Patent Grant
8993618
The present invention relates to a composition for treating influenza A (H1N1) virus and the preparation method therefor. In particular, it relates to a composition extracted from Ferula assa-foetida for treating influenza A (H1N1) virus and the preparation method therefor.
Influenza occurs with seasonal variations and reaches peak prevalence in winter, with many people killed worldwide every year. Until now, only a few organic compounds including amantadine, rimantadine and ribavirin have been used for influenza therapy. However, drug-resistant influenza viruses are generated quickly. The mutation of coat protein genome of influenza viruses severely influenced the antigen expression thereof and new influenza virus mutants are generated. Appearance of Influenza A (H1N1) virus is one of the examples in recent years.
Influenza A (H1N1) virus belongs to an influenza virus type A and is grouped as subtype H1N1 according to haemagglutinin and neuraminidase of the surface antigens. At present, most patients infected with influenza A (H1N1) virus are dosed with Tamiflu® (oseltamivir), the preferred therapeutic drug. Oseltamivir is a neuraminidase inhibitor, which inhibits the neuraminidase activity of influenza virus types A and B. It prevents the release of new virus particles made from the infected host cells, so that replication and transmission of influenza virus are stopped. At present, Tamiflu-resistant viruses are also appeared. Therefore, preparation of pharmaceutical composition particularly on treating influenza A (H1N1) virus becomes an important issue.
In addition to the chemical drugs from synthesis, the natural products obtained from medicinal plants also are the opportunity for developing anti-influenza virus drug. According to the anti-virus screen, the extracts of F. assa-foetida (Umbelliferae or Apiaceae) showed significant potency against influenza A virus (H1N1). The roots of this plant originally were an important remedy for “Spanish flu” (type A influenza, H1N1 subtype) in 1918. However, there is no further researches to explore anti-influenza A virus (H1N1) activity of the pure compounds from F. assa-foetida on within nearly one hundred years.
It is therefore attempted by the applicant to deal with the above situation encountered in the prior art.
In order to overcome the defect that the commercial drugs cannot efficiently inhibit influenza A (H1N1) virus, newly extracted sesquiterpene coumarins and diterpenes from Ferula sp. are found to efficiently inhibit influenza A (H1N1) virus activity and show cytotoxicity on human cancer cells. By bioassay-guided fractionation of the methanol extract of F. assa-foetida, our finding demonstrates the anti-influenza A virus (H1N1) activity of this medicinal plants is originated from sesquiterpene coumarins.
A pharmaceutical composition including an effective amount of a sesquiterpene coumarin represented by formula I or II and/or an effective amount of a diterpene represented by formula III is provided in the present invention.
Preferably, the sesquiterpene coumarin and the diterpene are extracted from Ferula sp., and the Ferula sp. preferably is Ferula assa-foetida. The sesquiterpene coumarin represented by formula II inhibits an influenza virus, and the influenza virus includes an influenza A (H1N1) virus.
Preferably, the pharmaceutical composition of the present invention further can inhibit the growth of a cancer cell line, and the cancer cell line preferably includes a human cancer cell line. Further, the human cancer cell line includes human lung cancer cell line (A549), human breast cancer cell line (MDA-MB-231 and MCF-7), human liver cancer cell line (Hep G2 and Hep 3B) and a human oral cavity cancer cell line (Ca9-22).
A method for preparing a pharmaceutical composition is provided in the present invention. The method includes: (a) extracting a Ferula sp. with an methanol to obtain a first extract; (b) partitioning the first extract with an n-hexane-methanol to obtain an methanol extract; (c) partitioning the methanol extract with a chloroform-water to obtain a chloroform extract; and (d) chromatographing the chloroform extract to obtain the pharmaceutical composition.
Preferably, the pharmaceutical composition includes at least a fraction, and the at least a fraction is a sesquiterpene coumarin or a diterpene. The chloroform extract inhibits an activity of an influenza A (H1N1) virus.
An purpose of the pharmaceutical composition prepared from the aforementioned method is provided in the present invention, and the pharmaceutical composition is performed on inhibiting an activity of an influenza A (H1N1) virus or a growth of a cancer cell line.
Preferably, the pharmaceutical composition includes 10′R-acetoxy-11′-hydroxyumbelliprenin, 5′-hydroxyumbelliprenin, 10′R-karatavicinol, 8′-acetoxy-5′S-hydroxyumbelliprenin, methyl galbanate, galbanic acid, farnesiferol C, farnesiferol A, conferol, ligupersin A and epi-conferdione when the pharmacetical composition inhibits the activity of the influenza A (H1N1) virus, and the pharmaceutical composition includes conferol when the pharmaceutical composition inhibits the growth of the cancer cell line.
The above objectives and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings, in which:
The present invention will now be described more specifically with reference to the following Embodiments. It is to be noted that the following descriptions of preferred Embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
I. Experimental Instruments:
Optical rotations were taken on a JASCO-P-1020 polarimeter (cell length 10 mm) UV spectra were measured on a JASCO V-530 UV/vis spectrophotometer. Infrared resonance (IR) spectra were recorded on a Mattson Genesis II FT-IR spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on Varian Gemini-20000 (200 MHz), Varian Unity-plus (400 MHz), and Varian Unity-plus (600 MHz) FT-NMR NMR spectrometers. Chemical shift (δ) values are in ppm (part per million) with deuterated chloroform (CDCl3) as internal standard, and coupling constants (J) are in Hz. High resolution fast atom bombardment mass spectrum (HRFABMS), high resolution electrospray ionization mass spectrometry (HRESIMS), and electrospray ionization mass spectrometry (ESIMS) measurements respectively were performed on JEOL JMS-700, Bruker APEX II, and Finnigan POLARISQ mass spectrometers. Thin layer chromatography (TLC) was performed on Kieselgel 60 F254 (0.20 mm, Merck), and spots were viewed under UV light at 254 nm and 356 nm and/or stained by spraying with 50% H2SO4 and heating on a hot plate. For column chromatography, silica gel (Kieselgel 60, 70-230, and 230-400 mesh, Merck) and Sephadex LH-20 were used. The instrumentation for the reverse phase-medium performance liquid chromatography (RP-MPLC) experiment was composed of a Supelco VersaFlash flash chromatography apparatus and VersaFlash C-18 cartridges (40×150 mm). Further purification of some of compounds obtained was achieved by preparative high performance liquid chromatography (HPLC), using a Shimadzu LC-10ATvp/Shimadzu SCL-10Avp UV-VIS detector and Thermo columns (analytical: 5 μm, 250×4.6 mm; preparative: 8 μm, 250×10 mm; C18) were used. For the preparation of Mosher ester derivatives, (S)-(+)- and (R)-(−)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride were used as the reagents.
II. Material:
Ferula assa-foetida resin (3.23 kg) was purchased from a Chinese herb shop in Taipei, Taiwan.
III. Extraction and Isolation:
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The chemical formulas, physical and chemical properties of brand new Compounds 1 (Formula I), 2 (Formula II) and 3 (Formula III) in the present invention are illustrated as follows.
Physical and chemical properties: yellow oil; [α]25D +8.6 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 211 (4.54), 318 (3.95) nm; IR (neat) νmax 3440, 1729, 1613, 1555, 1233, 836 cm−1; 1H and 13C NMR (CDCl3, 400 MHz), see Table 1; ESIMS m/z 463 [M+Na]+; HRESIMS m/z 463.2099 [M+Na]+ (calcd for C26H32O6Na, 463.2096).
Physical and chemical properties: yellow oil; [α]25D +11.2 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 213 (4.77), 321 (4.39) nm; IR (neat) νmax 3480, 1731, 1613, 1555, 1234, 836 cm−1; 1H and 13C NMR (CDCl3, 400 MHz), see Table 1; ESIMS m/z 465 [M+Na]+; HRFABMS m/z 443.2433 [M+H]+ (calcd for C26H34O6+H, 443.2434).
Physical and chemical properties: yellow oil; [α]25D −14.6 (c 0.16, CHCl3); UV (MeOH) λmax (log ε) 243 (3.95), 271 (3.74) nm; IR (neat) vmax 3417, 1696, 1613, 1514 cm−1; 1H and 13C NMR (CDCl3, 400 MHz), see Table 2; ESIMS m/z 337 [M+Na]+; HRESIMS m/z 337.1784 [M+Na]+ (calcd for C20H26O3Na, 337.1780).
IV. Anti-Influenza A virus (H1N1) Bioassay:
Madin-Darby canine kidney (MDCK) cells (ATCC CCL34) were used as target cells for viral infection in the XTT (tetrazolium hydroxide salt) assay. MDCK cells were grown as adherent cells in MEM medium supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. In the antiviral assay, the medium was supplemented with 2% FCS and the above-mentioned antibiotics. Virus titers were determined by the cytopathic effect in MDCK cells and expressed as 50% tissue culture infective dose (TCID50) values per mL All viruses were stored at −70° C. until use. The antiviral activity against influenza A virus (H1N1) was evaluated by the XTT method. MDCK cells, treated by trypsin, were seeded onto 96-well plates with a concentration of 1.0×105 cells/mL and a volume of 70 μL per well. After incubation at 35° C. with 5% CO2 for 24 hours, 20 μL of test virus solution were added and incubated for another 1 hour. Different concentrations of test substances were then added to culture wells in triplicate. Amantadine was used as a positive control. After incubation at 35° C. with 5% CO2 for 3 days, XTT reagent was added and incubated for 3 hours. The viral inhibition rate (%) was calculated as [100−(OD492/OD690)×100]%. The antiviral concentration of 50% inhibition (IC50) was defined as the concentration achieving 50% cytoprotection against virus infection.
V. Cytotoxicity Bioassays:
Fractions and isolates were tested against lung (A549), breast (MEA-MB-231 and MCF7), liver (HepG2 and Hep3B), and oral (Ca9-22) human cancer cell lines using an established colorimetric MTT (diphenyltetrazolium bromide) assay protocol. Doxorubicin was used as a positive control. In brief, freshly trypsinized cancer cell suspensions were seeded in 96-well microtiter plates at densities of 5,000-10,000 cells per well with test compounds added from a dimethyl sulfoxide (DMSO) stock solution. After 3 days in culture, the attached cells were incubated with MTT (0.5 mg/mL, 1 hour) and subsequently solubilized in DMSO. The absorbance was measured at 550 nm using an ELISA reader. The IC50 is the concentration of agent that reduced cell growth by 50%, under the experimental conditions used.
Experimental Results:
For conveniently illustrating Compounds 1 to 13 extracted in the present invention, the structural formulas (I to XIII) corresponding to Compounds 1 to 13 are listed as follows.
A MeOH extract of the resin of F. assa-foetida was partitioned between n-hexane-MeOH (1:1) and then the MeOH layer was partitioned between CHCl3—H2O (1:1) to obtain a CHCl3 extract, which showed significant anti-H1N1 activity (IC50<3.4 μg/mL) and cytotoxicity for lung, breast and oral human cancer cell lines (IC50<20 μg/mL) Initial fractionation of the CHCl3 extract was carried out by open liquid chromatography on silica gel to give 11 fractions. Chromatographic fractionation of these active subfractions provided two new sesquiterpene coumarins (Compounds 1 and 2), a new diterpene (Compound 3), and 27 known compounds.
HRESIMS of Compound 1 exhibited a [M+Na]+ ion at m/z 463.2099 (C26H32O6Na). The IR spectrum showed absorptions for hydroxy (3440 cm−1), acetoxy (1729 cm−1), and aromatic (1613 and 1555 cm−1) functional groups. UV absorptions at 211 and 318 nm also indicated a coumarin nucleus oxygenated at the C-7 position. In the 13C NMR spectrum, Compound 1 displayed 26 carbon signals, with nine being typical for an umbelliferone skeleton [δ 101.5 (C-8), 112.5 (C-10), 113.1 (C-3), 114.2 (C-6), 128.7 (C-5), 143.4 (C-4), 155.8 (C-9), 161.2 (C-2), and 161.9 (C-7)] and the remaining 17 signals ascribable to a sesquiterpene moiety [δ 18.0 (C-13′), 13.1 (C-14′), 17.1 (C-15′), 25.9 (C-12′), 34.2 (C-9′), 44.9 (C-4′), 65.2 (C-1′), 68.9 (C-5′), 75.9 (C-8′), 119.5 (C-10′), 122.1 (C-2′), 123.3 (C-6′), 135.5 (C-11′), 137.6 (C-3′), 141.9 (C-7′)] with an acetoxy group (δ 21.2 and 170.3). In the 1H NMR spectrum, signals for two main moieties, a coumarin and a sesquiterpene, were revealed. The coumarin moiety appeared as 5 signals [δH 6.24 and 7.63 (each 1H, d, J=9.4 Hz), 7.35 (1H, d, J=8.4 Hz), 6.82 (1H, dd, J=2.4, 8.4 Hz), 6.79 (1H, d, J=2.4 Hz)]. In turn, the sesquiterpene moiety displayed signals for 4 methyls [δH 1.62, (3H, brs), 1.71 (3H, brs), 1.72 (3H, brs), 1.81, (3H, s)], 2 methylenes [δH 2.23 (2H, m), 2.28 (1H, d, J=6.0 Hz), 2.45 (1H, dd, J=7.6, 13.6 Hz)], an oxygenated methylene [δH 4.57 (2H, d, J=6.2 Hz)], 3 olefinic methines [δH 5.08 (1H, brt, J=6.4 Hz), 5.41 (1H, d, J=8.8 Hz), 5.50 (1H, brt, J=6.2 Hz)], and 2 oxygenated methines [δH 3.99 (1H, brt, J=6.4 Hz), 5.70 (1H, m)]. Compound 1 gave the same molecular formula, C26H32O6, and similar 1D NMR data to Compound 6 (8′-acetoxy-5′-hydroxyumbelliprenin) with the only difference being due to the placement of an acetoxy group and a hydroxy group. The key heteronuclear multiple bond correlation (HMBC) correlations suggested that the acetoxy and hydroxy groups could be positioned at C-5′ and C-8′, respectively (
A [M+H]+ ion at m/z 443.2433 (C26H35O6) was present in the HRFABMS of Compound 2. IR spectrum showed absorptions for hydroxy (3480 cm−1), acetoxy (1731 cm−1), and aromatic (1613 and 1555 cm−1) functional groups. UV absorptions at 213 and 321 nm indicated a coumarin nucleus oxygenated at the C-7 position. In the 1D NMR spectrum (Table 1), the signals indicated a carbon skeleton with two main moieties, a coumarin and a sesquiterpene. On the basis of HMBC correlations [δH 4.59 (H-1′)/δC 162.1 (C-7), 118.4 (C-2′), and 142.2 (C-3′); δH 4.77 (H-10′)/δC 171.2 (OCOCH3); δH 1.17 (H-12′)/δC 79.5 (C-10′), 72.4 (C-11′), and 26.7 (C-13′); δH 1.18 (H-13′)/δC 79.5 (C-10′), 72.4 (C-11′), and 24.8 (C-12′)], the sesquiterpene unit could be attached to C-7 (δ 162.1) of the coumarin moiety via an ether linkage, and the acetoxy and hydroxy groups were positioned at C-10′ (δ 79.5) and C-11′ (δ 72.4), respectively. Compound 2 was elucidated as 10′-acetoxy-11′-hydroxyumbelliprenin.
To determine the only chiral center at C-10′, Compounds 2 and 5 (10′-karatavicinol) possess the same biogenetic origin is speculated. Compound 5 was treated separately with (R)- and (S)-α-methoxy-α-(trifluoromethyl)-phenylacetyl chloride [(R)- and (5)-MTPA-Cl] in the presence of C5D5N, to yield the (S)- and (R)-MTPA esters (
Compound 3 gave the molecular formula, C20H26O3, as determined by HRESIMS (m/z 337.1784 [M+Na]+), indicating 8 degrees of unsaturation. Its IR spectrum showed absorptions attributable to hydroxy (3417 cm−1), carboxylic acid (1696 cm−1), and aromatic ring (1613 and 1514 cm−1) functions. Based on its 13C NMR and DEPT (distortionless enhancement by polarization transfer data), Compound 3 showed 20 carbon signals, including 4 methyls (δC 17.7, 20.7, 31.6, 31.6), 3 methylenes (δC 18.3, 35.2, 35.6), 6 methines (δC 46.3, 121.6, 122.8, 123.7, 128.3, 130.0), and 7 quaternary (δC 37.1, 46.0, 72.4, 132.4, 145.9, 146.6, 183.5) carbons (Table 2). Among the seven quaternary carbons, one was assigned as a carbonyl carbon at δ 183.5. Therefore, the data supported the presence of one carbonyl, 4 olefins, and 3 ring moieties to fulfill the 8 degrees of unsaturation apparently, and Compound 3 was postulated to be an abietane-type diterpene. The HMBC correlations [δH 1.57 (H-16)/δC 72.4 (C-15), 31.6 (C-17), and 146.6 (C-13); δH 1.57 (H-17)/δC 72.4 (C-15), 31.6 (C-16), and 146.6 (C-13); δH 1.40 (H-19)/δC 35.6 (C-3), 46.0 (C-4), 46.3 (C-5), and 183.5 (C-18)] suggested the hydroxy and carboxylic groups to be located at C-15 (δ 72.4) and C-4 (δ 46.0), respectively (
The absolute configuration of Compound 6 (8′-acetoxy-5′-hydroxyumbelliprenin) at C-5′ was still unknown at the time of the present invention. Compound 6 was treated separately with (R)- and (S)-MTPA-Cl in C5D5N to yield the (S)- and (R)-MTPA esters (Compounds 6a and 6b, referring to
As to other Compounds 4, 7 to 13 extracted from F. assa-foetida in the present invention, according to the literatures, they are assigned as 5′-hydroxyumbelliprenin (formula IV, Appendino et al., 1994), methyl galbanate (formula VII, Appendino et al., 1994; Yang et al., 2006), galbanic acid (formula VIII, Yang et al., 2006), farnesiferol C (formula IX, Abd El-Razek et al., 2007; Yang et al., 2006), farnesiferol A (formula X, Abd El-Razek et al., 2007), conferol (formula XI, Zhou et al., 2000), ligupersin A (formula XII, Iranshahi et al., 2009), epi-conferdione (formula XIII, Abd El-Razek et al., 2007).
Regarding the in vitro anti-influenza A (H1N1) virus assay, the test drugs are the pure 7-O-sesquiterpene coumarins, including Compounds 2 and 4 to 13, with amantadine as the positive control. The results are shown in Table 3. In Table 3, most of these compounds exhibited a higher antiviral potency than amantadine, except for Compounds 2, 5 and 12. Because the structures of many these compounds are quite similar, the results in Table 3 are further discussed as follows. For example, Compound 6 (OH-5′, OAc-8′) has an additional acetoxy group in comparison with Compound 4 (OH-5′), but Compound 6 was much less potent than Compound 4. Compound 2 (OAc-10′) has the same skeleton as Compound 5 (OH-10′), and these two compounds showed a similar potency to the positive control, amantadine. Between Compounds 7 (COOCH3-3′) and 8 (COOH-3′), Compound 7 showed better potency than Compound 8, and therefore indicated that methyl esterification of C-3′ enhanced the activity in the bioassay used. For the bicyclic-sesquiterpene coumarins, Compounds 12 and 13, a C-3′-carbonyl afforded greater potency than a C-3′-OH in this kind of skeleton. Overall, the present invention has determined that sesquiterpene coumarins from F. assa-foetida may serve as promising lead compounds for new drug development against influenza A (H1N1) viral infection. A standardized plant extract of F. assa-foetida, may also be worthy of being further investigated as a new phytomedicine.
Furthermore, please refer to Table 4, the compounds in the present invention were screened in a cytotoxicity assay on the cancer cells with doxorubicin as the positive control. Compound 11 exhibited the best potency (IC50 0.51, 2.6, and 3.4 μg/mL) against HepG2, Hep3B, and MCF-7 tested cancer cell lines, respectively. In addition, Compound 11 also showed high potency influenza activity (IC50 0.47±0.05 μg/mL) The remaining compounds were all inactive for all cancer cell lines (IC50>4 μg/mL)
Accordingly, the chloroform extract extracted from F. assa-foetida and the further extracted and chromatographed compounds in the present invention can effectively inhibits influenza A (H1N1) virus, and have cytotoxicity on human cancer cell lines.
While the invention has been described in terms of what is presently considered to be the most practical and preferred Embodiments, it is to be understood that the invention needs not be limited to the disclosed Embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
1H and 13C NMR Data of Compounds 1 and 2 (400
1′
2′
3′
4′
5′
6′
7′
8′
9′
1H and 13C NMR Data of Compound 3 (400
| Number | Date | Country | Kind |
|---|---|---|---|
| 99104501 A | Feb 2010 | TW | national |
This application is a division of U.S. patent application Ser. No. 13/888,516, filed May 7, 2013, which is a division of U.S. patent application Ser. No. 12/858,595, filed Aug. 18, 2010 now issued as U.S. Pat. No. 8,476,311, and claims priority to Taiwanese Application No. 099104501 filed Feb. 11, 2010, all of which are incorporated herein by reference as if fully set forth.
| Entry |
|---|
| Abd El-Razek et al., “Sesquiterpene Coumarins from Ferula foetida,” Journal of the Chinese Chemical Society, 54: 235-238 (2007). |
| Appendino et al., “Sesquiterpene Coumarin Ethers from Asafetida,” Phytochemistry, 35(1): 183-186 (1994). |
| Yang et al., “Sesquiterpene Coumarins from the Roots of Ferula sinkiangensis and Ferula teterrima,” Chem. Pharm. Bull, 54(11): 1595-1598 (2006). |
| Zhou et al., “Coumarins and bicoumarin from Ferula sumbul: anti-HIV activity and inhibition of cytokine release,” Phytochemistry, 53: 689-697 (2000). |
| Iranshahi et al., “Sesquiterpene coumarins from the fruits of Ferula badrakema,” Pharmaceutical Biology, 47(4): 344-347 (2009). |
| Lee et al., “Influenza A (H1N1) Antiviral and Cytotoxic Agents from Ferula assa-foetida,” J. Nat. Prod., 72: 1568-1572 (2009). |
| Number | Date | Country | |
|---|---|---|---|
| 20140329897 A1 | Nov 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13888516 | May 2013 | US |
| Child | 14336341 | US | |
| Parent | 12858595 | Aug 2010 | US |
| Child | 13888516 | US |
