TWI304342B - An herbal extract having anti- enterovirus activity and preparation of same - Google Patents

Description

1304342, IX. Description of the Invention: [Technical Field] The present invention relates to a herbal extract having anti-enteric activity, which is obtained by extracting at least one solvent from Polygonum cuspidatum or Rhubarb rhizome or a mixture thereof. The extract is purified to obtain an active ingredient emodin 0. [Prior Art] The enterovirus is a small RNA virus (Piconaviridae) with more than 60 species, including 23 types of A-type oxalic acid, 6 B-type saxch virus, '3 polioviruses, 30 kinds of echo viruses and enteroviruses (10) to 71 found in recent years. Enteroviruses are highly active in warm weather and are a common infectious disease in infants and young children. Human being is the only host of enterovirus. The infection mode enters the human body through the feces or droplets. It stays in the oropharynx and part of the intestines. It enters the bloodstream through the lymph and infects all organs of the body including the skin, mucous membrane, heart, central nervous system. Liver, respiratory tract, incubation period of about 1_5 days, more serious meningeal and myocardial invasion is about 7-10 days 'virus stays in the oropharynx about $ 7 _ days, about 6-8 weeks from the intestinal tract. Symptoms caused by enterovirus include fever, oral ulceration, and skin complications. More than 90% of patients have mild or no symptoms. There is no specific drug for the treatment of the virus. Only supportive therapy can be taken. Will heal itself. 'For a small number of patients with severe enteroviruses, the method is adopted. Immunoglobulin therapy only smashes complications such as encephalitis, brain = pulmonary edema, and myocarditis to relieve symptoms, but the nine methods improve the prognosis. In the past few years, the enterovirus II y non-toxic 71 type occurred in Taiwan, and the clinical characterization was by hand; ^ mouth disease, two $ for enteropathy encephalitis, and occasional death sickness. ",, inflammation and 1304342; currently there is no vaccine to prevent enterovirus, nor can it be once-life immunization, only by strengthening personal hygiene, such as wearing a mask and washing hands frequently and 'environmental hygiene, such as strengthening chemical disinfection, and Public health policy is prevented for high-risk groups. Since there is no vaccine or no specific medicine for enteric virus treatment, there is an urgent need for the treatment of enterovirus in clinical medical research. Polygonum cuspidatum (/^/ygcwwm is a scorpion Plants, commonly used in the Chinese medicine industry to promote blood stasis, pass through pain, and good at Tongli meridians, to treat the itching caused by wind, cold and dampness, can be used for bruises to heal the pain. Polygonum® is also clear The effect of dampness and heat can treat jaundice and stones caused by hepatobiliary. The knotweed is bitter and cold, and it is good for heat-clearing and detoxifying. It can be used internally or externally. The knot can still relieve phlegm and stasis, because it is cold and cold, it is used for hot cough. Studies have found that Polygonum cuspidatum inhibits Staphylococcus, Escherichia coli, Rhizoctonia streptococci, etc., and also kills Leptospira, in addition to elevated white blood cells and platelets It has the functions of calming, diuretic, lowering blood sugar, stopping bleeding and reducing inflammation. Rhubarb (Rheum palmatum L·; R. officinale Baill.; R. tanguticum Maxim· exBalf·) is a plant of the family Polygonaceae, which is bitter and cold. It can be used for diarrhea, detoxification, blood stasis, heat and dampness. It is used for gastrointestinal stagnation, constipation, pain, abdominal pain, urgency, heat, suffocation, vomiting, blood, and redness. Pain, heat, sores, abdominal pain, erysipelas, scald, postpartum stagnation, abdominal pain, blood stasis, irregular menstruation, bruises, stagnation, jaundice, chest, etc. Modern use for viral hepatitis, acute gallbladder Inflammation, pancreatitis, etc. 曰Patent No. JP60250A discloses that the cytomegalovirus (Chinese herbal medicine compound) contains the Polygonum cuspidatum. The Chinese Patent No. CN1106690 discloses that the Chinese herbal compound for treating hepatitis also contains Polygonum cuspidatum. There is no specific medicine to treat, so if you can apply Polygonum cuspidus 1304342 to enterovirus suppression or prevention, it will be of great help for the treatment of enterovirus in clinical medicine. SUMMARY OF THE INVENTION In view of the prior art that there is no effective drug for treating enterovirus, one of the objects of the present invention is to provide a herbal extract having anti-enteric activity, the main purpose of which is to prevent, inhibit or kill enterovirus. Another object of the present invention is to provide a method for in vitro anti-enteric activity, which utilizes extracts of Polygonum cuspidatum, Rhizome rhizome or a mixture thereof to inhibit enterovirus activity. A further object of the present invention is to provide an anti-enteric activity active herbal extract. a method for extracting a chemical substance having an activity of inhibiting enterovirus among rhizome, Rhizome rhizome or a mixture thereof. To achieve the above object, the present invention provides an anti-enteric virus activity By the knotweed (/\ kisser (10) Sieb et

Zucc·), Rhubarb (Rheum PalmatUmU; R ()fficinaleBaill.; R·

And got it. Solvent extraction

7 l3〇4342 is prepared by the method of the following steps: (a) extracting the rhizome of rhizome or rhubarb or a mixture thereof with acetone to obtain an extract; (b) taking the acetone layer of the extract of the aforementioned step (a), Digesting methane with water to obtain another extract; and (c) taking the dichlorosilane layer of the extract of the above step (b). The dioxane layer of the above step ((;) contains a compound of the formula (I). The present invention also provides a pharmaceutical composition comprising an effective amount of a compound of the following formula (I), and a medicament for use together with the compound Acceptable carrier or diluent:

It is used to prevent, treat or kill enterovirus. R=H of the above chemical formula (1), chemical formula (1)

Yellow (Rheum palmatum L··, R· officinale Bam··, R·tanguticum

The rhizome of Maxim·ex Balf·) or a mixture thereof, obtained by extracting with at least one solvent. In a preferred embodiment, the solvent for extraction comprises methanol, acetone, dimethyl burn, alcohol, water or a mixture thereof. In another preferred embodiment, the compound (I) is prepared by a method comprising the steps of: (a) extracting the rhizome of the knotweed or rhubarb or a mixture thereof with an acetone to obtain an extract; (b) taking the foregoing Step (a) extracting the acetone layer of the extract, extracting with methylene chloride and water to obtain another extraction; (c) taking the methylene chloride layer of the above step (b) extract 1304342; and (d) purifying the above two gases The methane layer is obtained to obtain the compound (I). Purification of step (4) can be accomplished by, for example, but not limited to, over-extrusion or gel chromatography. Those skilled in the art can also use other purification methods to purify compound (I). The present invention also provides a method for in vitro anti-enteric activity, which comprises contacting the aforementioned herbal extract with a virus, thereby inhibiting the activity of the virus. The invention further provides a method of in vitro anti-enteric activity comprising contacting a pharmaceutical composition as described above with a virus, thereby inhibiting the activity of the virus. The present invention utilizes an extract of Polygonum cuspidatum, rhizome of rhubarb or a mixture thereof, and a pharmaceutical composition containing the formula (I), which is effective for inhibiting enterovirus activity for clinically preventing or treating enterovirus. [Embodiment] The present invention provides an extract containing humectin (Emodin), a root of rhubarb or a mixture thereof, and a pharmaceutical composition containing the chemical formula (1) for treating or preventing enterovirus. In order to achieve a better extraction effect, in the extraction step of the present invention, the above-mentioned Chinese herbal medicine material can be made into smaller particles by physical means such as mashing, grinding, chopping, etc., preferably by grinding. In a manner, it is more preferable to grind one or more of the above-mentioned Chinese herbal medicines into a powder form for subsequent stroke. After extracting the above-mentioned Chinese herbal medicines in a solvent, the extract can be formulated for various applications. However, in order to increase the purity of the active ingredient in the extract, various purification steps may be carried out after the extraction step of the present invention as needed. The method of purifying the extract is not specifically taught, and 1304342 • is well known to those skilled in the art, and methods which can be used include, for example, chromatography, crystallization, filtration, precipitation, etc., should be regarded as Determined for the purpose to be achieved. In a preferred embodiment of the present invention, the above-mentioned Chinese herbal medicine material may be subjected to a purification step after extraction with at least one polar solvent, for example, by filtration to remove insoluble components. In another preferred embodiment, the purifying step comprises using a silicone gel in the presence of dichloromethane and ethyl acetate (preferably 4:1) as the extract. The herbal extract having antiviral activity prepared by the present invention may be used in 10 purified and/or unpurified form, or preferably, with a carrier, a diluent, an excipient or a traditionally used in the formulating technique. Adjuvant is used together. For this purpose, they are suitably formulated in a conventional manner as emulsifiable concentrates (for example, as hand lotions, strippers, laundry detergents, shampoos), coatable pastes (for example , as a coating), a solution that can be sprayed directly (for example, as a spray), a diluted solution (for example, as a beverage, a health food), a fillable ingredient (for example, as a toy, a wipe), and a carrier The mixed powder, soluble powder, dust, granules, and m can also be coated in a suitable coating (eg, as an air filter, water filter, mask contents, or filter). As the type of the composition, it can be selected depending on the intended purpose and the main environment, for example, application methods such as spraying, atomizing, spraying, spreading, coating or emulsifying. The composition may also contain additional adjuvants such as stabilizers, antifoaming agents, viscosity modifiers, binders or tackifiers or other formulations for achieving particular effects. The herbal extract having antiviral activity prepared by the present invention may be formulated into a pharmaceutical composition for treating or preventing enterovirus to treat or prevent enterovirus infection or severe enterovirus infection, if necessary. The herbal extract having anti-enteric activity prepared by the present invention may be administered in a single dose or in multiple doses, either alone or in combination with a carrier or excipient which is acceptable on the medicine 1304342. Pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, may be formulated according to conventional techniques, for example, see Remington's Pharmaceutical Sciences, 19th edition, Gennaro Editor, Mack Publishing Company, Easton, PA (1995).

The pharmaceutical compositions can be formulated specifically for any suitable route of administration, for example, orally, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intra-cell, intraperitoneal, vaginal and parenteral. (including subcutaneous, intramuscular, intraspinal, intravenous, and intradermal) pathways. It will be appreciated that the preferred route of administration will depend on the general condition and the age of the patient being treated, the characteristics of the condition to be treated, and the active ingredient chosen. Pharmaceutical compositions for oral administration can include solid dosage forms, for example, capsules, lozenges, dragees, pills, powders, and granules. Suitably, they may be prepared with a film coat (e.g., a casing) according to methods well known in the art, or may be formulated to provide controlled release of the active ingredient, e.g., 'continuous or extended release. Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups, and panacea. Pharmaceutical compositions for parenteral administration include sterile aqueous solutions of non-aqueous solutions: m suspension (d), and sterile powders which are previously dissolved in a solution or dispersion. Other suitable forms of administration include suppositories, spray gels, inhalants, dermal patches, implants, etc. Guangjun" pore phase, the more effective dose of anti-enteric activity prepared by the present invention, will be based on the frequency of administration And the mode 2 is determined by the age, the weight, the site to be treated, and any accompanying diseases, etc., the weight 1304342 - the following provides a detailed description of the embodiments of the present invention, the technology and features of the present invention, The present invention is not intended to limit the invention, and any person skilled in the art can make various modifications and retouchings without departing from the spirit and scope of the invention. Embodiments Example 1, Polygonum cuspidatum or Rhubarb crude extract Preparation p The preparation process of the crude extract of Polygonum cuspidatum of the present invention is as shown in the first figure. The Polygonum cuspidatum used in this embodiment is the root of the perennial herb Polygonum cuspidatum (Potygom Sieb· et Zucc·), which is 100 grams of Polygonum cuspidatum. After grinding into powder, it was extracted 3 times with 300 mL of acetone, the acetone layer was POCU-A, the non-acrylic layer was extracted 3 times with 300 mL of methanol, the sterol layer was POCU-M, and the non-methanol layer was 300 mL. The extract was extracted twice, and the water layer was POCU-W. The rhubarb base used in this example was rhubarb (Rheum palmatum L·; R. officinale Baill·; R. tanguticum Maxim· ex Balf·) I rhizome. 100 g, after grinding into powder, extract 3 times with 300 mL of 95% alcohol, the alcohol layer is RHPA-Et, the non-alcohol layer is extracted twice with 300 mL of water, and the water layer is RHPA-W. Example 2: The tiger stick of the invention Or rhubarb crude extract for enterovirus test RD cells (embryonic human rhabdomyosa corma) were cultured on the day before the screening, which was planted into 4*104 cells in a 96-well cell culture dish. After 24 hours, after 12 1304342 • culture solution was aspirated, it was easy to rinse twice with PBS. Finally, each well was added to DMEM medium of ΙΟΟμΙ, and then into the incubator. Take the crude extract of Polygonum cuspidatum in Example 1 ( The original concentration is 200mg/ml, DMSO): POCU-A, POCU-M and P0CU-W are prepared into 300pg/ml and 100pg/ml respectively. Further, rhubarb crude extracts RHPA-Et and RHPA- are taken. W, separately formulated into SOOgg/ml and lOOpg/ml concentration for subsequent The virus infection rate of enterovirus 71 is valence 100 TGID I 50/0·1 mL, and 50 μl of enterovirus is applied to each well. Drug treatment can be divided into four conditions: 1. The drug to be tested is treated separately 〇D) After aspirating the old DMEM medium, 50 μl of fresh DMEM and 50 μl of the diluted drug to be tested were taken. 2. Pretreatment of the drug to be tested 1 day infection with virus (Dlh+v): After aspirating the old DMEM medium, 50 μl of the diluted drug to be tested was added, and 50 μl of virus was added 1 hour later. 3 · Simultaneous treatment of the drug to be tested and virus (v+d): After aspirating the old P DMEM medium, add 5 μl of the diluted drug to be tested and 50 μl of virus. 4. Virus adsorption 1 hour when the drug to be tested (Vlh+D) was added: After the old DMEM medium was aspirated, 5 〇μ1 virus was added for adsorption for 1 hour, and 50 μl of the diluted drug to be tested was added. After four days of treatment, when microscopic observation of apoptosis in the virus-controlled group was about 75% or more, the sputum test was performed, and the absorbance was compared with the blank group (M〇ck), and the 记录 was recorded when the cell survival rate was less than 25; Recorded as + when the cell survival rate is greater than 25, less than or equal to 50; recorded as ++ when the cell survival rate is greater than 5〇, and the small 13 1304342 is equal to 75; when the cell survival rate is greater than 75, less than or equal to 1 (K) It is +++; when the cell survival rate is greater than 100, it is recorded as ++++. It should be noted that the virus control group must be 50 or less. The drug to be tested has a recovery effect on the apoptosis caused by the virus, and the cell survival rate is +++ is valid for music. The experimental results are shown in Table 1. Table 1----- 15〇Rg/ml 50pg/ml Dl+V Vl+D DV D Dl+V Vl+D DV D ^P〇CU-A + +++ +++ +++ + ++ + +++ +++ JPOCU-M +++ ++ ^POCU-W + + RHPA-Et ++ ++ ++ +++ +++ ++++ +++ ++++ RHPA-W +++ ++++ +++ ++++ ++++ +++ +++ ++++ The experimental results show that p〇cu_A and p〇CU-M extracts are at a concentration of 150 pg/ The inhibition of enterovirus 71 by ml can reach 75%_1〇〇% of sputum cell survival rate, while POCU-W has only 25% _50% cell survival rate. However, the main component that can actually prevent or inhibit the enterovirus is still located in the P0CU-A of the same layer of Polygonum cuspidatum, which can achieve a cell survival rate of 75%_1〇〇%. The experimental results show that RHPA-Et (rhubarol alcohol extract) and RH^A-^ (large water extract) can inhibit the intestinal tract 7135 at a concentration of 5 〇 (4) and 150 pg/ml. %_1〇〇% of cell viability. Preparation of Purified % of Extracts It can be seen from the second embodiment that the components mainly inhibiting enterovirus are located in the two layers of the tiger. Therefore, the preparation of the large-scale extraction of Polygonum cuspidatum of the present invention 14 1304342 The flow is as shown in the second figure. The Polygonum cuspidatum used in this embodiment is the rhizome of the perennial herb Sieb· et Zucc. After 5.4 kg of Polygonum cuspidatum, after grinding into powder, it was extracted 4 times with acetone, and then the C-extract was concentrated to obtain 210.7 g of a dry extract. The dried extract is subjected to partition extraction using a two-phase extractant (dioxane/water). The digas methane layer was taken out and concentrated to give a dry extract of 157 g. The dried extract was purified by silica gel column chromatography and mixed in a ratio of dioxane/ethyl acetate 4:1 to elute POCU-k02 (Emodin) and POCU-kOl (Physcion). ) and other components [References 1 to 2]. Example 4: Enterovirus test was carried out with the purified product of Example 3 of the present invention. RD cells (embryonic human rhabdomyosa corma) were subjected to secondary cell culture one day before the screening, and each line was cultured in a 96-well cell culture dish. Into 4*104 cells. After 18-24 hours, after the culture solution was aspirated, it was easily washed twice with PBS, and finally, each well was added to a DMEM medium of ΙΟΟμΙ, and then placed in an incubator. # The purified product in Example 3 (original concentration 50 mg/ml, DMSO) was formulated into l〇〇pg/ml and 20 pg/ml. The amount of virus infection of enterovirus 71 was iootcid 50/0.1 mL, and 50 μl of enterovirus was applied per well. The drug treatment can be divided into four conditions: 1. The drug to be tested is treated separately (D): After the old DMEM medium is aspirated, 50 μl of fresh DMEM and 50 μl of the diluted drug are taken. 2. Pre-treatment of the music to be tested 1 day infection virus (D1 h+V): suction

15 1304342 Too test: After the sputum culture solution, add the diluted 1 to the sputum. 3. Add the human _ virus after the drug 1 to be tested. DMElvt^[, virus simultaneous treatment (V+D): after taking the old 50 μ1 disease ^ nutrient solution, 'add 5 〇 μ 1 diluted drug to be tested and I 1 time to add the drug to be tested (Vlh+D) ): After the hour of absorption, the culture solution is added, and 50 drugs are added to absorb the drug to be tested for 4 days. When about 75% or more, when the microscope observes the apoptotic light value of the virus control group, when the cell survives the ^Μττ test' and compares with the blank group (M°ck), the absorption is 25% less than the material^ less than 25%. + / _; when the cell survival rate is equal to 7 recorded as ++ cell survival rate is greater than 5 (10) less than recorded as +++; when (10) six cell survival rate is greater than 75% less than or equal to 1%, guangdong 2 soil cell A survival rate greater than 100% is recorded as + + + +. It should be noted that ::; with:: J is less than or equal to 5〇% ‘the drug to be tested is caused by the virus. From the ^: recovery effect, the cell survival rate is +++ or more for effective music. The experimental results are shown in Table 2. Table 2: Test concentration of Emodin (POCU-k02) (gg/ml) D1+V V1+D DV D Virus cell blank group 50 +/- +/- +/- +/- +/- +++ 25 + /- +/- +/_ +/- +/- ++++ 12.5 ++ + ++ ++ +/- 9.375 ++ +++ ++++ +++ +/- +++

16 1304342 6.25 ++ +++ +++ +++ +/- ++++ 4.68 +++ ++ +++ +++ +/- ++++ 3.125 +++ +++ +++ +++ +/ ++++ 1.56 ++ ++ ++ ++ +/- +++ The experimental results show that the emodin (Ein〇din) in the extract of the present invention has an effective prevention or inhibition of enterovirus The ingredients. When infected with sputum (Dlh+V) 1 hour after pretreatment of the drug to be tested, • P〇CU-k02 with a concentration of 3.125pg/ml to 4.68pg/ml can reach 75% to 1% of cell viability, showing Emodin has the effect of preventing enterovirus infection or inhibiting enterovirus. After the cells were adsorbed for 1 hour after the virus was added, the drug to be tested (Vlh+D) was added, and 3.125pg/ml to 9.375pg/ml of emodin (Emodin) reached a cell survival rate of 75% to 100%, indicating that the rod extract was extracted. The emodin (] 51110 (1111) can effectively inhibit enterovirus activity. When the (V+D) cells are treated with the drug to be tested, 3·125μ§/ιη1 to 9.375|Llg/ml rhubarb It can reach 75% to 100% of cell viability, indicating that Emodin, an extract of Polygonum cuspidatum extract, inhibits the apoptosis caused by enterovirus. The extract of Polygonum cuspidatum L. Emodin was analyzed to have the following chemical formula 1:

R = H, formula (I). In the preparation of the invention, the extract of the grass 17 1304342 is effective against the enterovirus, and therefore, the medicinal composition containing the emodin (Emodm) of the present invention or the mixture thereof can be used for clinical application. Prevention or treatment of the knotweed or rhubarb extract of the present invention can also be applied to air hand washing milk, water filter material, paint, rubbing cloth to shrub two: f 丄 丄 - aspects of the disease separated from the above materials ίί virus adsorption ^ In any case, to avoid infection of the virus in contact with the human body, on the other hand, materials with antiviral activity can deactivate the virus adsorbed on it, so that the disease can lose its infection, thereby eliminating the transmission route of the virus. It will be of great help to prevent and control the spread of the virus. Other embodiments of the present invention have been described in detail in the foregoing embodiments, and any person skilled in the art can modify and modify the present invention as needed without departing from the spirit and scope of the present invention. The invention is therefore also included in the scope of the patent application of the present invention. 〃 ^ 1304342 ; [Simplified description of the drawings] The first figure is a flow chart of the crude extraction of Polygonum cuspidatum in the first embodiment of the present invention. The second figure is a purification flow diagram of the Polygonum cuspidatum extract in the third embodiment of the present invention. [Component Symbol Description] None [Reference] • 1. Tsukida K, Yonemichi Μ (1954) Constituents of polygonaceous plants. III. Constituents of Ko-jo-kon (Polygonum cuspidatum). J Pharm Soc Jpn 74: 379-382. 2. He Ly, Luo SR (1980) Studies on the analysis of anthraquinone derivatives of Chinese medicinal herbs. I. Separation and determination of constituents of Chinese rhubarb- Acta Pharm Sin 15:555-562.

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Claims (1)

1304342 "",————一...".一^~! ""· ν一? .·〇>, ". . ;; .... .... X. Patent application scope: 1. A herbal extract with anti-enteric activity, which is obtained by using the knotweed (Mum cm) or rheumatism (Rheum palmatum L.; officinale Baill.; R. tanguticum Maxim, The rhizome of ex Balf·) or a mixture thereof is obtained by solvent extraction. The special solvent of the present invention includes alcohol, alcohol, acetone, dichlorosilane, water. Or a mixture thereof. _ 3. The herbal extract according to claim 1, wherein the extract contains at least the following compound of the formula (I): + ^ ^ ^ ^ ^ ^ ^ ^ R=H, Chemical Formula (I). The herbal extract according to item 1 is prepared by the method comprising the following steps: (8) $13⁄4 ketone to take the rhizome of rhizome or rhubarb or a mixture thereof to obtain an extract; (other): _ layer n-domain water extraction, obtained as a stroke liquid, and (C) take the methylene chloride layer of the above step (b) extract. 20 1 t application t ^ ^ in the middle of the grass The extract of the above step (6) contains the compound of the formula (1). The compound composition of the formula (1) contains a compound and a substance used together with the compound '1304342 〇H3 R=H, chemical formula (I), used to prevent, treat or kill enterovirus. 7. The pharmaceutical composition according to claim 6, wherein the compound of the aforementioned chemical formula is a rhizome r/i/ziwia or rhubarb (Rheum palmatum L.; R. officinale Baill.; R The roots of tanguticum Maxim, ex Balf·) or mixtures thereof are the source of extraction. 8. The pharmaceutical composition according to claim 6, wherein the compound of the above Chemical Formula 1 is obtained by at least one > gluten-free cadmium CM & r/uzoma or large page (Rheum palmatum L.; The rhizome of R. officinale Baill.; R tanguticum Maxim· ex Balf·) or a mixture thereof is isolated and purified. 9. The pharmaceutical composition according to claim 8, wherein the solvent comprises: alcohol, sterol, propylene, methylene chloride, water or a mixture thereof. 10. The pharmaceutical composition according to claim 6, wherein the compound (1) is prepared by a method comprising the steps of: (8) extracting a new or read root or a mixture thereof to obtain an extract, ( b) the step (8) extract the acetone layer of the extract, and the other extract; 卞# 〇又竹 (0 takes the step (b) of the extract of the dichlorohydrazine sound _ the aforementioned dichloro layer 峨 obtained compound; )'. 11. The application of the (4) composition described in the application of the paste _ 1G, the capping step (6) 21 1304342 • the purification is achieved by filtration or gel chromatography. • A method for in vitro anti-enteric activity, which comprises contacting a herbal extract of any one of claims 1 to 5 with a virus, thereby inhibiting the activity of the virus. A method for in vitro anti-enteric activity, which comprises contacting a pharmaceutical composition according to any one of claims 6 to 11 with a virus, thereby inhibiting the activity of the virus. / twenty two