CA1264474A - Pharmaceutical composition using pdi for increasing immunity function - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A new pharmaceutical composition for treatment and prevention of side effects of anticancer chemotherapy and rediotherapy and increasing the immune function con-tains Ferulic acid, Ginsenoside, Anethole and sodium Cinnamate. Processes for producing these components and related pharmacological effect are proveded.

Description

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PHARMACEUTICAL COMPOSITION USING PDI FOR INCREASI~G
IMMUNITY FUNCTION

Bac~ground ~f the Inventlon .

Field of the Invention This invention relates to a new pharmaceutical composition for decreasing side effects of anticancer chemotherapy and increasiny the immune function in human body, Specifically, this invention provides a new composition of four major active ingredients.
A. Ferulic acid extracted from Levisticum Officinale Koch or Angelica sinensis ~iels.
B. Ginsenoside extracted from Panax quinque~olium L.
or Panax ginseng C. A. Mey.
C. Anethole extracted from FoenicuLurn Vulyare Mill.
D. Sodium cinnamate extracted from Cinnamomuin Cas~ia Blume or Cinnamomum Cassla Presl.
Four herbs: ~evistlcum Of~icinale, Foeniçulum Vulgare Mill, Panax quinque~olium L~or Panax yinseng C.
A. Mey and Cinnamomum Cassia Blume are regarded by F.D.A~ of U.S. as recognized as safe.
Desçriptio~ o Prior Art A lot of anticancer medicine includiny chemical and antLbiotics have e~fect to kill off cancer cells. But it also kills off some normal human cells. E'or examp:Le, 44~

highly effective against a wide range o~ human cancer~.
Cy~lophosphamide established a role in the treatment of some major cancer types includin~ Lymphomas, Acute Ly~phatic Leukemias, Chronic Lymphatic Leukemias, Breast, Pulmonal, Ovarial cancer and Tumor o~ marrow mulciple, Osseous, Sarcoma etc.. ~nfortunately, Cyclophosphamide has toxicity, for example, it dves damag~ to hemotopoietic organs, alimentary tract and decreasing immunue ~UnGtiOn. The toxicity of other anticancer medicine, ~or example, Fluorouracil, ~5ustine and 6-Mércaptopurine, etc. is higher than Cyclophosphamide.
Some antibiotics are e~fect~ve anticancer drugs, for example, Adriamycin is used for treatment to some caneers which include leukemias, gastric pancreatlc, breast cancer, etc.. A prime limiting Pactor to the administration of adriamycln is cardiotoxicity. The most serious side effec~ of adriamycin administrati3n is myocardial deg~neration causing congestive heart failure. This acute cardiomyopathy may mani~est pericarditis-myocarditis, acute left ventricular .. :
dys~unction, arrhy~hmia and myocardial in~arction.
Adriamycin induced cardiomyopathy was thought to he permanent and rapidly progressive. Late cardiomyopathy develQps in weeks, months or even years. Some pa-tients were reported to have developed pro~ressive cardiomyopathy two and two-ha}f years a~ter recei~ln~

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this drug.
Obviously, to overcome toxicity of anticancer i6 very important. Many agents have been suggested to reduce or prevent toxicity of anticancer meclicine. For example, Vitamin E and ~-acetyl-L-Cysteine have also been reported to be effective in preventing adriamycin cardiotoxiclty. Although more recent research showed evidence to contradict these findin~s, in that neither of vitamin E and N-acetyl-L-Cysteine prevent adriamycin-induced cardiotoxicity.
So far, no drugs have been succeeded to overcome toxicity of anticancer drugs.
The basics in research of decreasing side effect of radiation therapy are the same as in chemotherapy.
For reason given above, effective treatment o cancer is very much hampered.
ry_~ Invention ~ ~
There is a need to provlde a composition comprislng several aotive ingredients which, in combination, are ~
useful in treatlng and~preventing tOXiCIty of anticancer drugs in human body and~in increasing i~nmunue function.
This invention provides a pharmaceutical compositlon referred to as PDI.
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PDI comprises (A) Ferulic Acid, ~Bt Ginsenoside, (Ct Anethole, (D) Sodium Cinnamate. The composition of PDI and the sources o~ its components are llsted below:

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TABL~ 1 Component Source ______.___~______ _____~_________________________________ (A) Ferulic Acid Roots of Levisticum Qfficinale Koch or Angelica Sinensis Diels (B) Ginsenoside Roots o~ Panax quinquefolium L or Panax ginseng C. A. Mey, (C) Anethole Fruits of Foeniculum Vul~are Mill (D) Sodi~m Cinnamate Stem of Cinnamonnum Cassia Blume or Cinnamomum Cass:La Presl ________________~____ _______________.__________________ The process for producin~ PDI comprises sep~rately extracting ground of the above natural materials with approprlate solvents such as alcohol or water, removin~
lipids by extraction with ether or petroleum ether, crystalization or chromatographic fractionation and then mixing its components in the desired preportion.
The chemical structures of the components of PDI
are shown in Figures I-V: :

CH = CHCOOH
,.

cll 1, FeFulic acld ~644~7~

~, o \ ~C 1~3 C~3 Cl~

When . : R1 = Glucose 6 1 ~lucose R2 = Glucose 2 l Glucos~e R3 ~ H
The compound is ~insenoside b1.
: Melting point, 198-202VC.
When R1 = Glucose ~2 H
R3 = -0- Glucose The campound .is Ginsenoside g1.
Melting point, 192-194C.
II Chemical structure of Ginsenoside.

c~ o~ ~26~74 1-~ Oj~

~0 I~ ol~ C

D~ ~ Glucose 6 _ 1 Glucose ,,~ olt C ~1LC~
~ o ~

D1~

~0 . Glucose Z 1 Glucose (.`t~DI~ ¦
~--0\o ~ , 0~ y ~ 0~ Glucose 2 _ 1 Rh~mnose t~
~) Dll ~
III chemical structure of substituents af Ginsenoside ~2~44~74 fH2CH = CH2 ~ ~ //J
y : ~ IV chemical structure of substituents of: Anethole : .. .

CH ~ C~-COONa~ ~ ~

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~: : V::chemical structure o~ sub~ti~u~nts of ~: : : Sodlum Cinnamate ~, ~2~4~4 Detailed DescriPtion The invention is further illustrated by the following examples and tests but not :Limited by the following examples:
Example 1: Ferulic acid extracted from Levisticum Officinale Koch or An~elica sinensis diels.
1 kg o~ dried powder of Levisticum Officinale Koch or Angelica sinensis Diels is extracted with 5000 ml of boiling water at 1 hour. Repeat 3 times. Combine extractives. Distilled to 1000 ml by reduced pressure distillation (about 1~ mm Hg is better). Add 2000 ml 95%
ethanol to 1000 ml the residue with stirring. Set ~4 hours. Pour filter. Save the filtrate. Filtrate is conce~trated to syrup under reduced pressure distiIlatl~on (about 17 mm ~g absolute).~ Add 300 g silica gel to syrup. Cool the syrup gel mixture to room temperature and dry the same at 60C. Dried powders is placed in a percola~or. These powders are washed with 95~ ethanol until ethanol become light color. Ethanol is ~combined and di~tilled at 17 mm H~. E-thanol is recovered and a still residue ia obtained. Thls still residue is dissolved in 200 ml of distllled water. This water solution is mixed with polyamide. Se~t column. Wash with di~tilled water, then wash Wlth 50X ethanol. 50X ethanol is combined and distilled at 1~ mm H~. The residue i8 dissolved In 100 ml o~ methanol. This methanol is mixed wi~h 5 g silica ~el. Dried. Set column ( 4 x 22 cm) . Wash , ,, .~....

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column with benzol - acetone ~7:3). The benzol - acetone is combined and distilled at 17 mm Hg. Crystals are obtained. Crystals are crystalized again in chloroform -methanol (1:1). White crystals are obtained.
Melting point is 169.5 -1~1C. Yield i6 about 0.1%.
Example 2: Extraction and Purificatlon of Ginsenoside lOoo gms. o~ dried ginseng powder is extracted with 2000 ml of 95~ ethanol at room temperature for 24 hours.
The powder is recovered by filtration. Filtrate A is saved and the powder filtercake is refluxed with an additional 2000 ml of 95% ethanol on a steam bath. The mixture is flltered again. Filtrate B is saved and the powder filtercake i5 refluxed two more times for 6 hours with additlonal 20ao ml batches of 95% ethanol and filtered, providlng f1ltrates C and D. Filtrates A,B,C, and D are comb1ned and distilled at 17 mm Hg absolute, whereby ethanol is recovered and a still residue i5 obtained. ~
This still residue is dissolved in 500 ml of dIstilled water. This water solutio~ is extracted five times with 500 ml of a lipophilic solvent, e.g. diethyl ether or petroleum ether, whereby liplds are re~oved from the solution.
To thls acqueous raffinate is added 500 ml of water-saturated n-butanol and the mixture is distilled at 17 mm Hg absolute to dryness, whereby a powder re~idue is obtained. This powder 15 dissolved in 500 ml `
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of anhydrous ethanol, and 2000 ml of acetone are added with agitation while a precipitate forms. The precipitate is recovered by filtration and washed twice with acetone and twice with diethyl ether and dried.
About 60 yms. of a white to liyht yellow powder are recovered. This is Ginsenoside.
Example 3: Anethole (P-allyl-methoxy benzenel extracted from Fennel (Foeniculum Vulgare Mill).
1 kg Fennel oil is Fractional-distilled over an oil bath in distillation apparatus. Collecting distillate at 229 -237 C. The resul~ing light-yellow liquor is anethole. d~5 O.9B3-O.g87. Refractive index n ~ 1.~8B-1.561. Yield is 60%.
Example 4: Sodium Cinnamate The Sodium Cinnamate are already on sale in the mar~et. The Sodium Cinnamate which extracted from Cinnamomum Cassia~Blume or Clnnamomum Cassia Presl is expensive.
Example 5: Preparation of PDI ~
On a dry basis, the composltio~ of PDI may vary as follows:

Wei~ht percent Preferred composition ` weight percent __ _ __ __ _________ Ferulic acid 15-60 25 Glnsenoside 5-50 20 Anethole 20-65 30 Sodium Cinnamate 15-60 25 ~X;

12~;~474 The dry ingredients of PDI, prepared in accordance with the present invention, may be incorporated in tablets, capsules and syrups by conventional methods.
Eaoh oral do ~ for an adult is 2G mg ~20-200 mg) injected dose for an adult is 10 mg (5-50 mg).
This invention will now be described with reference to its beneficial effects, as illustrated by the following tests:
~xample 6: The influence of PDI on the survlval rate of myocardial call Materials and methods:
Hearts were removed from 11 days embryos and were dissociated at 37C for 45 minutes with 0.25% tryps.ln (sigma, type III), 0.025% collagenase (sigma, type I), and 0.005% pancreatin(NBCo) prepared:in calcium and magnesium free saline G containing 4% chicken serum.
Then the tissUe is disperæed~into a single cell suspension in culture medium~:containlng~5~g/ml~DNAse sigma). Viable cell counts werz det rmined by hemocytometer counting. Cells~were~dispersed into 60 mm oulture diaher (surface area 2000~mm2)~at densities of 20D cells/mm2.
: Culture;~were maintained;in Ham's~F-12K cln~
modi~ied by Clark tlg8l]~and supplemented with 5% fetal bovine serum, gentamicin~(Smg/lOOml~. Tissue culture plates were incubated under at constant 5% C02 and 95 air at 3~lC.
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Cells counted - all cells were counted in 20 randomly selected fields across the entlre dish. A Zeis~
microscope 25X objictive havi~g a field of view of 0.32 mm2 was used for cell counting.
Immunostainin~:
I. Reagents 1. CMF solution: 8.0g NaCl, 0.4 g KCl, 0.15 g K~ PO~
0.29 Na2HP04.7H20, 1000 ml distilled water, solution is sterilized by passage throuyh a 0.22 millipore.

2.,Phosphat-buffered saline (PBS): 10 ml lM P04 buffer, 8g NaCl, H20 1 liter, pH to 7.6.

3. PBS/BSA: PBS plus 1% bovine serum albumin.

4. Biotinylated Antibody ~BA): ABC kit, l drop BA
in 20 ml PBS/BSA.
: 5. Avidî~ Biotin:Complex, ABC stain: ABC kit.
: 2 drops, Avidin DH, 2~ drops Biotin-perox~dase, 10 ml PBS/BSA.
6. Diaminobenzidin~ substaate, DAB: 5 mg diaminobenzidine tetrahydrochloride, 10 ml PBS, : 0.004 ml 30% H202.
: 7. 0.3% H202 in methanol: 0.1 ml 0% H202 in 10 ml.
8. ABC kit purchased from Vector Laborator~es, Burlin~ame, CA.
9. Adriamycin purchased from Adria Laboratories, Inc., Columbus, OH.

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II. Immunostaining procedure Monoclonal antibodies (diluted 1:1000 in PBS/BSA) were used Immunostaining procedure uses modified method of vectastain ABC immunoperoxidase staining procedure, briefly, 1. Culture plates were added ant:ibody of cardiac myosin before 3 hr. for staining. Then culture plates continuous incubated 3 hr. at 3~C.
2. Rinse cell culture plates in CMF saline.
3. Fi~ sections for 5 min. in S0% methanol and 50 , acetone.
4. Rinse 3 times in methanol.

5. Incubate sections for 10 min. in 0.3% ~l202 in methanol.

6. Incubate sections for 30 min. in BA antibody solutlon.
. Rinse sections in PBS for 5 min. with 2 times.
8. Incubate sectiona in ABC stain for 1 hr.
9. Rinse sections in PBS for 5 min. with 3 times.
10. Incubate sections in DAB-peroxide substrate solution for 5 min.
; 11. Rinse sections for 10 min. in water.
12. Counter-stain for 30 sec. in ~ a~eous fast green, soak in distilled water with 3 ~imes.
13. Air dry cell cultures.
Cell determined by recording the color and the nu~ber o~ microscopiC fields (250X). Norm~l cell is ~.2~7~L

light yreen and dead cell is brown or very dark green.
Results:
The influence of PDI on adriamyci~ induced damage o~ chick myocardial cell:
~ fter chick myocardial cell was put into culture for 4 days, adriamycin was added and remained in culture for 24 hours. Then ABC immunoperoxidase was used to determine survival rate. Under the above condition PDI
can clearly be shown to increase the survival rate of chick myocarclal cell.
TABL~ 3 The influence of PDI on the survival rate of myocardial cell Chick myocardial cell was put in culture for 4 days. Then Adriamycin was added. The concentration of each dish was lO~g Adriamycin/ml medium. In the PDI
group, PUI was added. In the Adriamycin ~roup, equal :
~ ~ ~value CMF solution was;adde~d. After~24 hours the - , survival rate was determined.
: + t t Survival rate (living/total X 100%) ~_____ __ _ __ ___ ____ +_ __ _ __ _ _____ __ +
I~Adriamycin (10~ 3~,3 + 3.1 (*16) +________ _____ ___ _ _ __+_ ____ _______ ______+
I Adriamycin (10~) + PDI l 61.0 + 3.0 (*15~ l + _+ _____ _ _ __ _ ____ _+
P < O.001 * indicate number of sampling Concentration of PDI is 150~g/ml.

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~xample ~: Effects of PDI on hemopoietic system Effects of PDI on hemopoietic system were investigated. Results showed that PDI (ip) could markedly improve the recovery rate of hemopoieses in treatment mice by cyclophosphamide.
With increased nucleated cells in bone-marrow (~MC), endogenous colonies in spleen and higher 3H-TdR
Uptake in ~arrow and spleen. The Ievel of serum colony stimulating factor (CSF) increased after injection. It is found that PDI protect the stem cells of bone marrow in mice from the killing efPect of cyclophosphamide.
Method of animal model is regular which i9 not part of this invention. Pharmacological effects as illustrated by the following table: by means of the spleen colony assay technique the action of PDI on bone marrow stem ce1ls (C~U-S).

+_________ ___________~__ _ ______~________ ____~______*
Group I Number l Mean CFU-S I P
of sample 1 + SD
+ ________ ___ _ ___ + _____ ___ ,~, ___ ______ +______+
I Control ~l 10~ 1 10.0 ~ O.Bl l ---- I
+ --_--____+ __ __+
I Cyclophosphamide j 10 i 4-0 + 0-~1 ¦ 0,001l *_____ _ __ _______ + ____ __ +________ _. +_ _ __~
IPDI+Cyclophosphamide l 10 ~ 7 + 0.80 1 0.05 1 :
.
Experimental procedure: ~
Male mice weight 18-20 g were used in the experiments and were divided into treated (PDI) and control groups. The dosage of PDI is 5.5 mg/kg in~ected intraperitoneally. The con-trol mice were injected with ~64~4 same volume of normal saline. These injections were repeated daily for 3 days. On the last day, both treated and control group were inJected intraperitoneally with cyclophosphamide. The dosaye of cyclophosphamide i5 4.5 mg/kg.
The same experimental procedure ~or example 8, 9, and 10 in testing mice was used.
Example 8: The ef fect of PDI on phagocytosls of peritoneal macrophage of mice Method:
Ad~ 0.02 ml of 5% washed chick red blood cell suspension to 0.5 ml of the peritoneal exudate, shake gently to mix and incubate at 37 C for 5 minutes. Dip two cover-slips, close to each other, in the above mlxture and incubate for 30 m1nutes for the migration of the macrophages along the cover slips, Fi~ and stain with Sharma stain. Examine microscopically for:
Phayocytic rate - number of macrophages with phagocytized chick red blood cells per 100 macrophages counted.
Method of animal model is regular.
Resul t5:
Pharmacological effects a5 illustrated by the following table.

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TABL~ 5 +_____________________~___________+___________+_______.1.
Groups I Nwnber I Phagocyticl P
o~ sample l rate+SD(~) +_____________________+___________+___________+_______+
I Control I 10 1 35.10*2.01l --+_______________~_____+___________+__________~+_______+
I Cyclophosphamide I 10 1 8.00+0.36l 0.001l +.______________ _____+___________.~___________+_______+
I Cyclophosphamide+PDII 10 1 33.00+1.14l 0.05 1 ~____________________+_ _________~_______~___+_______+
* concentration o~ PDI and cyclop~osphamide is the same as example 7.
~xample 9: The effect of PDI on white blood cells in rats treated by cyclophosphamide Action of PDI and cyclophosphamide on white blood cells was inve~tigated by means o~ white blood cells assay. It was revealed that PDI protect white blood cells in rats from the killing effect o~
cyclophosphamide. Method o~ testing in animal is standard. The dosage of PDI and cyclophosphamide is the ~same a~ in example ~. Time of:treatment is 10 days. The : ~ results are listed below table:

:
i ~ I Groups IWhite blood cellslNo. of I P
x 103~cm3~SD ISample Normal j 14.6 + 2.1 1 10 + _ _.. __ ____ __+ _ _ _ ______+~~_____ ~___ _ +
~: ~ ICyclophosphamide I 5 3 + 1.:0 1 10 l 0.001l :
*_ _ ______ ___~_~ ~ _~ _________~~_+_ __ ___~______+

ICyclophosphamide+PDII ~12.0 + 1.8 1 io I :o . 05 1 .,.___ ________ ______ *_ _ _____=_______+_ ______+______+
Example 10: The e~fect of PDI on }ymphoblastoid trans~ormation By means of H-TdR liquid scintillation assay :

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technique, the action of PDI on lymphoblastold transformation was investigated method:
(1) Experimental procedure of animal is the same as in example ~.
(2) ~ymphoblastoid transformation test:
I. Reagents and conditions for c211 culture a. Culture media - RPMI 1640, medium 199 minimal essential medium (Eagle).
b. Buffer - Hepes buffer, the final concentratio~
at 3~ C was 25 mM, to malntain the pH of the ,medium at ~.31 c. Serum - generally 15-20% fetal bovine sarum was incorporated, for lymphocytes from mice, 5% was used.
~ d. Gaseous pha~e - 5% CO~ in air~
: ~ e. Cell concentration -~generally 1-2xlO /ml.
f. Stimulants - 20~1/ml for phytohemagglutinin containing polysaccahride (PHA-M) or 10~1/ml for polysaccahride-free purifled phytohemagg:lutinin (PHA-P).
II. Measured by li~uid scintillation : a. The~condit;ions of cell cultllre are the same a~
above. 3 H-TdR was added after 48 hours of incubation at a final concentra~ion of l~ Ci/ml and continue the incubation for 24 ; ~ hours.
~ b. Wash the cells twice with cold normal saline ~ .
~ ,~

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and the erythrocytes were lysed by addition of distilled-water and equal volume of 3.6~ NaCl was then added. The intact lymphocyte~ were again washed once with cold saline. Spin down the lymphocyte~ and add 2 ml of 10~ trichloroac-etic acid to precipltate the protein. Wash twice with normal saline. Add 2 ml oP
ethanol:ether (1:1) to wash once. 0.2 ml of formic acid was then added for digestion till the precipitate was dissolved.
c. Add ~ ml of scintillation fluid to 0.1 ml o~
the final sample and count in a liquid scintillation counter.
Re~ults are listed in the following table:
(concentration of PDI and cyclophosphamide used is the same as that of ~xample ~.) TABL~ 7 +_________~___ __ ___+_ _ _____~____ ____ +~_______+
Group ~ ! Number I CPM + SD j P
o~ sample + + ____ ___ ~_______ __~________+
~ INormal l ~ 10 1 13~0+51 I ~~~
~______ ____________~______ _ __+__________+____ ___~
ICyclophosphamide l 10 ~l 69~+3a 1 0.001 1 t----------------------------------------+--__ _ _____+_._ _ ___ +_____ __+
ICyclophosphamide+PDII~ 10 1 1253*50 1 0.05 + _.__ __ _ __ + ____+_ = __ +___ _ _+
Exampie 11: Toxicity o~ PnI
1. ~.D.50: 1321 m~/kg injection in ab~ominal cavity in mice.
2. Each dose for an adult is 20 mg. Usin~ 50 kg as the ~26~47~

avera~e weight of an adult the dosage i5 0.4 mg/kg, therefore it is very safe.
Results and experimental procedure of PDI to overcome toxicity of radiotherapy would be the 6ame as in the example above chemotherapy.
The embodiment of the invention described here can be modified withirl the spirit and~scope o~ the present invention. Numerous modifications and variations of the present invention are possible in light of the above teachings.
A pharmaceutical composition referred to as PDI, a process for producing PDI and furthermore its effect on decreasing side effec.ts of anticancer chemotherapy, radiotherapy and increasing the immune function as well as other effects studied by tests carried out by :
approved procedures have been described~

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Claims (8)

1. A pharmaceutical composition for treatment of side effects of anticancer chemotherapy using a drug selected from a group consisting of adriamycin and cyclophosphamide comprising, in weight percent: ferulic acid 15 60%; ginsenoside b, 5-50%;
anethole 20-65%; and sodium cinnamate 15-60%.

2. A pharmaceutical composition for increasing the immune function comprising, in weight percent: ferulic acid 15-60%;
ginsenoside b, 5-50%; anethole 20-65%; and sodium cinnamate 15-60%.

3. The pharmaceutical composition of claim 1 wherein ferulic acid is extracted from the plant group consisting of levisticum Officinale Koch and Angelica Sinensis Diels.

4. The pharmaceutical composition of claim 2 wherein ferulic acid is extracted from the plant group consisting of Levisticum Officinale Koch and Angelica Sinensis Siels.

5. The pharmaceutical composition of claim 1 wherein anethole is extracted from Foeniculum Vulgare Mill.

6. The pharmaceutical composition of claim 2 wherein anethole is extracted from Foeniculum Vulgare Mill.

7. The pharmaceutical composition of claim 3, wherein ferulic acid is refined by chromatographic fractionation.

8. The pharmaceutical composition of claim 4 werein ferulic acid is refined by chromatographic fractionation.

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