CN1301535A - Use of curcumin to treat hepatitis B - Google Patents

Abstract

Curcumin extracted from curcuma root contains mainly three components, including curcumin not less than 70 % demethoxy curcumin not less than 10-20 %, and didemethoxy curcumin not less than 5-10 %. It is prepared into oral preparation for treating hepatitis B, especially chronic persisting and active hepatitis.

Description

The application of curcumin to treat hepatitis B

Involved in the present invention is a kind of medicine that is used for the treatment of hepatitis B, particularly the application of curcumin treatment hepatitis B.

Hepatitis B is one of modal infectious disease in the world.Therapeutic Principle clinically adopts composite treatment, should emphasize antiviral therapy theoretically, because this is to effect a permanent cure.Aspect medicinal application, Chinese medicine, Western medicine kind are a lot, the part patient is had certain curative effect, but most drug does not have antivirus action, or its antivirus action wait further research.

Chronic hepatitis B antiviral therapy medicine, mainly contain interferon, nucleoside analog and some Chinese medicine preparation at present, as hepatitis spirit, lentinus edodes polysaccharide injecta and ZHULINGDUOTANG ZHUSHEYE etc., these medicines cost an arm and a leg because of the effect of paying and each XOR of curative effect, remain further research and improvement.Up to now, the curcumin that Rhizoma Curcumae Longae (curcuma longa L) is extracted both at home and abroad is that universally acknowledged food coloring, research is a lot of as food additive.And national capital permissions to use such as FAO (Food and Agriculture Organization of the United Nation) of The World Health Organization (WHO) (FAO), the European Economic Community, the U.S., Canada.Curcumin is a lot of as the report of medicinal research, relate generally to blood fat reducing, CN1143518A, function of gallbladder promoting, anticancer, anti-cancer, anti-radiation, antioxidation, antiinflammatory and effect such as antibiotic, but, comprise curcumin (curcumln) and then do not appear in the newspapers so far as pharmacology, drug efficacy study and the clinical application research of antiviral hepatitis about Rhizoma Curcumae Longae extract.

The inventor is through for many years basic pharmacology, pharmacodynamic study, effect to curcumin antiviral property hepatitis B, done deep research, its objective is traditional Chinese medicine Rhizoma Curcumae Longae flavochrome total extract from China, develop a kind of medicine that is used for the treatment of hepatitis B, especially to the active drug of chronic persistent hepatitis and chronic active hepatitis.

Technical scheme of the present invention realizes by following research step:

Curcumin is used for the treatment of the application of hepatitis B, it is characterized in that the class flavochrome total extract that extraction separation goes out from plant Rhizoma Curcumae Longae (curcuma Longa L) rhizome, wherein comprise three monomeric compounds, i.e. curcumin, demethoxycurcumin, bisdemethoxycurcumin

Contain above-claimed cpd monomer or total extract, make oral agents, be used for the treatment of the medicine of hepatitis B.

Another feature of the present invention is the total extract of this class flavochrome, and wherein the content of curcumin is not less than 70%, also can be the medicinal application that monomeric compound is used for the treatment of hepatitis B.

It is this class flavochrome total extract that the present invention also has a feature, or the oral medication of monomeric compound preparation, is mainly used in the medicinal application of treatment chronic persistent hepatitis and chronic active hepatitis.

The extraction separation method of curcumin of the present invention, be by, confirm plant Rhizoma Curcumae Longae (curcuma Longa L) kind, make oral formulations through pulverizing → organic solvent extraction → crude extract → solvent refining → pure product (the curcumin total extract contains curcumin more than 70%) with containing the total extract (abbreviation curcumin) of curcumin more than 70%.Contain curcumin as the every capsules of capsule and always extract the medication that 50mg is used for the treatment of Type B viral hepatitis.

In order to understand essence of the present invention better, will its new purposes in pharmaceutical field be described with pharmacological testing and the results of pharmacodynamic test that curcumin carries out below.

1, curcumin inhibitory action that antigen of hepatitis B virus is produced:

Method: Hep G ₂T ₁, the growth conditions of cell, culture medium is that DMEM (Glbco) contains 10% hyclone (Glbco), each 100 μ g/ml of G-418 (400 μ g/ml) mycillin, 5%CO ₂Cultivate under 37 ℃ of conditions, curcumin then is dissolved in the dimethyl sulfoxide, can be diluted in during use in the cell growth medium, with cell inoculation in 48 well culture plates after 3 days, change the growth-promoting media that variable concentrations contains curcumin, continue to cultivate 3 days, take out culture supernatant then and put-20 ℃ of preservations, after the repeated experiments, detect HBV antigen with the ELISA method, the test box that detects HBs Ag and HBe Ag provides measurement result to see Table 1 by Huamei Bio-Engrg Co..

Table 1 curcumin is to the inhibitory action (%) of HBs Ag and HBe Ag generation

Drug level (μ g/ml)	Suppression ratio (%) to HBs Ag	Suppression ratio (%) to HBe Ag
			?10 ?5 ?2.5 ?1.25 ?0.62 ?0.31 ?0.15	???????66.1 ???????58.0 ???????41.2 ???????40.0 ???????36.6 ???????33.0 ???????14.3	?????20.0 ?????17.9 ?????15.7 ?????12.9 ?????10.0 ?????10.0 ?????5.0

The result confirms that curcumin to Hep G2T1 cell, does not produce cytotoxicity, and can reach 66.1% and 20% respectively to the suppression ratio of HBs Ag and HBe Ag when 10 μ g/ml.

2, curcumin is to the inhibitory action of HBV-DNA polymerase activity

The method that adopts is, with HBV-DNAP positive serum+anti-HSB → centrifugation HBV granule → increase the shell agent to make HBV-DNPA expose → add 37 ℃ of 30 minutes → enzyme-added effect substrate → mensuration HBV-DNAP activity of curcumin, the results are shown in Table 2.

Table 2, curcumin is to the active inhibitory action of HBV-DNAP

Dosage (ng/ml)	Suppress activity rate (%)
		??????????80 ??????????40 ??????????20	????47 ????37 ????11.7

The result shows: curcumin presses down the antigenic expression of HBV may have the active function that suppresses HBV-DVAP relevant with it.

3, the therapeutic effect of curcumin duck hepatitis B virus infection in duck body test

This test is finished by Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences.

This experiment is carried out the therapeutic test verification the verifying results to curcumin in duck hepatitis B virus infection duck body.Experiment adopts an age in days Beijing duck intravenous injection duck second to hepatitis virus, begins after 7 days to the oral curcumin of duck, and 1 day 2 times, toxicity and the Sanguis Anas domestica clear DHB DNACDHBV-DNA of medicine to duck observed in administration 10 days (Bld * 10)) influence.Experiment shows, 62.5mg/Kg, and 125mg/Kg, the 250mg/Kg group is oral, and 1 day 2 times 10 days, avirulence.125mg/Kg, the 250mg/Kg group all can reduce DHBV infected duck serum DHBV-DVA level, and doing in groups with the virus control group, statistical analysis compares the 5th day (T of administration ₅) and the 10th day (T ₁₀), three batches of experiments of these two dosage groups are repeated to rare two batches of differences that remarkable (P＜0.05) or highly significant (P＜0.01) are arranged.The inhibition effect that significantly (P＜0.05) or highly significant (P＜0.01) are also arranged in drug withdrawal the 3rd day (P3) simultaneously.

The result also shows, curcumin 125mg/Kg, and 250mg/Kg, dosage has clear and definite inhibitory action to the clear DHBV-DNA of Sanguis Anas domestica, and dose-effect relationship is arranged.

Table 1 curcumin in the duck body to the inhibitory action of DHBV-DNA OD value

The experiment batch	Group	Duck number (only)	??????T0	???????OD 490Value T5	?????T10	??????P3
							?Ⅰ	Virus control curcumin 62.5mg/Kg 125mg/Kg 250mg/Kg	??6 ??6 ??6 ??5	??1.15±0.09 ??1.54±0.53 ??0.79±0.15 ??1.13±0.22	??1.09±0.19 ??1.24±0.48 ??1.08±0.15 ??0.60±0.26	?1.27±0.09 ?1.12±0.24* ?0.43±0.07** ?0.67±0.26*	??1.26±0.08 ??1.00±0.75* ??0.81±0.30 ??0.55±0.29
?Ⅱ	Virus control curcumin 62.5mg/Kg 125mg/Kg 250mg/Kg	??6 ??6 ??6 ??6	??0.59±0.08 ??0.61±0.0?5 ??0.63±0.11 ??0.85±0.13	??0.50±0.08 ??0.50±0.07** ??0.59±0.06 ??0.57±0.06**	?0.57±0.06 ?0.57±0.06* ?0.59±0.05 ?0.54±0.06**	??0.61±0.12 ??0.66±0.10 ??0.64±0.06 ??0.44±0.06**
							?Ⅲ	Virus control curcumin 62.5mg/Kg 125mg/Kg 250mg/Kg	??5 ??5 ??5 ??5	??0.63±0.04 ??0.54±0.05 ??0.53±0.07 ??0.68±0.04	??0.68±0.05 ??0.53±0.04 ??0.56±0.08 ??0.48±0.09*	?0.69±0.06 ?0.53±0.08 ?0.41±0.04* ?0.45±0.06**	??0.73±0.09 ??0.59±0.05 ??0.40±0.05** ??0.42±0.03**
	??ACV ??0.1g/Kg	??6	??0.66±0.09	??0.43±0.06**	?0.50±0.06**	??0.69±0.15

(T0) 0D value comparison (paired t-test) before different time after the administration (T5, T10, P3) 0D value and the administration on the same group

*P＜0.05,**P＜0.01。

Table 2. curcumin treatment group and virus control group

In the duck body to the comparison of DHBV-DNA suppression ratio

The experiment batch	Group	Duck number (only)	T5	Suppression ratio (%) T10	P3
Ⅰ	Virus control group curcumin 62.5mg/Kg 125mg/Kg 250mg/Kg	6 6 6 5	5.0 16.0 -42.3 41.4	-11.5 25.3 ** 44.5 ** 40.7 **	-10.7 38.6** -3.5 45.5*
Ⅱ	Virus control curcumin 62.5mg/Kg 125mg/Kg 250mg/Kg	6 6 6 6	14.1 18.2 5.3 32.3 *	2.8 7.5 3.3 34.2 **	-5.2 -7.0 -3.5 46.8 **
Ⅲ	Virus control curcumin 62.5mg/Kg 125mg/Kg 250mg/Kg	6 5 5 6	-8.8 -0.3 -5.3 29.1**	-9.9 0.4 21.8 32.6**	-16.6 10.5 24.3** 38.1**
	ACV 0.1g/Kg	6	33.5**	24.2**	-5.3

Identical with the virus control group respectively time D HBV-DNA suppression ratio of drug treatment group different time DHBV-DNA suppression ratio is (t check in groups) relatively, * P＜0.05, * * P＜0.01.

4, the reducing enzyme and treating jaundice effect of curcumin test.

(1) curcumin is to the effect of D-Gal amine induced mice liver injury protection.

Curcumin oral administration 3 days, dosage 20,100mg/Kg, (800mg/Kg, lp) SGPT of induced mice hepatic injury raises certain reduction effect to D-Gal amine.(seeing Table 3)

Table 3, curcumin are to the protective effect of hepatic injury due to the mice Ga/N

Group	Dosage (mg/Kg)	Number of animals (only)	??SGPT(u/dl) ????x±SD	Reduction rate (%)	The P value
						Normal control contrast curcumin curcumin	???? ???? ????20 ????100	???20 ???20 ???20 ???20	????27.8±8.7 ????82.9±44.6 ????63.7±46.7 ????46.8±13.8	???? ???? ????23.1 ????43.5	???? ???? ????＞0.05 ????＜0.01

(2) curcumin is to the protective effect of rats'liver damage due to the isothiocyanic acid (APIT).

Curcumin oral medicine 4 days, dosage 100,200mg/Kg, (PO, 100mg/Kg) the rats'liver damage due to has significant protective effect, can obviously reduce glutamate pyruvate transaminase, the heavy coefficient of serum total bilirubin regulating liver-QI can to make APIT.The results are shown in Table 4.

Table 4, curcumin is to the effect of rat experiment jaundice model

Group	Dosage (mg/Kg)	Number of animals (only)	??ALT(U/dl) ????x±SD	??SB(mg/dl) ????x±SD	Heavy coefficient (g/100 body weight) X ± SD of liver
						Normal control contrast curcumin curcumin	???? ???? ???100 ???200	??10 ??10 ??10 ??10	??28.3±11.3 ??129.3±13.5 ??97.4±35.6* ??95.9±28.7*	??0.12±0.04 ??2.09±1.87 ??0.51±0.50* ??0.36±0.37*	??4.07±0.66 ??5.87±0.69 ??4.80±0.55* ??4.87±0.49*

* compare P＜0.01 with matched group

5, curcumin is to the effect test of rat chronic hepatic injury due to the carbon tetrachloride

Experiment purpose is observed curcumin rat chronic hepatic injury due to the CCl4 is had or not protective effect.

Method: imitate according to bureau of drug administration of Ministry of Health of the People's Republic of China " new drug (Western medicine) preclinical study guideline compilation " (pharmacy, pharmacology, toxicology) antihepatitis drug and to learn guideline and design.

With rat wlstar strain; female rats body weight 140-180g; male rat body weight 160-200g; totally 108 (female 48; male 60) be assigned as 6 groups by the body weight layering; every group 18 (male 10; female 8); these 6 groups are respectively the normal control group; model group, positive drug control group (are the bifendate group, are the hepatoprotective of national approved listing because of the positive contrast medicine of bifendate; and have and report that hepatic injury has protective effect to rat chronic for it), curcumin high dose group, low dose group and control group.Each group is subcutaneous injection 25%CCl respectively ₄₂Ml/Kg, 2 times weekly (Tuesday, five mornings) are continuously to CCl _{4 6}Begin treatment after week, the positive control drug group is pressed 50mg/Kg dosage, irritates stomach bifendate suspension, and curcumin high dose group and low dose group are irritated stomach curcumin suspension 200mg/Kg and 100mg/Kg respectively, and administration is 6 times weekly, treats for 6 weeks altogether.Curcumin control group is from animal subcutaneous injection CCl ₄The time, irritating stomach curcumin dosage every day simultaneously is 50mg/Kg, 6 times weekly, is total to 12 weeks of administration.Every Mus of normal control group and model group is irritated stomach tap water 2ml every day, also gives for 6 weeks, and each treated animal is weighed weekly 1 time, gives CCl to adjust ₄And dosage, after time administration 20 hours, every group of blood (male 8 female 4) of all getting 12 rats, separation of serum is measured ALT, AST, TP, A and A/G.Get a liver part and survey the liver hydroxyproline, all the other hepatic tissues are done pathological observation, the results are shown in Table 4 and table 5.

Table 4 curcumin is to the influence of chronic hepatic injury rat blood serum ALT and AST

Group	Number of animals (n)	Dosage regimen	ALT(u/dj) ??X±SD	??AST(u/dj) ????X±SD
					Normal control model bifendate curcumin curcumin curcumin (control)	???? ???? ???12 ???12 ???12 ???12 ???12 ???12	?50?mg/Kg×6w,lg ?100mg/Kg×6w,lg ?200mg/Kg×6w,lg ?50mg/Kg×6w,lg	?27.2±8.8** ?263.8±51.6 ?190.3±82.4* ?196.5±80.4* ?260.3±26.5 ?193.1±81.5*	?85.9±17.1** ?251.5±48.0 ?207.2±75.4 ?214.2±77.1 ?226.3±52.2 ?217.0±71.5

* compare P＜0.05, * * P＜0.01 with model group

6 weeks of curcumin low dose group (100mg/Kg) treatment and the effect of remarkable reduction glutamate pyruvate transaminase (ALT) is all arranged in control group (50mg/Kg) 12 weeks of administration as seen from Table 1, similar to biphenyl two (50mg/Kg) treatment 6 all effects, more all present significant difference with model group, though and AST is had certain reduction effect but through t test, not seeing has significant difference.This description of test curcumin has certain hepatoprotective effect to chronic hepatic injury, and showing has the reduction effect to ALT.

Table 5 curcumin is to the influence of chronic hepatic injury rat blood serum TP, A and A/G

Group	Number of animals (n)	Dosage regimen	ALT(u/dj) ????X±SD	??AST(u/dj)???A/G ????X±SD
					The normal group model	???12 ???12		?7.46±0.54 ?6.62±1.59	??3.85±0.02**1.07 ??2.76±0.83??0.72
Bifendate curcumin curcumin curcumin (control)	???12 ???12 ???12 ???12	50mg/Kg×6w,lg 100mg/Kg×6w,lg 200mg/Kg×6w,lg 50mg/k×6w,lg	?7.22±0.63 ?7.05±0.47 ?7.30±0.60 ?7.22±0.61	??3.13±0.30??0.77 ??2.93±0.58??0.71 ??3.16±0.52??0.76 ??3.33±0.28*?0.86

* compare P＜0.05, * * P＜0.01 with model group

Curcumin is the effect that all improves of albumen (TP) and albumin (A) to the chronic hepatic injury rat blood serum as can be seen from Table 2, wherein improving certain dose-effect relationship of using of A, and the effect of control group is better, with model group significant difference (P＜0.05) is arranged relatively, though the A/G of each group still has phenomena of inversion, but the control group is the most near the normal control group, this tests bright curcumin can improve the synthetic albuminous ability of impaired liver recovery, thereby prompting helps recovering the nutriture of body.

6, the choleretic effect of curcumin test:

Method, curcumin be to mice oral administration 7 times, dosage 250, and 500mg/Kg, or to rat single duodenal administration, dosage 100,250mg/Kg shows choleretic effect preferably, if see Table 6,7.

Table 6 curcumin is to the choleretic effect test of mice

Group	Number of animals (only)	The weight of animals (g) X ± SD	Bile amount (mg) X ± SD	Bile amount (mg/10g body weight) X ± SD	Increment rate (%)	The P value
							Contrast	???10	??30.6±1.4	?28.4±13.5	??9.24±4.25
Curcumin 250mg/Kg	???10	??29.5±1.6	?42.6±10.6	??14.4±3.2	???55.8	??＜0.01
							Curcumin 500mg/Kg	???12	??30.0±1.2	?42.4±7.0	??14.2±2.3	???53.7	??＜0.01

Table 7 curcumin is tested the choleretic effect of rat,

Group	Number of animals	Body weight (g) x ± SD	Preceding 1 hour secretory volume (ml) of administration x ± SD	Choleresis after the administration (ml) x ± SD			The highest increment rate (%)
								1h	2h	3h
				Contrast		312		0.524	0.482	0.470	0.470
NS	10	±23.0	±0.181	±0.145	±0.108	±0.105	-8.0
(10ml /Kg				(-8.090)	(-10.3％)	(-9.5％)
Curcumin	10	304	0.505	0.583	0.592	0.530
(100mg /Kg		±23.4	±0.094	±0.112 (15.4％)	±0.103 (17.2)	±0.116 (5.0％)
Curcumin	10	302	0.508	0.669 *	0.654 *	0.607	31.7
250mg /Kg		±30.8	±0.118	±0.114 (31.7％)	±0.136 (28.7％)	±0.114 (19.5％)
Dehydrocholic acid (1g/Kg)	10	346 ±32.0	0.745 ±0.197	1.150 ** ±0.273 (54.4％)	1.320 ** ±0.417 (77.2％)	1.180 * ±0.447 (58.4％)	77.2

* with administration before choleresis relatively P (0.05, * * P＜0.01

Bracket inner digital be with administration before the % ratio that increases of choleresis relatively.

7, the toxicity test of curcumin:

(A) acute toxicity test: mice single oral curcumin 1Og/Kg, do not see poisoning symptom to occur.So inferring its LD5O answers＞10g/Kg.

(B) long term toxicity test: the oral curcumin 500mg/Kg of rat, totally 12 weeks, the oral curcumin 200mg/Kg of Canis familiaris L., 14 weeks are to animal behavior, hair color, appetite, hematochrome, red, leukocyte.Platelet count, leukocyte differential count, clotting time, SGPT, SGOT, BUW, Cr, AKP, BLS, Ch, TP, A, SB, uroscopy (glucose in urine, urine protein, microscopy) ECK, acropetal coefficient, etc. the detection of index, all no abnormal, only the male rat body weight gain is influential slightly, the rarely seen lung of pathologic finding, kidney individual animal have slight interstitial inflammation, and all the other do not see variation.

8. curcumin is treated chronic hepatitis B 71 routine clinical observation on the therapeutic effect

Diagnosis basis is diagnosed according to diagnosis and typing standard that the whole nation viral hepatitis academic conference of nineteen ninety Shanghai is worked out.Observe 71 examples altogether, 50 examples in addition, women 21 examples, minimum 17 years old, maximum 61 years old, chronic persistent hepatitis (CPH) 39 examples wherein, chronic active hepatitis 32 examples, wherein 13 routine liver puncture are made a definite diagnosis.Medication: obey a capsules every day 3 times, every capsules contains curcumin 50mg at every turn, and a day clothes total amount is 150mg, and serveing on 3 months is a course of treatment.

Take main liver function index situation of change behind the curcumin medicine

Lab work	Unusual routine number before the treatment	Positive common practice number is recovered in the treatment back	Effective percentage (%)
				??ALT ??AST ??BIL ??TTT ??HBs?Ag(+) ??HBe?Ag(+) ??DNA-P ??HBV-DNA	????58 ????38 ????9 ????16 ????71 ????38 ????18 ????16	??????43 ??????31 ??????9 ??????14 ??????19* ??????22 ??????12 ??????7	???74.1 ???81.6 ???100 ???87.5 ???26.8 ???57.9 ???66.7 ???43.8

More than every data result card man, oral curcumin treatment chronic hepatitis B has the symptom of improvement, alleviates body, the jaundice eliminating effect of reducing enzyme levels is apparent in view, it suppresses hepatitis B virus duplication and makes HBe Ag negative conversion rate that effect preferably all be arranged, and medication person does not have any toxic reaction, does not also have irritated phenomenon.

Claims (4)

1, the application of curcumin to treat hepatitis B, it is characterized in that the class flavochrome total extract that extraction separation goes out from plant Rhizoma Curcumae Longae (curcumaLonga L) rhizome wherein comprises three monomeric compounds, be curcumin, demethoxycurcumin, two born of the same parents' methoxyl group curcumin

The monomer or the total extract that contain above-claimed cpd.

2, the application of curcumin to treat hepatitis B according to claim 1 is characterized in that this class flavochrome total extract, and wherein the content of curcumin is not less than 70%, can be monomeric compound also, is used for the treatment of the medicinal application of hepatitis B.

3,, it is characterized in that this medicine is used for the treatment of chronic persistent hepatitis and chronic active hepatitis according to the application of claim 1 and 2 described curcumin to treat hepatitis B.

4,, it is characterized in that to make oral agents according to the application of claim 1 or 2 or 3 described curcumin to treat hepatitis B.