CN116375776A - Dendrobium candidum sesquiterpene glycoside compound with anti-tumor activity and preparation method and application thereof - Google Patents
Dendrobium candidum sesquiterpene glycoside compound with anti-tumor activity and preparation method and application thereof Download PDFInfo
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- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
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Abstract
The invention belongs to the technical field of medicines, and discloses a sesquiterpene glycoside compound separated from traditional Chinese medicine dendrobium candidum, a preparation method thereof and application thereof in resisting tumors. According to the invention, through carrying out systematic deep research on chemical components of traditional Chinese medicine dendrobium candidum, the analysis of data such as wave spectrum, mass spectrum and the like shows that the novel sesquiterpene glycoside compound is separated from dendrobium candidum. MTS experiments show that the compound separated by the invention has certain anti-tumor activity on various tumor cells including human liver cancer cells, human lung cancer cells and human breast cancer cells, and is expected to be developed into a novel anti-tumor drug.
Description
Technical Field
The invention belongs to the field of natural pharmaceutical chemistry, relates to a sesquiterpene glycoside compound with anti-tumor activity and a preparation method and application thereof, and in particular relates to a novel sesquiterpene glycoside compound obtained by separating from traditional Chinese medicine dendrobium (Dendrobium findlayanum Par et Rchb.f.), a preparation method and application thereof in anti-tumor medicines.
Background
Cancer is one of the leading causes of morbidity and mortality in the world, and the global burden of cancer will continue to increase as the population ages. The tumor seriously damages the health of people, and the tumor is in the defect of lack of effective medicines at present, and the toxic and side effects of partial effective medicines are intolerable. The natural products play an important role in the anti-tumor active molecules, so that the search of anti-tumor active ingredients from natural medicines has become the focus of more and more medicine researchers.
Dendrobium (Dendrobium) is the second largest genus of Orchidaceae, and is mainly distributed in Asian tropical and subtropical regions, australian and Pacific islands, and China is mainly distributed in southwest, south China, etc. The dendrobium nobile is a cultivated product of dendrobium nobile Dendrobium nobile, dendrobium huoshanense D.huoshanense, dendrobium chrysotoxum or dendrobium fimbriatum D.fimbriatum which are orchid plants in 2020 edition of Chinese pharmacopoeia, and is a fresh or dried stem of similar species of plants of the same genus, and comprises dendrobium clavatum D.findlayanum. In the traditional Chinese medicine, the dendrobium is a common precious medicinal material, has long medicinal history, and has the effects of benefiting stomach, promoting fluid production, nourishing yin, clearing heat and the like. Modern pharmacological researches have proved that herba Dendrobii has the effects of resisting tumor and inflammation, enhancing immunity and treating cataract. Researches prove that the dendrobium nobile has various chemical component structure types and mainly contains sesquiterpenes (glycoside), bibenzyl, alkaloids and the like. Researches have reported that dendrobium has an anti-tumor effect and is a medicine and food homologous traditional Chinese medicine, but few sesquiterpene glycoside compounds with the anti-tumor effect in dendrobium are reported at present; the invention carries out intensive research on chemical components of dendrobium, and separates to obtain the novel sesquiterpene glycoside compound with anti-tumor activity.
Disclosure of Invention
The invention aims to: the invention aims to separate a sesquiterpene glycoside compound with anti-tumor activity from dendrobium candidum, a preparation method thereof and application thereof in preparing anti-tumor drugs. The invention discovers that the sesquiterpene glycoside compound has potential treatment effect on human non-small cell lung cancer cell strain (A-549), human breast cancer cell strain (MCF-7) and human liver cancer cell strain (SMM 7721) through researches.
The invention provides a dendrobium candidum sesquiterpene glycoside compound with anti-tumor activity, which has a structural formula shown in a formula I:
The sesquiterpene glycoside compound represented by the above formula I of the present invention is named Findlayanoside C.
A Chinese medicinal preparation with antitumor activity comprises sesquiterpene glycoside compound represented by formula I and its derivatives or pharmaceutically acceptable salts.
The traditional Chinese medicine preparation comprises solid preparations such as: tablets, capsules, pills, granules and the like; semisolid formulations, such as: ointments, suppositories, and the like; liquid formulations, such as: solutions, injections, sprays, and the like.
The invention provides a preparation method of dendrobium candidum sesquiterpene glycoside compound, which comprises the following steps:
(1) Pulverizing dried stem of Dendrobium Officinale, extracting with ethanol, filtering, mixing extractive solutions, and recovering ethanol under reduced pressure until no ethanol smell; adding proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, concentrating the extracts under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Taking the n-butanol extract in the step (1), performing MCI gel CHP 20P column chromatography, performing ethanol-water gradient elution, and detecting and combining the same fractions by TLC to obtain dendrobium candidum total sesquiterpene glycoside;
(3) Dividing the dendrobium candidum total sesquiterpene glycoside obtained in the step (2) by RP-18 column chromatography, eluting with methanol-water gradient, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; subjecting Fraction A to silica gel column chromatography, eluting with ethyl acetate-methanol, and mixing to obtain 4 fractions A1-A4; separating Fractiona1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions, wherein Fractiona 1.1.1-Fractiona 1.1.3; the Fractiona1.1.2 is taken and purified by semi-preparative HPLC to obtain the compound of the invention.
As a preferred scheme, the preparation method of the dendrobium candidum sesquiterpene glycoside compound comprises the following steps of:
(1) Taking dry dendrobium candidum stems, crushing, extracting for 2-4 times by using 95% ethanol in a cold leaching way, stirring every time for 1-5 days, filtering, and recovering ethanol under reduced pressure until no alcohol smell exists; adding a proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, extracting each time with 8L of organic solvent for 3 times, concentrating under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Subjecting the n-butanol extract obtained in the step (1) to MCI gel CHP 20P column chromatography, sequentially eluting with ethanol-water gradient of volume ratio of 10:90,30:70,50:50 and 70:30, and detecting and combining the same fractions by TLC to obtain herba Dendrobii total sesquiterpene glycoside;
(3) Taking the dendrobium candidum total sesquiterpene glycoside obtained in the step (2), separating by RP-18 column chromatography, sequentially eluting with methanol-water gradient with the volume ratio of 20:80,40:60,60:40 and 80:20, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; separating the Fraction A by silica gel column chromatography, eluting with ethyl acetate-methanol with volume ratio of 11:1, and mixing to obtain 4 Fraction A1-A4; separating the Fraction A1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions A1.1.1-A1.1.3; taking part Fr.A1.1.2 of the compound and purifying the part Fr.A1.2 by semi-preparative HPLC to obtain the compound; the semi-preparative HPLC purification conditions were: the mobile phase is methanol-water with the volume ratio of 40:60, the flow rate is 3mL/min, and the detection wavelength is 210nm.
Of course, the sesquiterpene glycoside compound is extracted from dendrobium candidum or can be artificially synthesized by adopting a drug synthesis method.
The beneficial effects are that:
1. the invention discloses a preparation method for extracting and separating the sesquiterpene glycoside compound Findlayanoside C from dendrobium candidum for the first time. Cold soaking at normal temperature, separating resin and separating by semi-preparative HPLC.
2. The sesquiterpene glycoside compound Findlayanoside C is a novel compound obtained by first separating and identifying dendrobium candidum through data analysis and identification such as spectrum, mass spectrum and the like.
3. Cell experiments prove that the sesquiterpene glycoside compound Findlayanoside C obtained by separation has obvious inhibition effect on a human non-small cell lung cancer cell strain (A-549), a human breast cancer cell strain (MCF-7) and a human liver cancer cell strain (SMM 7721), and can be used for preparing antitumor drugs.
Drawings
FIG. 1 is a diagram of Findlayanoside C according to the present invention 1 H NMR spectrum.
FIG. 2 is a diagram of Findlayanoside C according to the present invention 13 C NMR spectrum.
FIG. 3 is a HMBC spectrum of Findlayanoside C of the present invention.
FIG. 4 is a HMQC spectrum of Findlayanoside C in the present invention.
FIG. 5 is a diagram of Findlayanoside C according to the present invention 1 H- 1 H COSY profile.
FIG. 6 is a ROESY spectrum of Findlayanoside C of the present invention.
FIG. 7 shows the structural formula of Findlayanoside C in the present invention.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1: the invention relates to the extraction and separation of sesquiterpene glycoside compounds
(1) Taking 5kg of dried dendrobium candidum stems, crushing, carrying out cold leaching extraction for 4 times by using 95% ethanol for 5 days each time, stirring at intervals, filtering, and recovering ethanol under reduced pressure until no ethanol smell exists; adding proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, extracting with 8L of organic solvent each time for 3 times, concentrating under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract for later use.
(2) Subjecting the n-butanol extract obtained in the step (1) to MCI gel CHP 20P column chromatography, and subjecting to ethanol/water (volume ratio 10:90,30:70,50:50, 70:30) gradient elution, and combining the same fractions by TLC detection to obtain herba Dendrobii total sesquiterpene glycoside (310 g).
(3) Dividing the dendrobium candidum total sesquiterpene glycoside obtained in the step (2) by RP-18 column chromatography, and performing methanol/water gradient elution (volume ratio is 20:80,40:60,60:40, 80:20), wherein TLC detection is performed to combine identical Fractions to obtain 4 fraction sections of Fractions A-D. And performing silica gel column chromatography on the Fraction A, eluting with ethyl acetate/methanol in a volume ratio of 11:1, and combining to obtain 4 Fraction sections, wherein the Fractions A1-A4 are obtained. The Fractions A1 is taken and separated by Sephadex LH-20 column chromatography, eluted by methanol, and 3 Fractions are obtained by combining A1.1.1-A1.1.3. Fr.A1.1.2 (130 mg)
The preparation is partially carried out by semi-preparative liquid chromatography, and the chromatographic conditions are as follows: chromatographic column: YMC-Pack ODS-A (250 mm. Times.10 mm,5 μm, japan YMC Co.); mobile phase: methanol/water, 40:60 (V/V); detection wavelength: 210nm; flow rate: 3ml/min; column temperature: purifying at 30 deg.c to obtain the compound (5 mg, t) R ＝32min)。
The structure of the sesquiterpene glycoside compound is identified as follows:
the compound of the invention is white amorphous powder and is easy to dissolve in methanol. The excimer ion peak given by high resolution mass spectrometry (+) HR-ESI-MS was at m/z 439.2305 (C 21 H 36 O 8 Na, calculated: 439.2302 Determining the molecular formula as C 21 H 36 O 8 The unsaturation was 4. Hydroxyl groups (3425 cm) –1 ) And c=c double bond (1637 cm –1 ) Is not shown in the figure). Hydrolysis of the compound with 10% HCl in methanol, detection of glucose from thin layer chromatography in the hydrolysate, and further passage 1 The coupling constant of the terminal protons in the H NMR spectrum (j=7.8 Hz) determines that there is a β -D-glucosyl group in the compound. 13 C NMR, DEPT and HSQC spectra showed 21 carbon signals: 4 methyl groups, 4 methylene groups (1 of which is linked to oxygen), 10 methine groups (6 of which is linked to oxygen), and 3 quaternary carbons (1 of which is linked to oxygen), wherein the carbon atoms comprising a set of glucosyl groups are believedNumber [ delta ] C 98.3(C-1′),75.7(C-2′),77.7(C-3′),72.3(C-4′),78.1(C-5′),63.9(C-6′)]The compound may be a sesquiterpene glycoside. And from 1 Three sets of unimodal methyl signals delta were observed in the H NMR spectrum (Table 1) H 1.28(3H,s,H 3 -12)，1.21(3H,s,H 3 -13) and 1.71 (3H, s, H 3 -15) a set of bimodal methyl signals delta H 0.88(3H,d,J＝6.6Hz,H 3 -14) and a sugar end group proton signal delta H 4.52(1H,d,J＝7.8Hz,H-1′)。
The compounds of the present invention were analyzed for one-dimensional, two-dimensional nuclear magnetic data (Table 1) and HMBC spectra for signals related to H-15 and C-3, C-5, and H-5 and C-3. Compounds of the invention 1 Olefin proton H-5 (delta) in H NMR spectra (Table 1) H 6.36, s) is a set of unimodal signals, indicating that OH-1 and H-6 in the compounds of the invention are in opposite spatial directions; h-6 (delta) H 2.09, s) is a set of unimodal signals, indicating that H-6 is spatially oriented in the same direction as H-7. And in the ROSEY spectra, H-3/Ha-2, ha-2/H 3 The relevant signals for-14, H-10/H-6, H-10/H-7 and H-6/H-7 indicate that OH-3, H-6, H-7 and H-10 in the compounds of the invention are in the beta-configuration and OH-1 is in the alpha-configuration. Therefore, the structure of the compound is determined as muurol-4-ene-1 alpha, 3 beta, 11-triol 11-O-beta-D-glucopyranoside, and the compound is not reported, and is a novel compound named as Findlayanoside C.
Table 1: the novel sesquiterpene glycoside compound 1 H NMR 13 Data assignment by C NMR (methanol-d) 4 ,600MHz；δin ppm；J in Hz)
Example 2: evaluation of in vitro antitumor Activity of sesquiterpene glycoside Compounds
(1) Experimental materials
Human non-small cell lung cancer cells (A-549), human breast cancer cells (MCF-7), human liver cancer cells (SMM 7721).
(2) Principle of experiment
The inner salt Method (MTS) is a completely new MTT analogue, a yellow dye. The MTS method is similar to the MTT method in principle but superior to the MTT method. Succinate dehydrogenase in the mitochondria of living cells is capable of metabolizing and reducing MTS to form soluble Formazan (Formazan) compounds, the amount of which can be measured by an microplate reader at 490 nm. In general, the formazan production is proportional to the number of living cells, and the number of living cells can be estimated from the optical density OD value.
(3) The experimental method comprises the following steps: the MTS method detects cell activity.
(1) Inoculating cells: single cell suspension is prepared by culture solution (DMEM or RMPI 1640) containing 10% fetal bovine serum, 3000-10000 cells per well are inoculated into a 96-well plate, the volume of each well is 100 mu l, and adherent cells are inoculated and cultured in advance for 12-24 hours.
(2) Test compound solution (compound Findlayanoside C) was added: the compound was dissolved in DMSO and rescreened at concentrations of 40. Mu.M, 8. Mu.M, 1.6. Mu.M, 0.32. Mu.M, 0.064. Mu.M, 200. Mu.L per well final volume, 3 rescreens per treatment.
(3) Color development: after 48 hours of culture at 37 ℃, the adherent cells discard the culture solution in the wells, and 20 mu L of MTS solution and 100 mu L of culture solution are added to each well; suspension cells discard 100. Mu.L culture supernatant, add 20. Mu.L MTS solution per well; 3 blank wells (mixed solution of MTS solution 20. Mu.L and culture solution 100. Mu.L) were used, and incubation was continued for 2 to 4 hours to allow the reaction to proceed sufficiently.
(4) Colorimetric: selecting 492nm wavelength, reading light absorption value of each hole by multifunctional enzyme labeling instrument (MULTISKAN FC), recording, plotting cell growth curve with concentration as abscissa and cell survival rate as ordinate, and calculating compound IC by two-point method (Reed and Muench method) 50 Values.
(5) Positive control: in each experiment, two positive compounds of cisplatin (DDP) and Taxol (Taxol) are adopted, the concentration is taken as an abscissa, the cell survival rate is taken as an ordinate, a cell growth curve is drawn, and the IC of the compound is calculated by a two-point method (Reed and Muench method) 50 Values.
(4) The experimental results are shown in table 2:
TABLE 2 inhibition of tumor cells by sesquiterpene glycoside compounds of the invention (IC 50 ，μM)
The experimental results are shown in Table 2, and the dendrobium candidum sesquiterpene glycoside compound Findlayanoside C provided by the invention has good inhibition effect on human non-small cell lung cancer cell strain (A-549), human breast cancer cell strain (MCF-7) and human liver cancer cell strain (SMM 7721), so that the dendrobium candidum sesquiterpene glycoside compound provided by the invention has anti-tumor effect and potential.
Example 3
The sesquiterpene glycoside compound Findlayanoside C obtained in example 1 was taken and added with an injection solvent by a conventional method, subjected to fine filtration, and subjected to filling and sterilization to prepare an injection.
Example 4
Taking the sesquiterpene glycoside compound Findlayanoside C obtained in the embodiment 1, dissolving in sterile water for injection, filtering with a sterile funnel, packaging, freeze-drying at low temperature, and sealing in sterile manner to obtain powder for injection. Example 5
Taking the sesquiterpene glycoside compound Findlayanoside C obtained in the embodiment 1, adding various pharmaceutical excipients such as starch and cyclodextrin, and making into tablet or capsule.
Claims (7)
1. The dendrobium candidum sesquiterpene glycoside compound with anti-tumor activity is characterized in that the chemical structure of the sesquiterpene glycoside compound is shown as a formula I:
2. the method for preparing dendrobium candidum sesquiterpene glycoside compound according to claim 1, which comprises the following steps:
(1) Pulverizing dried stem of Dendrobium Officinale, extracting with ethanol, filtering, mixing extractive solutions, and recovering ethanol under reduced pressure until no ethanol smell; adding proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, concentrating the extracts under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Taking the n-butanol extract in the step (1), performing MCI gel CHP 20P column chromatography, performing ethanol-water gradient elution, and detecting and combining the same fractions by TLC to obtain dendrobium candidum total sesquiterpene glycoside;
(3) Dividing the dendrobium candidum total sesquiterpene glycoside obtained in the step (2) by RP-18 column chromatography, eluting with methanol-water gradient, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; subjecting Fraction A to silica gel column chromatography, eluting with ethyl acetate-methanol, and mixing to obtain 4 fractions A1-A4; separating Fractiona1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions, wherein Fractiona 1.1.1-Fractiona 1.1.3; the Fractiona1.1.2 is purified by semi-preparative HPLC to obtain the compound shown in the formula I.
3. The method for preparing dendrobium candidum sesquiterpene glycoside compound according to claim 2, comprising the following steps:
(1) Taking dry dendrobium candidum stems, crushing, extracting for 2-4 times by using 95% ethanol in a cold leaching way, stirring every time for 1-5 days, filtering, and recovering ethanol under reduced pressure until no alcohol smell exists; adding a proper amount of water into the concentrate to prepare a suspension, sequentially extracting the suspension with petroleum ether, ethyl acetate and n-butanol respectively, extracting each time with 8L of organic solvent for 3 times, concentrating under reduced pressure to obtain petroleum ether layer extract, ethyl acetate layer extract and n-butanol layer extract respectively for later use;
(2) Subjecting the n-butanol extract obtained in the step (1) to MCI gel CHP 20P column chromatography, sequentially eluting with ethanol-water gradient of volume ratio of 10:90,30:70,50:50 and 70:30, and detecting and combining the same fractions by TLC to obtain herba Dendrobii total sesquiterpene glycoside;
(3) Taking the dendrobium candidum total sesquiterpene glycoside obtained in the step (2), separating by RP-18 column chromatography, sequentially eluting with methanol-water gradient with the volume ratio of 20:80,40:60,60:40 and 80:20, and detecting and combining the same Fractions by TLC to obtain 4 Fractions A-D; separating the Fraction A by silica gel column chromatography, eluting with ethyl acetate-methanol with volume ratio of 11:1, and mixing to obtain 4 Fraction A1-A4; separating the Fraction A1 by Sephadex LH-20 column chromatography, eluting with methanol, and mixing to obtain 3 fractions A1.1.1-A1.1.3; taking part Fr.A1.1.2 of the extract, and purifying by semi-preparative HPLC to obtain a compound shown in formula I; the semi-preparative HPLC purification conditions were: the mobile phase is methanol-water with the volume ratio of 40:60, the flow rate is 3mL/min, and the detection wavelength is 210nm.
4. A Chinese medicinal preparation with antitumor activity is characterized by comprising the sesquiterpene glycoside compound shown in the formula I in claim 1 and derivatives or pharmaceutically acceptable salts thereof.
5. The Chinese medicinal preparation according to claim, wherein the Chinese medicinal preparation comprises a tablet, a capsule, a pill, a granule, an ointment, a suppository, a solution, an injection, and a spray.
6. The use of the dendrobe sesquiterpene glycoside compound according to claim 1 in the preparation of antitumor drugs.
7. The use of the dendrobe sesquiterpene glycoside compound according to claim 1 in the preparation of a medicament for treating human lung cancer, human breast cancer or human liver cancer.
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