TW201306856A - Extract of Erodium stephanianum Willd and use for suppressing tumor necrosis factor TNFα - Google Patents

Abstract

The present invention provides an extract of Erodium stephanianum Willd, which is capable of suppressing tumor necrosis factor TNF<alpha>. The present invention further provides an antagonist of the tumor necrosis factor TNF<alpha>, which includes the aforementioned extract of Erodium stephanianum Willd. The antagonist can be prepared as powders, capsules, pills, liquids, syrups, solutions, suspensions, emulsions, ointments, creams, lotions or patch Also, the extract of Erodium stephanianum Willd can be prepared into powders, capsules, pills, liquids, syrups, solutions, suspensions, emulsions, ointments, creams, lotions or patch, and utilized as medicine by oral, intravenous, parenteral or percutaneous dosing.

Description

The extract of Erium stephanianum Willd. and its use in inhibiting tumor necrosis factor TNFα

The invention belongs to the pharmacological use of a herbal extract. In particular, the present invention discloses the use of an extract of Erium stephanianum Willd . to inhibit tumor necrosis factor TNFα.

Press, Erodium stephanianum Willd . is a kind of traditional Chinese medicine, which has the effects of rheumatism, promoting blood circulation and relieving phlegm and relieving phlegm (see "Hannan Materia Medica"). The main chemical components of geranium include tannins, flavonoids, organic acids and volatile oils. So far, the pharmacological effects of geranium include antibacterial, antiviral, anti-oxidation, anti-liver disease, hemostasis and anti-tumor effects (see Journal of Pharmaceutical Practice, Vol. 19, No. 1, pp. 31-34, 2001). .

Tumor necrosis factor TNFα is a multifunctional monocyte that is released by macrophages, monocytes, and natural killer cells and plays a key role in acute and chronic inflammatory diseases, anti-tumor responses, and infections. The increase in TNFα synthesis is associated with the development of rheumatoid arthritis, psoriatic arthritis and intestinal inflammation. Clinical studies have confirmed that TNFα plays a major role in the pathogenesis of rheumatoid arthritis.

TNFα antagonists have been used to treat inflammatory diseases. For example, etanercept (Enbrel) has been used to treat rheumatoid arthritis and infliximab (Remicade) has been used to treat Crohn's disease and rheumatoid arthritis. Another anti-TNFα drug, adalimumab (Humira, Abbott), is also on the market. In addition, the drug D2E7 (i.e., a human anti-TNFα monoclonal antibody, Knoll Pharmaceuticals) is used to treat Crohn's disease and rheumatoid arthritis. The developing drug CDP 571 (Celltech) is used to treat Crohn's disease and the drug CDP 870 (Celltech) is used to treat rheumatoid arthritis. However, etanercept and infliximab have been found to present a potential risk of worsening congestive heart failure, and azumab is shown to increase the likelihood of developing lymphoma. In fact, all existing treatments using TNFα antagonists exhibit the disadvantages of toxicity, drug resistance and low efficacy.

This technology continues to find and develop TNFα antagonists with high efficacy and low toxicity. Herbs contain many unknown ingredients with potential pharmacological effects and are therefore always a source of drug development.

The invention discloses the use of the extract of Erium stephanianum Willd . for inhibiting tumor necrosis factor TNFα.

Brief description of the invention

In one aspect, the invention provides an extract of Erium stephanianum Willd . which inhibits tumor necrosis factor TNFα.

In another aspect, the present invention provides a tumor necrosis factor TNFα antagonist comprising the aforementioned geranium extract and optionally a pharmaceutically acceptable excipient.

The preferred embodiments of the present invention are set forth in the accompanying drawings, and in the claims

The present invention is an extract of Erodium stephanianum Willd. and its use in inhibiting tumor necrosis factor TNF. The specific embodiments of the present invention are illustrated for convenience of description and are not intended to limit the present invention. Variations in the design and needs of the specific embodiments of the invention may be made without departing from the scope of the invention.

As for the detailed composition of the present invention, since the tumor necrosis factor TNFα is a conventional inflammatory cytokine, it has a wide range of biological functions. TNFα is known to affect the growth, differentiation, survival, and physiological functions of a variety of cells. TNFα interacts with two distinct cell surface receptors, TNFR1 (p55) and TNFR2 (p75), both of which have high binding affinity to TNFα. The TNFα line is directly involved in the nervous system and inflammatory conditions and plays a major role in responsive to the damage caused by the virus or disease. TNFα also plays a major role in neuronal apoptosis.

Specific TNFα inhibitors provide the possibility of therapeutic intervention for diseases mediated by TNFα. The development of TNFα inhibitors is used to treat systemic diseases. For example, Immunex Corporation develops etanercept (Enbrel) to treat rheumatoid arthritis; Johnson & Johnson develops infliximab (Remicade) to treat Crohn's disease and rheumatoid arthritis; drug D2E7 ( That is, a human anti-TNFα monoclonal antibody, Knoll Pharmaceuticals) is used to treat Crohn's disease and rheumatoid arthritis; and Celltech develops the drug CDP 571 to treat Crohn's disease and the drug CDP 870 to treat rheumatoid arthritis. The successful development of therapeutic TNFα antagonists is a positive outcome in the treatment of TNFα-related diseases.

Despite the positive results of the above treatments, there are still many limitations in the use of biological TNFα antagonists, such as (1) increasing the likelihood of unwanted infections, especially in patients with latent tuberculosis, 2) limited reaction rate, (3) toxicity, drug resistance and/or low efficacy, and (3) high cost.

Herbs contain many unknown ingredients with potential pharmacological effects and are therefore always a source of drug development. This technique attempts to find and develop TNFα antagonists with high efficacy and low toxicity from herbs.

Herbs have been applied to humans and animals to treat a variety of different diseases and conditions. Herbs can be formulated into a variety of different forms including capsules, tablets and coated tablets; pellets; extracts and elixirs; powders; fresh or dried plants and plant parts; tea; juices; creams and creams; And any combination of the above. The herbal medicine can be administered by any conventional route including oral, rectal, parenteral, enteral, transdermal, intravenous, transdermal and topical delivery.

For the treatment of diseases mediated by TNFα, this technique requires a treatment that is anti-inflammatory and soothes pain and is based on herbal medicine, which has low toxicity and low side effects. Suitably, the method of treatment can be administered orally.

Erodium stephanianum Willd. is a kind of traditional Chinese medicine, which has the effects of rheumatism, promoting blood circulation and relieving phlegm. The pharmacological effects of the old geranium recorded in the literature include antibacterial, antiviral, anti-oxidation, anti-liver disease, hemostasis and anti-tumor effects.

When using the herbal wafer disclosed in U.S. Patent No. 6,645,719 to select an herbal active ingredient capable of binding to TNFα, the inventors have found that the geranium extract exhibits an activity of binding to TNFα, that is, in the geranium extract. There is at least one active ingredient which specifically binds to TNFα and inhibits the activity of TNFα. Further, through in vitro pharmacological analysis using L929 cells, the inventors surprisingly found that the geranium extract did inhibit the activity of TNFα and showed no cytotoxicity at all.

Accordingly, the present invention discloses the use of geranium extract for inhibiting TNFα. Further, the present invention provides an geranium extract which inhibits the activity of TNFα. The present invention also provides a TNFα antagonist comprising the aforementioned geranium extract.

To prepare the TNFα antagonist, the geranium extract can be mixed with a suitable pharmaceutically acceptable excipient. For example, the geranium extract can be mixed with a suitable powder base to form a dry powder, wherein the powder substrate is selected from the group consisting of lactose, starch, starch derivatives (such as polyglucan), hydroxypropyl methylcellulose or Polyvinylpyrrolidone (PVP). The geranium extract can also be mixed with a suitable pharmaceutically acceptable liquid carrier such as saline and dextrose solution.

A pharmaceutically acceptable additive such as a binder, a sweetener, a thickener, a fragrance, a disintegrant, a coating agent, a preservative, a lubricant, and/or a time delay agent may be added to the geranium extract in. Suitably, suitable sweeteners include sucrose, fructose, glucose, aspartame and saccharin; suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, yellow Raw gum, bentonite, alginic acid and agar; suitable fragrances include peppermint oil, whitegreen (wintergreen) oil, cherry flavor, citrus flavor and raspberry fruit flavor; suitable coating agents include acrylic acid and / or methyl Acrylic polymers or copolymers and/or their esters, waxes, fatty alcohols, corn bran, shellac and gluten; suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, Ascorbic acid, methyl paraben, propyl paraben and sodium hydrogen sulfate; suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride and talc; and, with appropriate time delay Agents include glyceryl monostearate and glyceryl distearate.

For the treatment of diseases mediated by TNFα, the administration mode of the geranium extract of the present invention includes oral, intravenous, parenteral and transdermal administration.

For oral administration, the geranium extract can be formulated into tablets, granules, powders, sachets, capsules, syrups, solutions, suspensions or emulsions. For intravenous and parenteral delivery, the geranium extract can be formulated as a pharmaceutically acceptable syrup, solution, suspension or emulsion. For transdermal delivery, the geranium extract can be formulated as an ointment, cream, lotion or patch.

The geranium extract is administered in an amount of 200 to 4000 mg/day, suitably 400 to 2000 mg/day. The dosage will depend on the age, weight, physiological state of the patient and the severity of the condition. It can be administered 1 to 4 times a day.

The following examples are provided to further illustrate the technical contents and features of the present invention. However, it should be understood that the invention is not limited to the embodiments described below.

Implementation Example 1. Method for preparing geranium extract

The geranium extract was prepared according to the flow chart shown in FIG. Specifically, geranium (100 kg) was added to a 50% aqueous ethanol solution (100 kg) at room temperature and soaked for 8 hours with stirring. Subsequently, the first filtrate and the first filter residue are separated by filtration. The first filter residue was added to a 50% aqueous ethanol solution (80 kg) at room temperature and soaked for 8 hours with stirring. Subsequently, the second filtrate and the second filter residue are separated by filtration. The second filter residue was added to a 50% aqueous ethanol solution (60 kg) at room temperature and soaked for 8 hours with stirring. Subsequently, the third filtrate and the third filter residue were separated by filtration. The first filtrate, the second filtrate, and the third filtrate were combined and the combined filtrate was concentrated under reduced pressure. The resulting concentrate is dried under reduced pressure or vacuum freeze-dried to obtain an extract.

Example 2. Screening on herbal wafers

A high-throughput method for the discovery of active ingredients in herbal medicines has been described in the specification of U.S. Patent No. 6,645,719. The method is described as follows:

(a) Classification of geranium extract by high performance liquid chromatography (HPLC)

The geranium (50 g) was ground to a fine powder and soaked for 3 days at room temperature with a 50% aqueous solution of ethanol (1 L). After filtration, the filtrate was concentrated under reduced pressure (BUCHI R200) to 25-30 ml. The concentrate was freeze-dried under vacuum (Beijing Sihuan LGJ-10D). One-50 of the amount of the obtained lyophilized powder was taken and dissolved in a 50% aqueous ethanol solution (1 ml) to obtain a solution equivalent to 1 g of a crude drug/ml. The solution was centrifuged (12000 rpm; Shanghai Anting TGL-16B) for 20 minutes. The clarified supernatant was collected and diluted 5 times with a 50% aqueous solution of ethanol, followed by filtration through a syringe filter (0.45 μm). The obtained filtrate was subjected to HPLC fractionation, and the process was as follows:

LC name: Agilent 1100

String: Hypersil ODS (250 x 4.6 mm, 5-Micron)

Temperature: 30 oC

VWD: 254 mm

Flow rate: 0.75 ml/min

Processing time: 96 minutes

Maximum pressure: 400 bar

Sample volume: 50 μl

Mobile phase: ethanol/water gradient solution (0 min: water; 90 min: 50% aqueous ethanol; 96 min: ethanol)

The results are shown in Figure 2. A tube of fractionation fluid was collected every minute and a total of 96 tubes were collected. The 96 tube fractions were separately loaded into respective wells of a microtiter plate containing 96 U-bottomed wells. The microtiter plate was dried by centrifugation under reduced pressure (Thermo Speed Vac).

(b) Loading the sample onto the coated plastic slide as a microarray

The fractionated liquid sample obtained in the above (a) was placed on the coated plastic slide using a microarray (Taiwan led Biodot A101) as described in the specification of U.S. Patent No. 6,645,719. The dried components of the geranium in the microtiter plate were first dissolved in 20% DMSO/0.1 M phosphate buffer (30 μl/well). The sample was loaded from the 96-well microtiter plate to the groove of the coated plastic slide using a 2-needle (0.8 mm diameter) tool. Biotin conjugates at concentrations of 4, 10, 50, and 250 ng/ml were also placed on the slides as a control group. After the spot sample on the groove of the plastic carrier was dried for 2 to 3 hours, the plastic slide was immersed in 1 M ethanolamine (pH 8.0) for 1 hour.

(c) Signal detection

The hybridization reaction was carried out using biotinylated tumor necrosis factor-α (B-TNFα) and Cy5-labeled streptavidin as probes. Cover the 2 grooves of each plastic slide with 2 glass cover sheets (22 mm x 22 mm), then add 20 μl TBST buffer (50 mM Tris-HCl/0.15 M NaCl to the first groove). /0.05% Tween 20, pH 7.3) As a blank control group, 20 μl of B-TNFα in TBST buffer (10 μg/ml) was added to the second groove. The reaction was then carried out in the dark for 1 hour at room temperature. After the reaction is completed, the plastic slide is rinsed 4 times with the TBST buffer, then washed 4 times with double distilled water, then dried at 37 ° C, and 2 of the plastic slides are covered with a glass cover sheet as described above. Groove. 20 μl of Cy5-labeled streptavidin in TBST solution (2.5 μg/ml) was added to the two grooves, and the reaction was protected from light for 1 hour at room temperature. After the reaction was completed, the plastic slide was washed 4 times with the TBST buffer, and then washed 4 times with double distilled water, followed by drying at 37 °C. Subsequently, a laser scanner (GenePix 4000, Axon) was used for scanning. The results are shown in Figure 3, in which the development of a red fluorescent spot indicates that the fractionated liquid system binds to B-TNFα.

Example 3. Evaluation of antagonism and cytotoxicity of TNFα by in vitro L929 cell proliferation/cytotoxicity assay

In vitro L929 cell proliferation/cytotoxicity assays were performed on microtiter plates. L929 cells were cultured in Eagle's minimal essential medium (EMEM) containing 10% bovine serum, 1% PBS, and 1% non-essential amino acids. Confluent L929 cells were washed with 2 ml PBS solution and trypsinized and subsequently resuspended in intact medium. A 200 μl cell suspension was aspirated to count the cell density, and the remaining suspension was centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and the intact medium was added to the diluted cells to form a final concentration of 1.5 x 10 ⁵ cells/ml. 100 μl of the cell suspension was added to each well of a 96-well trough flat-bottomed microtiter plate and incubated at 37 ° C and 5% CO ₂ atmosphere for 24 hours.

Antagonistic activity of TNFα

The geranium extract prepared in Example 1 was dissolved in a 1 x PBS solution to obtain a solution equivalent to 1 g of a crude drug per ml. The solution was further diluted 16, 1 , 64, 256 and 1024 times in 1 x PBS solution to obtain 62.5, 15.6, 3.9 and 0.98 mg crude drug/ml solutions, respectively. The resulting dilutions were separately mixed with an equal volume of TNFα solution and incubated for 1 hour. The medium was removed from the 96-well flat-bottomed microtiter plate of the L929 cells cultured for 24 hours before the end of the 1 hour pre-culture, followed by the addition of 50 μl of fresh 4 μg/ml actinomycin D (Act D). Medium. 50 μl of each of the four mixtures was taken and added to the 96-well flat-bottomed microtiter plate of the L929 cells cultured for 24 hours. The final concentration of actinomycin D (Act D) in the culture medium was adjusted to 2 μg/ml and the final concentration of TNFα was 0.1 ng/ml. Only the wells containing 2 μg/ml Act D or 0.1 ng/ml TNFα were used as the positive control group and the blank control group. The flat-bottomed microtiter plate was gently shaken for mixing, and incubation was continued for 24 hours at 37 ° C and 5% CO ₂ atmosphere. OD value readings were taken at 490/630 nm using an ELISA reader. The formula for the inhibition of TNFα is as follows:

The results are shown in Figure 4, which demonstrates that the geranium extract has an activity of inhibiting TNFα and acts as an antagonist of TNFα. Among them, the geranium extract showed a 78.9% inhibitory activity at a concentration of 3.9 mg of the crude drug/ml solution.

Cytotoxicity

The geranium extract prepared in Example 1 was dissolved in a 1 x PBS solution to obtain a solution equivalent to 1 g of a crude drug per ml. The solution was further diluted 16, 1 , 64, 256 and 1024 times in 1 x PBS solution to obtain 62.5, 15.6, 3.9 and 0.98 mg crude drug/ml solutions, respectively. The obtained 4 dilutions (50 μl) were separately added to the 96-well trough flat-bottom microdroplet of the above-mentioned 24-hour-bred L929 cells, followed by the addition of 50 μl of 4 μg/ml actinomycin D (Act D). Fresh medium was used to adjust the final concentration of actinomycin D (Act D) in the culture solution to 2 μg/ml. The 96-well trough flat bottom droplets were incubated at 37 ° C and 5% CO ₂ atmosphere for 24 hours. Subsequently 50 μl of XTT (ie 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide ()) was added to each well and incubated for an additional 4 hours. Readings were taken at 490/630 nm using an ELISA reader. The calculation formula for cytotoxicity is as follows:

The results are shown in Figure 4, which confirmed that the geranium extract did not exhibit cytotoxicity.

In this way, it can be understood that the present invention is an excellent creation, in addition to effectively solving the problems faced by the practitioners, and greatly improving the efficiency, and the same or similar product creation or public use is not seen in the same technical field. At the same time, it has the effect of improving the efficiency. Therefore, the present invention has met the requirements for "novelty" and "progressiveness" of the invention patent, and is filed for patent application according to law.

Figure 1 is a flow chart showing the preparation of the geranium extract of the present invention.

Figure 2 is a high performance liquid chromatography (HPLC) spectrum showing the geranium extract of the present invention.

Fig. 3 shows the results of screening of active fractions of the geranium extract of the present invention which bind to tumor necrosis factor TNFα.

Figure 4 shows the in vitro TNFα inhibitory activity of the geranium extract of the present invention and its cytotoxicity as revealed on L929 cell proliferation/cytotoxicity assay.

Claims (10)

An extract of Erium stephanianum Willd . is used for the manufacture of a medicament for inhibiting tumor necrosis factor TNFα. The application of the first aspect of the patent application, wherein the geranium extract is an extract prepared by the following method: (1) stirring and soaking geranium in an aqueous solution of 30-70% C _1-6 alkanol, After filtering and removing the residue, the filtrate is collected; (2) repeating step (1) for 2 to 4 times, and combining the filtrates collected in steps (1) and (2); (3) the filtrate collected in step (2) Concentrated under reduced pressure and then centrifuged to collect the supernatant; (4) The supernatant obtained in the step (3) is subjected to high performance liquid chromatography (HPLC) to obtain all the fractionated liquid obtained by time separation; (5) Combining the first 1/6 grade of all the fractions obtained in the step (4), the first 1/6 fraction can bind to the tumor necrosis factor TNFα; and (6) the first 1/6 fraction can optionally be pharmaceutically Acceptable carriers, excipients or adjuvants. The use of the first or second aspect of the patent application, wherein the medicament is administered orally, intravenously, parenterally or transdermally. The use of the second aspect of the patent application, wherein the HPLC eluate is a C _1-6 alkanol aqueous solution. The use of the second aspect of the patent application, wherein the C _1-6 alkanol is ethanol. The use of the medicinal herb extract according to claim 1 or 2, wherein the geranium extract is prepared into a powder, a capsule, a tablet, a liquid, a syrup, a solution, a suspension, an emulsion, an ointment, a cream, a lotion or a sticker. sheet. For the use of the first or second aspect of the patent application, wherein the geranium extract is administered in an amount of from 200 to 4000 mg/day, suitably from 400 to 2000 mg/day. A tumor necrosis factor TNFα antagonist comprising the geranium extract as described in claim 1 or 2. An antagonist of claim 8 which inhibits the activity of TNFα. An antagonist of claim 8 which is formulated as a powder, capsule, tablet, liquid, syrup, solution, suspension, emulsion, ointment, cream, lotion or patch.