CN1837226A - Isolation of apigenin derivatives from Pteris vulgaris and their use - Google Patents

Abstract

The present invention is a new glycoside of apigenin obtained from Pteris chinensis—apigenin-4′-α-L-rhamnoside and apigenin-4′-α-L-rhamnose -7-β-D-glucoside and behenic acid have a strong inhibitory effect on the proliferation of cultured prostate cells, and are expected to be developed into new drugs for the treatment of benign prostatic hyperplasia and prostatitis.

Description

Isolation of apigenin derivatives from Pteris vulgaris and their use

technical field

The present invention relates to the separation of active ingredients from Chinese herbal medicines, more specifically to the separation of apigenin derivatives from Pteris vulgaris and their application.

Background technique

Benign prostatic hyperplasia (BPH) is a common senile disease in men, and its incidence is increasing year by year with the aging of society. According to statistics, about 50% of males aged 60 to 80 can be confirmed to have BPH through histopathological examination, and its etiology is not yet fully understood. In recent years, relevant experts have put forward three hypotheses on the matrix of BPH occurrence and development, namely, the theory of sex hormones, the theory of embryonic reawakening, and the theory of expansion of prostate epithelial cell populations. Endocrine-related substances such as sex hormones are the external factors that regulate the growth of the prostate, and their biological effects on the prostate are controlled by various peptide growth factors such as epithelial growth factor (EGF), keratinocyte growth factor (KGF), fibroblast growth factor ( FGF) and transforming growth factor (FGF), and the imbalance of prostate cell proliferation and apoptosis is the root cause. The interaction between fibroblasts and epithelial cells is very important in the normal development and hypertrophy of the prostate and in the development of tumors. External and internal factors run through the pathogenesis of BPH. Moreover, the presence of bacteria has been shown to be linked to benign prostatic hyperplasia. Bacteria itself can be used as an antigen to release certain substances, directly or indirectly induce changes in the morphology and growth mode of the prostate epithelium and mesenchyme. The regulation mechanism of prostate growth is complex, and it maintains a relative balance under normal conditions. Once it is out of balance, it can produce hyperplasia , prostatitis may be an important way to break this balance leading to benign prostatic hyperplasia.

Drugs for the treatment of BPH are mainly divided into α ₁ -AR blockers, 5α-reductase inhibitors, LHRH promoters and antagonists, as well as anti-estrogen drugs and plant drugs.

Prostate cell culture is currently one of the effective methods for screening drugs for the treatment of BPH. In this study, immature rat prostate cells were cultured without serum, using the interaction between epithelial cells and fibroblasts, and adding insulin, EGF, transferrin, prolactin and other cell growth factors to simulate the process of prostate cell proliferation. The compounds or monomers screened by this model can effectively inhibit the proliferation of prostate cells, which is of great significance for further research and development of new drugs for treating benign prostatic hyperplasia and prostatitis.

Contents of the invention

The purpose of the present invention is to provide the isolated celgenin-4'-α-L-rhamnoside, celgenin-4'-α-L-rhamnose-7-β-D-glucose Glycosides and behenic acid, palmitic acid, luteolin.

Another object of the invention is to disclose their preparation.

Another object of the present invention is to provide the use of such compounds.

Ferntail grass, also known as thin-leaf ferntail grass and small-leaf ferntail grass, is Pteris multifida Poir., aliases of Pteris multifida Poir. Tail fern, five finger grass and so on. It is a perennial herbaceous plant, mostly born in semi-shady rocks and crevices in corners, distributed in Yunnan, Sichuan, Guangdong, Guangxi, Hunan, Jiangxi, Zhejiang, Anhui, Jiangsu, Fujian, Taiwan and other places. When used as a traditional Chinese medicine, it tastes light and slightly bitter, and is cold in nature. Function can clear heat and dampness, cool blood to stop bleeding, reduce swelling and detoxify.

We have isolated new glycosides, apigenin-4'-α-L-rhamnoside and apigenin-4'-α- L-rhamnose-7-beta-D-glucoside, and behenic acid, palmitic acid, luteolin.

Detailed ways

The present invention is implemented through the following steps.

Dried Pteris vulgaris is extracted three times with 20% ethanol, the combined extracts are concentrated under reduced pressure to a thin ethanol-free extract, passed through a styrene HPD100 macroporous resin column, and firstly eluted with water to remove sugars, proteins and inorganic salts, etc. eluted with 50% water ethanol and ethanol, collected and concentrated, and then respectively passed through LH-20 gel column chromatography, and eluted with methanol and methanol: chloroform = 7: 3 respectively, thin-layer detection, Collect and combine fractions containing the same spot on the silica gel thin layer plate, concentrate under reduced pressure, and then use silica gel (produced by Qingdao Ocean Chemical Factory, 200-300 mesh) column chromatography with CHCI ₃ : MeOH: H ₂ O = 40:10: 1 and CHCI ₃ : MeOH: H ₂ O = 70: 25: 5 elution, further separation and purification to obtain crude PM-7 and PM-8, and then refined with methanol to obtain pure PM-7 and PM-7 as yellow or light brown powder PM-8, the Rf value of their silica gel thin-layer chromatography (thin-layer plate is also produced by Chromatography Qingdao Ocean Chemical Factory) is about 0.7 and 0.3 (the developing agent used in thin-layer chromatography is CHCI ₃ : MeOH: H ₂ 0=70:30:5).

After identification by chemical, spectroscopic and other methods, the structure is determined as:

Compound PM7

Compound PM8

PM8 is a yellow powdery substance, mp.223-224°C, [α] _D ²⁰ -116.8° (c=0.23, pyridon). IR spectrum shows that there are -OH (3384.5cm ^-1 ), carbonyl (1662.4cm ^-1 ) and characteristic vibrations of benzene ring double bonds (1606.4cm ^-1 , 1496.5cm ^-1 ) in the molecule. FAB-MS gave quasi-molecular ion peak [M+H] ⁺ m/z: 579 (22%); HR ESIMS (m/z579.1712[M+H] ⁺ ) confirmed its molecular formula as C ₂₇ H ₃₀ O ₁₄ (calculated 579.1708 [M+H] ⁺ ). ¹³ C NMR gave a total of 25 carbon lines, among which CH with _δC 128.8 and 117.4ppm not only showed a high abundance, but also a pair of symmetrical ddd peaks appeared in ¹ H NMR, each represented by two H According to the integration of atoms, it is believed that there are 27 C in the compound, which is consistent with the molecular formula provided by ESI high-resolution mass spectrometry. It can also be seen from ^{the 13} C NMR spectrum that there should be two sugar groups in the molecule, one of which is β-D-glucose (δ _C : 101.9 d, 71.3 d, 78.6 d, 75.0 d, 79.4 d, 62.5 t ), the other sugar should be α-L-rhamnose (δ _C : 100.0 d, 71.9 d, 72.6 d, 73.7 d, 71.3 d, 18.7 q), except for the sugar moiety, the chemical shift of the aglycone has The characteristics of flavonoids, so it is speculated that compound PM8 is a flavone glycoside with two sugar groups. According to the integration of ¹ H NMR, except for two sugar groups, the aglycone part has a total of 7 hydrogen integrations. Since the C-5 hydroxyl group gives a broad singlet with 1/2 hydrogen in the low field area, in addition to the other ^1H NMR gives a set of singlets with integral 1H (δ7.04), and the two groups are d of 1H The peak (the long-range coupling constant ⁴ J is 2.1Hz) and a pair of ddd peaks, each of which is 2H integral, are deduced from the basic skeleton of flavonoids. The two d peaks are respectively C-6 and C-8 H, a group of single peaks It should be H at C-3 position, and a pair of ddd peaks, each of which is 2H integration, should be the result of mutual coupling and long-distance coupling of 3', 5' and 2', 6' symmetric H after substitution at 4' position of C ring. Glycosylation should be at two positions, C-7 and C-4', and which position is connected to which sugar group can be proved by 2D NMR. The HNBC spectrum gives clear evidence for this.

From the HNBC spectrum, you can find the relevant points where all flavone aglycone, β-D-glucosyl and α-L-rhamnosyl should appear, and it can be seen that the two sugars are connected to C-7 and C-4 respectively ' position, because the hydrogen (δ5.94 (1H, d, J=7.5Hz)) on the bridgehead carbon of the β-D-glucosyl group has an obvious correlation point with the C-7 quaternary carbon (δ164.3), The hydrogen on the α-L-rhamnosyl bridgehead carbon (δ6.24 (1H, d, J=1.3Hz)) also has an obvious correlation point with the C-4' carbon (δ160.4), thus Compound PM8 was identified as 4H-1-Benzopyran-4-one, [(2-β-D-glucopyranosyl)oxy]5-hydroxy-2-(4-α-L-rhamnosyl)oxy]phenyl-, namely: parsley See Table 1 for the C and H assignments of 7-β-D-glucose-4'-α-L-rhamnoside.

Correlation revealed by the HMBC spectrum of compound PM 8

(Only the key related points for determining the position of the glycosyl connection are listed, other related points are omitted, and the direction of the arrow is from H to C)

PM7 is a yellow-brown powdery substance, mp. 281°C (decomposition point), [α] _D ²⁰ -61.3° (c=0.13, DMSO). IR spectrum shows that there are -OH (3232.2cm ^-1 ), carbonyl (1656.6cm ^-1 ) and characteristic vibrations of benzene ring double bonds (1608.4cm ^-1 , 1579.4cm ^-1 , 1502.3cm ^-1 ) in the molecule. ESIMS gives quasi-molecular ion peak [M+H] ⁺ m/z: 417.1 (100%), [MH] ⁺ m/z: 415.2 (100%); HR ESIMS (m/z 417.1182[M+H] ⁺ ) was determined to have a molecular formula of C ₂₁ H ₂₀ O ₉ (calculated value 417.1180[M+H] ⁺ ). In its carbon spectrum, it can be clearly seen that the molecule contains an α-L-rhamnosyl group, and the three -OH groups in the sugar group give three groups of d peaks with an integral of 1/2H in ¹ H NMR. The constant is about 5Hz, combined with the information given by the carbon spectrum and the hydrogen spectrum, the compound is also an acetin-α-L-rhamnoside compound, because both the hydrogen spectrum and the carbon spectrum indicate that the molecule should contain and The same aromatic hydrogen symmetry structure as PM 8, that is, two CHs overlap at _δC 128.3 and 116.7ppm, and two groups at _δH 8.01 (d, J=9.0Hz) and 7.19 (d, J=9.0Hz)ppm The peaks are 2H integration, and a broad single peak of 1/2 hydrogen integration of the C-5 hydroxyl group also appeared in the downfield area. The substitution position of α-L-rhamnose was also determined by HMBC.

The relationship between the definite glycosyl linkage positions revealed by the HMBC spectrum of compound PM7

Compound PM7 was finally identified as: 4H-1-Benzopyran-4-one, 2-[(4-α-L-rhamnosyl)oxy]phenyl-, that is, apigenin-4'-α-L-rhamnoside, The assignment of C and H is shown in Table 1.

The NMR spectrum assignment (400MHz, TMS, δppm) of C, H in the structure of table 1 compound PM7 and PM8: h ^1H NMR C ¹³ C NMR 8 ^ 7 ^ 8 ^ 7 ^ 23456789101'2', 6'3', 5'4'1"2"3"4"5"6"1"'2"'3"'4"'5"'6"'5-OH7-OH2" -OH3”-OH4”-OH 7.04 (1H, s) 6.95 (1H, d, J = 2.1Hz) 7.21 (1H, d, J = 2.2Hz) 7.99 (2H, ddd, J = 9.0, 1.9Hz) 7.44 (2H, ddd, J = 8.9 , 1.8Hz) 5.94 (1H, d, J = 7.5Hz) 4.46 (1H, m) 4.51 (1H, m) 4.44 (1H, m) 4.33 (1H, dd, J = 5.3, 2.0Hz) 4.68 (1H, dd, J=12.2, 2.2Hz) 4.49 (1H, m) 6.24 (1H, d, J=1.3Hz) 4.79 (1H, dd, J=3.3, 1.7Hz) 4.73 (1H, dd, J=9.2, 3.4 Hz) 4.46 (1H, m) 4.29 (1H, dq, J = 12.4, 9.4, 6.2, 3.2Hz) 1.67 (3H, d, J = 6.2Hz) 13.57 (1/2H, brs.) 6.84 (1H, s) 6.20 (1H, d, J = 1.8Hz) 6.50 (1H, d, J = 1.8Hz) 8.01 (2H, d, J = 9.0Hz) 7.19 (2H, d, J = 9.0Hz) 5.52 (1H, d, J = 1.6Hz) 3.85 (1H, dd, J = 3.1, 1.7Hz) 3.65 (1H, dd, J = 9.2, 3.4Hz) 3.32 (1H, dd, J = 14.8, 6.6Hz) 3.43 (1H, dd, J=9.4, 6.2Hz) 1.10 (3H, d, J=6.2Hz) 12.88 (1/2H, s) 10.83 (1/2H, hrs.) 5.10 (1/2H, d, J = 4.2Hz) 4.76 (1/2H, d, J = 5.8Hz) 4.89 (1/2H, d, J = 5.4Hz) 23456789101'2', 6'3', 5'4'1"2"3"4"5"6"1"'2"'3"'4"'5"'6"' 164.4s105.1d183.0s162.7s100.9d164.3s95.6d158.1s106.8s124.9s128.8d117.4d160.4s101.9d75.0d78.6d71.3d79.4d62.5t100.0d71.9d37d72.6d7.3 163.1s103.8d181.7s161.5s98.8d161.1s94.1d157.4s103.9s124.0s128.3d116.7d159.0s98.2d70.0d70.3d71.7d69.8d17.9q

^* All assignments confirmed by 2D NMR spectroscopy

^Use deuterated pyridine as solvent

^Use deuterated xylenol as solvent

Another dry Pteris vulgaris powder was extracted three times with acetone, the combined extracts were concentrated under reduced pressure to an extract, firstly passed through silica gel column chromatography, and the fractions were collected in sections, and then respectively passed through LH-20 gel column chromatography, and the oil Ether: acetone = 8: 2 and chloroform: methanol = 7: 3 elution, based on thin gel chromatography and the same fraction, concentrated under reduced pressure and then washed with CHCI ₃ : MeOMe = 7: 3 by silica gel column chromatography After further separation and purification, PM-15, PM-20, and PM-22 compounds were obtained respectively.

There are 6 hydrogens in the ¹ H NMR low field region of compound PM 15, and 15 carbon lines are given in the ¹³ C NMR spectrum. From the two carbon lines at δ147.51 and 151.41ppm, it is speculated that this compound should belong to Luteolin flavonoids The compound, compared with the literature, its spectral data is consistent with Luteolin, so the compound is identified as luteolin.

^{The 1} HNMR of compound PM 20 only gives four sets of proton signals, δ2.34 (2H, t, J=7.7Hz), 1.63 (2H, p, J=14.7, 7.3Hz), 1.25 (24H, brs.), 0.88 (3H,t,J=6.6Hz). Judging from the broad single peak at 1.25ppm, there should be a long fatty chain in the molecule, which is confirmed by the strong single peak at δ29.36 in its ¹³ C NMR spectrum. Except for the carbonyl group and the carbonyl group at δ180.34, the rest of the carbon lines are all CH ₂ , and the molecular weight given by EIMS is 256. Based on these information, the compound is identified as Palmitic acid, ie palmitic acid. Since the silica gel thin layer chromatography behavior of compound PM 22 is similar to that of PM 20, its molecular weight was determined to be 368 by mass spectrometry, and the NMR spectrum is basically similar to PM 20, thus identifying compound PM 22 as Behenic acid, ie behenic acid.

Pharmacological test:

1. Cell culture:

Take 40-day-old male SD rats, isolate the prostate under aseptic conditions, wash the culture medium and crush it, add collagenase and DNase, shake and digest in a constant temperature water bath at 37°C, stop the reaction after 18 hours, filter, and centrifuge at 1000rpm for 5 minutes Cells were collected, counted, and seeded in 24-well culture plates.

2. Drug treatment:

Drug treatment was started after 3 days of prostate cell culture. Two doses of 180umol/l and 360umol/l were set for each drug, and the solvent control was 0.1% DMSO. After 3 days of drug treatment, the cell viability was measured by MTT method, and the inhibition rate of each drug on prostate cell proliferation was calculated. In this experiment, drugs were screened under the conditions of adding growth factors in the culture medium and without growth factors in the culture medium. The growth factors used in the test were transferrin, insulin, epidermal growth factor, PRL prolactin and testosterone.

3. Inhibition rate calculation:

The following formula was used to calculate the inhibitory rate of the drug on rat prostate cells using the MTT measurement value.

Inhibition rate=(cell activity of blank control group-cell activity of administration group)/cell activity of blank control group×100%

4. Drug action evaluation:

With reference to the inhibition rate of the positive control drug Aprelide, the inhibitory effect of the drug on rat prostate cells was evaluated according to the following criteria.

Potent: inhibition rate greater than 50% at both doses

Effective: one of the two doses has an inhibitory rate greater than 50%, the other less than <50%

Weak: Less than 50% inhibition at both doses, but greater than 25% inhibition

Ineffective: less than 25% inhibition at both doses

Experimental results:

1. Screening results without growth factors in the culture medium (Table 1):

Compounds that have potent inhibitory effects on rat prostate cells cultured in vitro include: PM7, PM15 and PM22

Compounds that have an effective inhibitory effect on isolated cultured rat prostate cells include: PM20

Compounds with weak inhibitory effect on rat prostate cells cultured in vitro include: PM12, PM14, PM16, PM11+2.

Compounds that have no inhibitory effect on rat prostate cells cultured in vitro include: PM1, PM2, PM4, PM5, PM6, PM9.

2. Screening results under the conditions of growth factors in the culture medium (Table 2):

Compounds that have potent inhibitory effects on rat prostate cells cultured in vitro include: PM7, PM8, PM15, PM20 and PM22

Compounds that have an effective inhibitory effect on rat prostate cells in vitro: PM14

Compounds with weak inhibitory effect on rat prostate cells cultured in vitro include: PM12, PM16.

Compounds that have no inhibitory effect on rat prostate cells cultured in vitro include: PM1, PM2, PM4, PM5, PM6, PM9, PM11+2.

The inhibitory rate (%) of table 1 compound to prostate cell hyperplasia in growth factor-free culture fluid drug name Drug concentration (umol/l) molecular weight 180 360 Applet 53.29 95.86 PM1 0 0 396 PM2 8.38 7.64 396 PM4 9.46 8.73 380 PM5 0.74 2.94 396 PM6 0 0 380 PM7 60.39 81.08 41 6 PM9 3.46 0.74 234 PM12 41.3 41.3 306 PM14 41.3 43.48 270 PM1 5 79.32 89.66 286 PM16 8.7 28.26 272 PM20 37.95 93.07 256 PM22 52.17 57.29 368 PM11+2 41.07 43.91 234

The inhibitory rate (%) of table 2 compound to prostatic cell hyperplasia in growth factor culture fluid drug name Drug concentration (umol/l) molecular weight 180 360 Applet 55.6 100 PM1 4.73 2.4 396 PM2 5.9 9.06 396 PM4 8.17 6.36 380 PM5 1.2 3.12 396 PM6 4.39 4.32 380 PM7 76.31 100 416 PM8 54.55 95.45 578 PM9 4.32 5.67 234 PM12 36.36 34.09 306 PM14 50 50 270 PM15 79.66 99.32 286 PM16 9.09 34.09 272 PM20 63.03 93.84 256 PM22 50 76.47 368 PM11+2 8.9 10.89 234

Example

1. 1000 grams of dry Pteris pratense powder, extract three times with 40% ethanol, combine the extracts and concentrate under reduced pressure to obtain 395 grams of extract, pass through styrene series (including HPD100, HPD300, HPD400, etc.) macroporous resin or chromatography Polyamide column, first wash with water to remove sugars and inorganic salts, then use gradient elution with different concentrations of water and ethanol, collect 50% ethanol fractions containing flavonoids, combine and concentrate, and then pass through the LH-20 gel column Chromatography, using methanol, CHCl3 gradient elution, collecting the fractions at a single point on the polyamide thin-layer plate, concentrating respectively to obtain crude PM-7 and PM-8, and then purifying with aqueous ethanol or aqueous acetone to obtain yellow or light Pure PM-7 20mg and PM-8 120mg of brown yellow powder;

2. 1,000 grams of dry Pteris vulgaris, extract three times with acetone, combine the extracts and concentrate under reduced pressure to obtain 256 grams of extract, first pass through silica gel column chromatography, collect fractions in sections, and then pass through LH-20 gel column respectively Chromatography, PM-15 15mg, PM-20 23mg, PM-22 18mg were obtained respectively.

Claims (6)

1. A kind of apigenin derivative with the following structure

2. The preparation method of the apigenin derivative as claimed in claim 1 is: Dried Pteris vulgaris is extracted three times with 30-60% ethanol, and the combined extracts are decompressed to ethanol-free dilute extract, passed through styrene series (including HPD100, HPD300, HPD400, etc.) macroporous resin or polyamide for chromatography Column, wash with water to remove sugars and inorganic salts, then use gradient elution with different concentrations of water and ethanol, collect fractions containing flavonoids, combine and concentrate, and then pass through silica gel column or LH-20 gel column chromatography , with methanol, CHCl3 gradient elution, collect the fractions at a single point on the polyamide thin layer plate, concentrate respectively to obtain crude PM-7 and PM-8, and then purify with aqueous ethanol or aqueous acetone to obtain yellow or light brown yellow The pure PM-7 and PM-8 of the powder; another dry Pteris vulgaris coarse powder was extracted three times with acetone, the combined extract was decompressed to the extract, firstly passed through silica gel column chromatography, and the fractions were collected in sections, and then passed through LH -20 gel column chromatography, respectively to obtain PM-15, PM-20, PM-22 compounds. 3. The preparation method of apigenin derivatives according to claim 2, characterized in that the dilute extract is eluted with 50% H ₂ O and ethanol through a macroporous resin or polyamide column for chromatography and gradually drops to The fractions of flavonoids were collected by washing with ethanol, combined and concentrated to obtain the extract. 4. The preparation method of apigenin derivatives according to claim 2, characterized in that the above-mentioned extract is collected in sections by silica gel column or LH-20 gel column chromatography with methanol and methanol: chloroform = 7: 3 split. 5. The preparation method of apigenin derivatives according to claim 2, characterized in that the above-mentioned extract is subjected to column chromatography with CHCl ₃ : MeOH: _{H 2} O = 40: 10: 1 and chloroform: methanol: Water = 70: 25: 5 Washing off, concentrating and purifying under reduced pressure to obtain PM7-PM8. 6. The use of the apigenin derivative according to claim 7, characterized in that the isolated luteolin, palmitic acid and behenic acid are used in the preparation of drugs for the prevention and treatment of prostatitis and prostatic hypertrophy.