹ The high field region of the H-NMR spectrum gives 7 methyl signals Δ 0.53 (3H, s, H-19), 0.59 (3H, s, H-30), 0.64 (3H, s, H-18), 0.69 (3H, s, H-29), 1.05 (3H, s, H-28), 1.23 (3H, s, H-27) and 1.29 (3H, s, H-26); the terminal proton signal delta 4.62 (1H, d, J =7.9Hz, H-1') of 1 sugar is given, and the configuration of the sugar is judged to be beta according to the terminal proton coupling constant value of the sugar of 7.9 Hz; 1 disubstituted olefinic hydrogen proton signals delta 4.77 (1H, bs, H-21 a), 4.55 (1H, bs, H-21 b) and one trisubstituted olefinic hydrogen proton signal delta 4.93 (1H, m, H-24) are given. In that ¹³ In a C-NMR spectrum, 36 carbon signals are given, wherein chemical shifts of carbon at the 20, 25, 24 and 21 positions are delta 156.9, 132.6, 126.8 and 109.5 respectively, and 4 alkene carbons exist; the terminal carbon signal of the bound sugar was 107.9 (C-1') and the H-3 related signal in the HMBC spectrum, suggesting that the compound has 1 sugar attached to the C-3 position. Of dalbergin F ¹ H-NMR spectrum and ¹³ the C-NMR spectrum data are very similar to Darlin B. The compound damulin F was therefore identified as: 2 alpha, 3 beta, 12 beta-trihydroxydammarane-20 (21), 24-diene-3-O-beta-D-glucoside [2 alpha, 3 beta, 12 beta-trihydroxydammar-20 (21), 24-diene-3-O-beta-D-glucopyranoside]。

Darlin E in ¹ The high field region of the H-NMR spectrum gives 8 methyl signals Δ 0.53 (3H, s, H-19), 0.58 (3H, s, H-30), 0.64 (3H, s, H-18), 0.69 (3H, s, H-29), 1.05 (3H, s, H-28), 1.21 (3H, s, H-27), 1.25 (3H, s, H-26) and 1.46 (3H, s, H-21); the terminal proton signal delta 4.62 (1H, d, J =7.9Hz, H-1') of 1 sugar is given, and the configuration of the sugar is judged to be beta according to the terminal proton coupling constant value of the sugar of 7.9 Hz; gives 2 trisubstituted olefinic hydrogen proton signals delta 4.85 (1H, m, H-24), 5.13 (1H, m, H-22). In that ¹³ In a C-NMR spectrum, 36 carbon signals are given, wherein chemical shifts of carbon at 20, 25, 22 and 24 positions are delta 141.5, 132.6, 124.5 and 124.9 respectively, and 4 alkene carbons exist; the terminal carbon signals of the sugars, delta 107.9 (C-1'), correlated with 2.93 (1H, d, J =9.2Hz, H-3) in the HMBC spectrum, suggesting that the compound is linked to 1 sugar at the C-3 position. Of dalbergin E ¹ H-NMR spectrum and ¹³ the C-NMR spectrum data are very similar to Darlin A. The compound damulin E was therefore identified as: 2 alpha, 3 beta, 12 beta-trihydroxydammarane-20 (22), 24-diene-3-O-beta-D-glucoside [2 alpha, 3 beta, 12 beta-trihydroxydammar-20 (22), 24-diene-3-O-beta-D-glucopyranoside]。

Test example 1 anti-tumor Activity test of the Compounds of the present invention, damorin E (damulin E) and Damorin F (damulin F)

1) Experimental Material

Cell: human liver cancer cells (HepG 2 cell). Human gastric cancer cell SGC-7901, human lung cancer cell A549, human lung cancer cell H1299, human myeloneuroblastoma cell strain SH-SY5Y, human bladder cancer cell T24 and human myelogenous leukemia cell K562.

2) Experimental methods

Human gastric cancer cell SGC-7901, human lung cancer cell A549, human lung cancer cell H1299, human bone marrow neuroblastoma cell strain SH-SY5Y, and human bladder cancer cell T24 activity determination adopt MTT method; the activity of the human myeloid leukemia cell K562 adopts a CCK-8 method.

3) Test compounds: the compounds prepared in example 1 (damulin E and damulin F).

4) Results of the experiment

Table 2 results of the determination of the antitumor activity of the compounds of the present invention (Damulin E and Damulin F ).

Note: + indicates valid; -means invalid.

Note: + indicates valid; -means invalid.

Note: + indicates valid; -means invalid.

Note: + indicates valid; -means invalid.

Note: + indicates valid; -means invalid.

Note: + indicates valid; -means invalid.

The experimental results show that: the compounds of the formulae (III) and (IV) have stronger antitumor activity.

Claims

1. A compound represented by the formula (III):

2. a process for preparing a compound of claim 1, comprising:

(1) Treating coarse powder of dried whole plant of Gynostemma pentaphyllum Makino at 125 deg.C under 0.24MPa for 3 hr, reflux-extracting with ethanol, and recovering solvent from the extractive solution under reduced pressure to obtain ethanol extract;

(2) Passing the ethanol extract through HP20 resin, eluting with 20% ethanol and 50% ethanol, eluting with 95% ethanol, and concentrating 95% ethanol eluate under reduced pressure to obtain 95% ethanol eluate; subjecting 95% ethanol eluate to silica gel column chromatography, eluting with dichloromethane/methanol at volume ratio of 20: 1-2: 1, collecting eluate by stages to obtain 13 components, subjecting 8 th component to semi-preparative chromatographic column (250 × 2.5mm,5 μm) with flow rate of 1mL/min, and eluting with acetonitrile/water at volume ratio of 50: 50.