CN111470955B - Phenanthrene compound with anti-tumor effect, preparation method and application - Google Patents

Phenanthrene compound with anti-tumor effect, preparation method and application Download PDF

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CN111470955B
CN111470955B CN202010442375.9A CN202010442375A CN111470955B CN 111470955 B CN111470955 B CN 111470955B CN 202010442375 A CN202010442375 A CN 202010442375A CN 111470955 B CN111470955 B CN 111470955B
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ethyl acetate
petroleum ether
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phenanthrene compound
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张海龙
杨敏飞
张昌龙
鲍和
高阳
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Xian Jiaotong University
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Abstract

The invention belongs to an anti-tumor compound and a preparation method and application thereof, and provides a phenanthrene compound with an anti-tumor effect, a preparation method and application thereof, aiming at solving the technical problems that although the existing anti-tumor drugs are more in types, the price is high, the use burden of patients is heavy, the side effect is large, especially the extraction source and the yield of part of anti-tumor drugs are limited, and the large-scale production cannot be carried out through total synthesis, so that the mass production and popularization are difficult31H28O7The phenanthrene compound is a dihydrophenanthrene dimer, the preparation method comprises the steps of extracting a total extract from rhizoma gastrodiae, extracting the total extract and carrying out column chromatography separation on an ethyl acetate extraction layer, the preparation method is simple, convenient and effective, the phenanthrene compound is obtained, the purity is high, the cost is low, and the phenanthrene compound can be applied to anti-tumor drugs, particularly anti-liver cancer drugs.

Description

Phenanthrene compound with anti-tumor effect, preparation method and application
Technical Field
The invention belongs to an anti-tumor compound and a preparation method and application thereof, and particularly relates to a phenanthrene compound with an anti-tumor effect, a preparation method and application thereof.
Background
Tumors have now become the second leading killer of human health, with a malignant incidence of about 22.1% and a mortality rate of about 12.9%, with at least one in every 5 people having cancer and one in every 8 people dying from cancer! Cancer has the characteristics of high incidence, high mortality, youthfulness, regionalization and the like.
Although there are many anti-tumor drugs currently used in clinical practice, including chemotherapeutic drugs and targeted drugs, these anti-tumor drugs are generally expensive and have great side effects. In addition, although some antitumor drugs have good activity, the extraction source is limited, and the large-scale production and popularization cannot be carried out, for example, the chemotherapeutic drug taxol, although the activity is good, particularly for breast cancer and cervical cancer, the source and the yield are limited, so far, the full-synthetic method cannot be used for large-scale production, and meanwhile, the natural content is extremely low, so that the terminal price is high, and the burden of a patient is heavy. Therefore, there is still a need to develop antitumor drugs with good activity and low cost, especially natural antitumor drugs with low side effects.
Disclosure of Invention
The invention provides a phenanthrene compound with an anti-tumor effect, a preparation method and application, and aims to solve the technical problems that although the existing anti-tumor drugs are various, the price is high, the use burden of patients is heavy, the side effect is large, particularly, the extraction source and the yield of part of anti-tumor drugs are limited, and large-scale production cannot be performed through total synthesis, so that the mass production and popularization are difficult.
In order to achieve the purpose, the invention provides the following technical scheme:
the phenanthrene compound with the anti-tumor effect is characterized in that the structural formula of the phenanthrene compound is shown in the specification
Figure BDA0002504442680000011
The molecular formula of the phenanthrene compound is C31H28O7。
The invention also discloses a preparation method of the phenanthrene compound with the anti-tumor effect, which is characterized by comprising the following steps:
s1, extracting from rhizoma Kadsurae Coccineae to obtain total extract
Taking dried rhizome of radix et rhizoma Dispori Cantoniensis, extracting for multiple times by cold soaking at room temperature or heating reflux, mixing extractive solutions, and concentrating to obtain total extract;
s2, extracting the total extract
Sequentially extracting the total extract obtained in the step S1 with petroleum ether and ethyl acetate for at least one time to respectively obtain an organic layer, and removing the petroleum ether and the ethyl acetate under reduced pressure to respectively obtain a petroleum ether extraction layer and an ethyl acetate extraction layer;
s3, carrying out column chromatography separation on the ethyl acetate extraction layer
S3.1, loading the ethyl acetate extraction layer obtained in the step S2 on a silica gel column, and eluting with chloroform-methanol as an eluent according to the volume ratio of chloroform-methanol of 100:0 to 0:100, performing gradient elution, respectively collecting and combining same fractions to obtain a first column passing part;
s3.2, taking the flow part with the polarity of 80:20 in the first column passing part obtained in the step S3.1, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate, and respectively collecting the same flow part to obtain a second column passing part;
and S3.3, eluting the fraction with the polarity of 5:1 in the second column passing part obtained in the step S3.2 by Sephadex LH-20 column chromatography, and taking methanol-water as an eluent to obtain the phenanthrene compound with the anti-tumor effect.
Further, in step S1, a plurality of extractions are performed by heating reflux;
the heating reflux is specifically that methanol or ethanol with volume fraction of 75-95% is used as an extracting agent, and the material-liquid ratio of the dry rhizome of the root of the manyleaf paris rhizome to the extracting agent is 1 kg: 1-5L, heating, refluxing and extracting for 1-5 times, and each time lasts for 1-3 hours.
Further, in step S1, multiple extractions are performed by cold dipping at room temperature;
the cold soaking is carried out for 2-6 times at room temperature, and each time lasts for 8-24 hours.
Further, in step S2, the total extract obtained in step S1 is sequentially extracted at least once with petroleum ether and ethyl acetate, specifically:
suspending the total extract obtained in the step S1 in water, and sequentially extracting for 1-6 times by using petroleum ether and ethyl acetate; wherein the volume ratio of the total extract to water is 1: 1-1: 4.
Further, in step S2, the total extract obtained in step S1 is sequentially extracted at least once with petroleum ether and ethyl acetate, specifically:
and (4) sequentially carrying out ultrasonic solid-liquid extraction on the total extract obtained in the step S1 for 1-6 times by using petroleum ether and ethyl acetate.
Further, in step S3.3, methanol-water is used as the eluent, wherein the volume ratio of methanol to water is 100: 0-98: 2.
the phenanthrene compound with the anti-tumor effect can be applied to preparation of anti-tumor drugs.
In addition, the phenanthrene compound with the anti-tumor effect can be applied to preparation of anti-liver cancer drugs.
Compared with the prior art, the invention has the beneficial effects that:
1. the phenanthrene compound with the anti-tumor effect is a new phenanthrene compound separated from the rhizome of Maya seven which is one of Shanxi seven medicines for the first time, has a structure of a dihydrophenanthrene dimer, has the anti-tumor effect through experimental verification, has a good cytotoxic effect on HepG2 cells, and can be developed into a new anti-tumor medicine as an effective component;
the ' Maya Qiis one of ' seven medicines ' in Shaanxi, is a pseudo bulb and a whole grass of calanthe fimbristylis swingle in calanthe of orchidaceae, is mainly distributed in the southern Shaanxi mountain area, and is widely distributed under mountain forests with altitude of 1500 + 3500 m and on grass slopes, so that the use cost can be reduced to a certain extent, and the large-scale production and popularization are facilitated.
2. The preparation method of the phenanthrene compound with the anti-tumor effect is simple, convenient and effective in extraction and separation method and low in cost; experiments prove that the phenanthrene compound prepared by the preparation method has high purity; meanwhile, the preparation method is easy to popularize and use, and can be used for large-scale production, so that the phenanthrene compound with the anti-tumor effect is more feasible to popularize.
3. In the preparation method, the extraction of the dried rhizome of the aesculus kadsurae can adopt a heating reflux method or a room-temperature cold soaking method; meanwhile, the total extract can be extracted by suspending in water or by solid-liquid extraction. Provides a plurality of specific preparation methods, and is convenient to select according to the use requirement in the actual production.
4. The phenanthrene compound with the anti-tumor effect has better effect on the anti-tumor aspect, particularly on the anti-liver cancer aspect through experimental verification, so that the phenanthrene compound can be applied to anti-tumor medicines, particularly anti-liver cancer medicines.
Drawings
FIG. 1 shows the phenanthrene compounds with anti-tumor effect obtained in the first embodiment of the present invention1An H-NMR spectrum;
FIG. 2 shows the phenanthrene compounds with anti-tumor effect obtained in the first embodiment of the present invention13A C-NMR spectrum;
FIG. 3 is a DEPT map of the phenanthrene compound with anti-tumor effect obtained in the first embodiment of the invention;
FIG. 4 shows the phenanthrene compounds with anti-tumor effect obtained in the first embodiment of the present invention1H-1HCOSY spectra;
FIG. 5 is an HSQC spectrum of the phenanthrene compound with anti-tumor effect obtained in the first embodiment of the invention;
FIG. 6 is an HMBC map of the phenanthrene compound with anti-tumor effect obtained in the first embodiment of the present invention;
FIG. 7 is a NOESY map of a phenanthrene compound having an anti-tumor effect obtained in example one of the present invention;
FIG. 8 is an HR-ESI-MS spectrum of the phenanthrene compound with anti-tumor effect obtained in the first embodiment of the present invention
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments do not limit the present invention.
The seven medicines in Shaanxi are mainly from plant medicines in Shaanxi and surrounding areas, are important components of folk medicines in China, are folk herbal medicines with definite curative effects which are gradually determined by people in Qinba mountainous areas after hundreds of years of repetition and verification and in disease treatment practices, and the seven medicines in the seven medicines are odd variations, which means the herbal medicines with the odd and special curative effects. Shanxi 'seven drugs' is a precious medical cultural heritage in China and should be inherited and developed.
The "Maya Qin" as one of the seven herbs in Shanxi is the pseudobulb and whole grass of calanthe striata (Calanthefibricata Franch) in calanthe of Orchidaceae, mainly distributed in the mountain area of Shannan, and often grown under mountain forest and on grass slope with elevation of 1500-3500 m. It is light, slightly pungent and bitter, and cool in nature. Has little toxicity. It enters stomach, liver and lung meridians. Has the effects of clearing away heat and toxic materials, relieving swelling and pain, and removing blood stasis, and is mainly used for treating hepatitis, pharyngitis and gastric ulcer. No report of anti-tumor treatment exists in the traditional application, and a novel phenanthrene compound is found to have good cytotoxic activity in the research process of chemical components and biological activity of the phenanthrene compound, so that the phenanthrene compound has the potential of being developed into an anti-tumor medicament or a lead compound.
At present, the research on the whole calanthe plant at home and abroad mainly focuses on the aspects of ornamental value, cultivation technology, breeding method and the like, and the research reports on the effective components and the pharmacological activity are very limited.
The invention provides a preparation method of a phenanthrene compound with an anti-tumor effect, which comprises the following steps:
s1, taking a certain mass of dried rhizome of the root of Caulophyllum vulgare, and carrying out heating reflux extraction for 1-6 times at the respective boiling points for 1-3 hours each time by using methanol with the volume 1-5 times of the mass of the dried rhizome of the root of Caulophyllum vulgare or ethanol with the volume fraction of 75-95%;
or cold soaking at room temperature for 2-6 times at room temperature for 8-24 h;
mixing the extractive solutions, and concentrating to obtain total extract;
s2, sequentially extracting the total extract obtained in the step S1 with petroleum ether and ethyl acetate for at least one time;
liquid-liquid extraction can be carried out, namely, the total extract is suspended in water, the volume ratio of the total extract to the water is 1: 1-1: 4, extract liquid is obtained, and organic solvents with the same volume are sequentially used for extraction, wherein petroleum ether is used for carrying out the extraction with the same volume on the extract liquid during the first extraction, an organic layer obtained after the last extraction is separated out during each extraction, and the rest of the aqueous layer is used for carrying out the next extraction with the same volume of the organic solvents; extracting each solvent for 1-6 times, combining the extract liquor, and distilling under normal pressure or reduced pressure to remove the organic solvent to obtain each extraction layer and a water layer respectively. The organic solvent comprises petroleum ether, ethyl acetate and the like, and the extraction sequence is that the solvent with low polarity is used firstly, and then the organic solvent with high polarity is used;
or, performing solid-liquid extraction, namely performing ultrasonic solid-liquid extraction on the total extract for 1-6 times by sequentially using petroleum ether and ethyl acetate;
respectively obtaining organic layers, and removing petroleum ether and ethyl acetate under reduced pressure to respectively obtain a petroleum ether extraction layer and an ethyl acetate extraction layer;
s3, taking the ethyl acetate extraction layer, and obtaining the phenanthrene compound by adopting a separation method such as column chromatography purification.
The column chromatography purification comprises the following three stages:
the first stage is as follows: loading the ethyl acetate extraction layer on a silica gel column, taking a chloroform-methanol system as an eluent, carrying out gradient elution according to the volume ratio of (100:0) - (0:100), collecting and combining the same fractions to obtain 33 fractions with numbers of EA-1-EA-33, wherein the fraction is a first column passing fraction;
and a second stage: separating EA-11 flow parts obtained by the first column passing part by silica gel column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate (5:1, 3:1, v/v) to obtain 4 flow parts (EA-11-1-EA-11-4), wherein the flow parts are second column passing parts;
and a third stage: and (3) carrying out Sephadex LH-20 column chromatography on the EA-11-2 of the second column passing part, and eluting by using methanol-water as an eluent to obtain the phenanthrene compound.
And eluting by using methanol-water as an eluent in the third-stage Sephadex LH-20 column chromatography, wherein the volume ratio of methanol to water in a methanol-water system is 100: 0-98: 2.
Example one
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizomes of the root of Caesalpinia Spinosa, heating and refluxing for 3 times by using methanol with the volume of 5 times of the mass of the dry rhizomes of the root of the Caesalpinia Spinosa for 3 hours each time, combining extracting solutions, decompressing and recovering a solvent to obtain a total extract;
2) suspending the total extract in 4 times of water, extracting with petroleum ether for 4 times, extracting with ethyl acetate for 4 times in equal volume, and removing organic solvent under reduced pressure to obtain petroleum ether layer and ethyl acetate layer.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to the volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, combining the same flow portions through TLC (thin layer chromatography) detection to obtain 33 flow portions, wherein the 33 flow portions are respectively marked as EA-1 and EA-2 … … EA-33.
4) The 11 th flow part, namely EA-11, is separated by silica gel column chromatography again, petroleum ether-ethyl acetate (5:1, 3:1, v/v) is used for gradient elution, one flow part is collected every 300mL, and 4 flow parts (EA-11-1 to EA-11-4) are obtained after TLC identification and combination of the same flow parts.
5) Wherein, the 2 nd flow part EA-11-2 is eluted by Sephadex LH-20 column chromatography and methanol as eluent to obtain the phenanthrene compound.
Example two
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizomes of the root of Caesalpinia Spreng, heating and refluxing for 5 times by using ethanol with the volume amount being 3 times of the mass of the dry rhizomes of the root of the Caesalpinia Spreng for 1 hour each time, wherein the volume fraction of the ethanol is 75%, combining extracting solutions, and recovering the solvent under reduced pressure to obtain a total extract;
2) suspending the total extract in 3 times of water, extracting with petroleum ether for 5 times, extracting with ethyl acetate for 5 times with equal volume, and removing organic solvent under reduced pressure to obtain petroleum ether layer and ethyl acetate layer.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein, the 12 th flow part EA-12 is separated by silica gel column chromatography again, petroleum ether-ethyl acetate (5:1, 3:1, v/v) is used for gradient elution, one flow part is collected for each 300mL, and 4 flow parts (EA-11-1 to EA-11-4) are obtained after the same flow part is combined by TLC identification.
5) And (3) performing Sephadex LH-20 column chromatography on the 2 nd flow part EA-11-2, and eluting by using methanol-water as an eluent, wherein the volume ratio of the methanol to the water is 98:2 to obtain the phenanthrene compound.
EXAMPLE III
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizomes of aesculus kawakamii, heating and refluxing for extraction for 4 times and 3 hours each time by using ethanol with the volume amount being 1 time of the mass of the dry rhizomes of aesculus kawakamii, wherein the volume fraction of the ethanol is 95%, combining extracting solutions, and recovering the solvent under reduced pressure to obtain a total extract;
2) and sequentially carrying out ultrasonic solid-liquid extraction on the total extract for 6 times by using petroleum ether and ethyl acetate, and decompressing an extraction layer to remove the organic solvent to obtain a petroleum ether layer and an ethyl acetate layer respectively.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein the 11 th flow part EA-11 is separated again by silica gel column chromatography, petroleum ether-ethyl acetate (5:1, 3:1, v/v) is used for gradient elution, one flow part is collected for every 300mL, and 4 flow parts (EA-11-1 to EA-11-4) are obtained after the same flow part is combined by TLC identification.
5) And performing Sephadex LH-20 column chromatography on the 2 nd flow part EA-11-2, and eluting by using methanol as an eluent, wherein the volume ratio of the methanol to the water is 99:1 to obtain the phenanthrene compound.
Example four
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizomes of aesculus kawakamii, heating and refluxing for 1 time and 2 hours each time by using ethanol with the volume amount being 5 times of the mass of the dry rhizomes of aesculus kawakamii, wherein the volume fraction of the ethanol is 85%, combining extracting solutions, and recovering a solvent under reduced pressure to obtain a total extract;
2) and sequentially carrying out ultrasonic solid-liquid extraction on the total extract for 1 time by using petroleum ether and ethyl acetate, and decompressing an extraction layer to remove the organic solvent to obtain a petroleum ether layer and an ethyl acetate layer respectively.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein the 10 th fraction EA-10 was again separated by silica gel column chromatography using petroleum ether-ethyl acetate (5:1,3: 1, v/v) were subjected to gradient elution, and fractions were collected every 300mL, identified by TLC and combined to give 4 fractions (EA-11-1 to EA-11-4).
5) Wherein the 2 nd flow part EA-11-2 is subjected to Sephadex LH-20 column chromatography, and is eluted by using methanol-water as an eluent, wherein the volume ratio of the methanol to the water is 99:1, obtaining the phenanthrene compound.
EXAMPLE five
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizomes of the root of Caesalpinia Spinosa, heating and refluxing for 4 times by using methanol with the volume amount being 3 times of the mass of the dry rhizomes of the root of the Caesalpinia Spinosa for 2 hours each time, combining extracting solutions, decompressing and recovering a solvent to obtain a total extract;
2) suspending the total extract in 4 times of water, extracting with petroleum ether for 2 times, extracting with ethyl acetate for 2 times in equal volume, and removing organic solvent under reduced pressure to obtain petroleum ether layer and ethyl acetate layer.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein the 11 th fraction EA-11 was again separated by silica gel column chromatography using petroleum ether-ethyl acetate (5:1,3: 1, v/v) were subjected to gradient elution, and fractions were collected every 300mL, identified by TLC and combined to give 4 fractions (EA-11-1 to EA-11-4).
5) Wherein the 2 nd flow part EA-11-2 is subjected to Sephadex LH-20 column chromatography, and is eluted by using methanol-water as an eluent, wherein the volume ratio of the methanol to the water is 98:2, obtaining the phenanthrene compound.
EXAMPLE six
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizome of radix et rhizoma Rhei Palmatae, cold soaking at room temperature for 6 times for extraction, each time for 18h, mixing the extractive solutions, and recovering solvent under reduced pressure to obtain total extract;
2) suspending the total extract in 1 times of water, extracting with petroleum ether for 5 times, extracting with ethyl acetate for 5 times in equal volume, and removing organic solvent under reduced pressure to obtain petroleum ether layer and ethyl acetate layer.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein the 11 th fraction EA-11 was again separated by silica gel column chromatography using petroleum ether-ethyl acetate (5:1,3: 1, v/v) were subjected to gradient elution, and fractions were collected every 300mL, identified by TLC and combined to give 4 fractions (EA-11-1 to EA-11-4).
5) And (3) performing Sephadex LH-20 column chromatography on the 2 nd flow part EA-11-2, and eluting by using methanol-water as an eluent, wherein the volume ratio of the methanol to the water is 100:1 to obtain the phenanthrene compound.
EXAMPLE seven
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizome of radix et rhizoma Rhei Palmatae, cold soaking at room temperature for 2 times for extraction, each time for 24h, mixing the extractive solutions, and recovering solvent under reduced pressure to obtain total extract;
2) and sequentially carrying out ultrasonic solid-liquid extraction on the total extract for 4 times by using petroleum ether and ethyl acetate, and decompressing an extraction layer to remove the organic solvent to obtain a petroleum ether layer and an ethyl acetate layer respectively.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein the 11 th flow part EA-11 is separated again by silica gel column chromatography, petroleum ether-ethyl acetate (5:1, 3:1, v/v) is used for gradient elution, one flow part is collected for every 300mL, and 4 flow parts (EA-11-1 to EA-11-4) are obtained after the same flow part is combined by TLC identification.
5) Wherein, the 2 nd flow part EA-11-2 is eluted by Sephadex LH-20 column chromatography and methanol as eluent to obtain the phenanthrene compound.
Example eight
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizome of radix et rhizoma Rhei Palmatae, cold soaking at room temperature for 4 times for extraction, each time for 8h, mixing the extractive solutions, and recovering solvent under reduced pressure to obtain total extract;
2) suspending the total extract in 3 times of water, extracting with petroleum ether for 6 times, extracting with ethyl acetate for 6 times in equal volume, and removing organic solvent under reduced pressure to obtain petroleum ether layer and ethyl acetate layer.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein the 11 th fraction EA-11 was again separated by silica gel column chromatography using petroleum ether-ethyl acetate (5:1,3: 1, v/v) were subjected to gradient elution, and fractions were collected every 300mL, identified by TLC and combined to give 4 fractions (EA-11-1 to EA-11-4).
5) And (3) performing Sephadex LH-20 column chromatography on the 2 nd flow part EA-11-2, and eluting by using methanol-water as an eluent, wherein the volume ratio of the methanol to the water is 98.5:1.5, so as to obtain the phenanthrene compound.
Example nine
A preparation method of phenanthrene compounds with anti-tumor effect comprises the following steps:
1) taking 10kg of dry rhizome of radix et rhizoma Rhei Palmatae, cold soaking at room temperature for 5 times for extraction, each time for 12h, mixing the extractive solutions, and recovering solvent under reduced pressure to obtain total extract;
2) and sequentially carrying out ultrasonic solid-liquid extraction on the total extract for 3 times by using petroleum ether and ethyl acetate, and decompressing an extraction layer to remove the organic solvent to obtain a petroleum ether layer and an ethyl acetate layer respectively.
3) Taking 100g of an ethyl acetate extraction layer, firstly performing gradient elution by adopting silica gel column chromatography and chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC (thin layer chromatography) to obtain 33 flow portions (EA-1-EA-33).
4) Wherein the 11 th fraction EA-11 was again separated by silica gel column chromatography using petroleum ether-ethyl acetate (5:1,3: 1, v/v) were subjected to gradient elution, and fractions were collected every 300mL, identified by TLC and combined to give 4 fractions (EA-11-1 to EA-11-4).
5) Wherein the 2 nd flow part EA-11-2 is subjected to Sephadex LH-20 column chromatography, and is eluted by using methanol-water as an eluent, wherein the volume ratio of the methanol to the water is 98:2, obtaining the phenanthrene compound.
In the first to ninth embodiments, in step 3), the ethyl acetate extraction layer was collected and eluted by silica gel column chromatography to obtain 33 fractions; in step 4), the 10 th or 11 th or 12 th fraction is separated again by silica gel column chromatography; in step 5), the 2 nd fraction is eluted by Sephadex LH-20 column chromatography. The related numbers are all records in experimental verification, and in practical application, the judgment of the numbers all takes polarity as a judgment standard.
The phenanthrene compound with the anti-tumor effect is prepared by the preparation method, and the structure of the phenanthrene compound is determined by physicochemical constants and spectrum technology (HR-ESI-MS, 1D-NMR and 2D-NMR) by taking the phenanthrene compound obtained in the first example as an example:
the properties of the obtained compound are as follows: brown powder, readily soluble in methanol.
Results of the spectrum: as shown in FIG. 8, according to HR-ESI-MS spectrum, the peak of the excimer ion is M/z535.17282[ M + Na ]]+(calcd for 535.17272) and determining the molecular formula as C31H28O7The unsaturation degree was 18.
As shown in fig. 1 and 2, in1H-NMR and13proton signal delta in C-NMR spectrumH 6.60(1H,s)，δH 6.83(1H,d,J＝8.1Hz)，δH 7.04(1H,t,J＝7.4Hz)，δH 6.77(1H,d,J＝7.3Hz)，δH 6.20(1H,s)，δH 7.94(1H,s)，δH 6.39(1H,s)，δH 6.26(1H,s)，δH2.48-2.60(6H,m)，δH2.36-2.43(2H, m) and 24 unsaturated carbon signals and 4 methylene signals, which shows that the basic skeleton of the compound is a dimer of dihydrophenanthrene, and the two dihydrophenanthrenes are substituted by delta at 8 positionsH3.75(3H,s)，δH3.92(3H,s)，δH3.85(3H, s) it is known that three positions are substituted by methoxy groups and that the remaining three substituents are all hydroxy groups as deduced by carbon spectral chemical shift. As shown in FIG. 7, in the NOESY spectrum, H-9 'and H-8' are related, H-8 'and the proton of the hydroxyl group are related, and it can be determined that the substituent at C-7' is hydroxyl, H-6 'and the proton at the methoxy group are related, indicating that the substituent at the 5' position is methoxy, and H-4 'is other than 5' -OCH3And the proton on the other methoxy group, indicating that the substituent at the 3 'position is the methoxy group, and the 2' position is the connecting position of two dihydrophenanthrenes. In the NOESY spectra, H-9 and H-8 are related and are connected at positions 8, 7 and 6, as shown in FIG. 6HMBC mapping, both H-6 and H-7 are associated with C-5, indicating that the substituent at position 5 is a hydroxyl group. In the NOESY spectrum, the 5-OH proton is associated with another active proton, indicating that the substituent at position 4 is a hydroxyl group, the 4-OH proton is associated with H-3, and H-3 is associated with a proton on a methoxy group, indicating that the 2-position is a methoxy group, thus, it is determined that the 1-position and the 2' -position are connected through an oxygen atom. By combining the maps in fig. 3 to 5 of the present invention, the structural formula of the compound is finally determined as follows:
Figure BDA0002504442680000121
it was named as 4,5, 7-trihydroxy-2, 3, 5-trimethoxydihydrophenanthrene dimer. Is a novel compound which is not disclosed.
The nuclear magnetic data are shown in table 1:
TABLE 1 method for preparing phenanthrene compounds of the present invention1H-NMR and13C-NMR data
Figure BDA0002504442680000122
Figure BDA0002504442680000131
The phenanthrene compound has a good application prospect in the aspect of resisting tumors, particularly resisting liver cancer, and the obtained phenanthrene compound is subjected to a cytotoxicity test as follows:
the specific experimental method comprises the following steps:
HepG2 cells at 2X 105Inoculating each cell/well in a 96-well plate at the density, respectively adding samples with different concentrations after culturing for 24h at 37 ℃, wherein the samples comprise the phenanthrene compound and the doxorubicin positive drug, adding equal volume of DMSO into a blank group, adding 20 mu of LMTT solution into each well after culturing for 48 h, continuously culturing for 4h, then absorbing the culture medium, adding 150 mu of DMSO into each well to dissolve the generated formazan, measuring the OD value under the 490nm wavelength by using an enzyme labeling instrument after shaking uniformly, calculating the inhibition rate by using the following formula, and using the S formula to calculate the inhibition ratePSS software calculates the median Inhibitory Concentration (IC) of the samples tested50Value, μ M).
The inhibition ratio (%) [ 1- (sample group OD value/blank group OD value) ] × 100%.
The results of the cytotoxicity experiments are shown in table 2:
TABLE 2 results of the inhibition of HepG2 cells by the phenanthrene compounds of the present invention
Figure BDA0002504442680000132
Analysis of the result of the cytotoxicity experiment: the data in Table 2 show that the phenanthrene compounds of the present invention have very good cytotoxic effects and half inhibitory concentration IC50Is 8.3 +/-0.6 mu M, has the potential of being prepared into antitumor drugs or being used as antitumor drug lead compounds, and particularly has better application prospect for liver cancer.
The preparation methods in the second to ninth embodiments all obtained phenanthrene compounds with anti-tumor effects, and the results consistent with those in the first embodiment were obtained through corresponding spectrum analysis and cytotoxicity experiments.
The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to other related technical fields, are included in the scope of the present invention.

Claims (7)

1. A phenanthrene compound with an anti-tumor effect is characterized in that: the structural formula of the phenanthrene compound is shown as
Figure FDA0003075585180000011
The molecular formula of the phenanthrene compound is C31H28O7。
2. A process for producing the phenanthrene compound having an antitumor effect according to claim 1, which comprises the steps of:
s1, extracting from rhizoma Kadsurae Coccineae to obtain total extract
Taking dried rhizome of radix et rhizoma Dispori Cantoniensis, extracting for multiple times by cold soaking at room temperature or heating reflux, mixing extractive solutions, and concentrating to obtain total extract;
s2, extracting the total extract
Sequentially extracting the total extract obtained in the step S1 with petroleum ether and ethyl acetate for at least one time to respectively obtain an organic layer, and removing the petroleum ether and the ethyl acetate under reduced pressure to respectively obtain a petroleum ether extraction layer and an ethyl acetate extraction layer;
s3, carrying out column chromatography separation on the ethyl acetate extraction layer
S3.1, loading the ethyl acetate extraction layer obtained in the step S2 on a silica gel column, using chloroform-methanol as an eluent, and using the volume ratio of chloroform-methanol as 100:0 to 0:100, performing gradient elution, respectively collecting and combining same fractions to obtain a first column passing part;
s3.2, taking the flow part with the polarity of 80:20 in the first column passing part obtained in the step S3.1, separating by silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate, and respectively collecting the same flow part to obtain a second column passing part, wherein the volume ratio of the petroleum ether to the ethyl acetate is 5: 1-3: 1;
s3.3, eluting the fraction with the polarity of 5:1 in the second column passing part obtained in the step S3.2 by Sephadex LH-20 column chromatography, and taking methanol-water as an eluent to obtain the phenanthrene compound with the anti-tumor effect, wherein the volume ratio of methanol to water is 100: 0-98: 2.
3. the method of claim 2, wherein: in step S1, multiple extractions are performed by heating reflux;
the heating reflux is specifically that methanol or ethanol with volume fraction of 75-95% is used as an extracting agent, and the material-liquid ratio of the dry rhizome of the root of the manyleaf paris rhizome to the extracting agent is 1 kg: 1-5L, heating, refluxing and extracting for 1-5 times, and each time lasts for 1-3 hours.
4. The method of claim 2, wherein: in step S1, multiple extractions are performed by cold immersion at room temperature;
the cold soaking is carried out for 2-6 times at room temperature, and each time lasts for 8-24 hours.
5. The method of claim 2,3 or 4, wherein: in step S2, the total extract obtained in step S1 is sequentially extracted at least once with petroleum ether and ethyl acetate, specifically,
suspending the total extract obtained in the step S1 in water, and sequentially extracting for 1-6 times by using petroleum ether and ethyl acetate; wherein the volume ratio of the total extract to water is 1: 1-1: 4.
6. The method of claim 2,3 or 4, wherein: in step S2, the total extract obtained in step S1 is sequentially extracted at least once with petroleum ether and ethyl acetate, specifically,
and (4) sequentially carrying out ultrasonic solid-liquid extraction on the total extract obtained in the step S1 for 1-6 times by using petroleum ether and ethyl acetate.
7. The use of the phenanthrene compounds with anti-tumor effect according to claim 1 in the preparation of anti-liver cancer drugs.
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CN108191616A (en) * 2018-01-05 2018-06-22 四川大学 There is monomer component of selective butyrylcholine esterase inhibiting effect and application thereof in bletilla

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CN108191616A (en) * 2018-01-05 2018-06-22 四川大学 There is monomer component of selective butyrylcholine esterase inhibiting effect and application thereof in bletilla

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