樟芝液體發(fā)酵粉對肝癌 H22的體內生長抑制作用

許泓瑜,孔曉明,敖宗華,陸震鳴,許正宏,＊

1江南大學醫(yī)藥學院制藥工程研究室;2江南大學工業(yè)生物技術教育部重點實驗室,無錫 214122

樟芝液體發(fā)酵粉對肝癌 H22的體內生長抑制作用

許泓瑜1,孔曉明1,敖宗華1,陸震鳴2,許正宏1,2＊

1江南大學醫(yī)藥學院制藥工程研究室;2江南大學工業(yè)生物技術教育部重點實驗室,無錫 214122

本文采用肝癌 H22荷瘤小鼠的腫瘤模型,對樟芝液體發(fā)酵粉的體內抗腫瘤活性進行評價。結果表明,樟芝液體發(fā)酵粉 (500或 1000 mg/kg b.w.)能夠體內顯著抑制 H22腫瘤的生長,抑制率分別為 42.0%和46.4%。同時,與模型組相比,樟芝發(fā)酵粉能夠延長荷瘤小鼠的生存周期,延長率為 29.4%。組織病理學研究結果表明,模型組小鼠的腫瘤細胞生長旺盛,而樟芝發(fā)酵粉處理組的小鼠腫瘤細胞具有明顯的皺縮和壞死癥狀。樟芝發(fā)酵產物具有較好的體內抗腫瘤活性。

樟芝;抗腫瘤;肝癌 H22;深層培養(yǎng)

Introduction

Edible mushrooms have been used as flavorful foods and as health nutritional supplements for several centuries.For the Chinese,some mushrooms are especially regarded as medical substances to increase health and longevity.Nevertheless,systemic studies of its bio-function were not performed until the last third of this past centurywhen biochemical technology for dissecting these traditionalmedicinal mushrooms and isolating their most active anticancer constituents became available.Antrodia cam phorata,a new basidiomycete distributed only in Taiwan province of China,is well known under the name“Niu-chang-ku”or“Niu-chang-chih”due to its potent biologic activities.It grows slowly on the inner hear twood wall of“Niu-chang”,an endemic evergreen,Cinnam om um kanehirai,which is native to Taiwan only.Investigations reveal thatAntrodia cam phoratahas extensive biological activities,such as hepatoprotective effect,anti-hepatitis B surface antigen,antioxidation and anticancer activities[1].Chemical ingredients found inAntrodiacam phoratainclude polysaccharide,sesquiterpene lactone,steroids,triterpenoids,phenol compounds,adenosine,cordycepin and ergosterol[2,3].

Because its host wood is a local species that is getting scarce,Antrodia cam phoratais difficult to find in theforest and is also very expensive.Thus,artificial cultivation was developed as a substitute.Recently,Antrodia camphorata is commercially available in Taiwan in the for m of fermented wine or pure culture in powdered, tablet,or capsule form[4].It therefore is worthwhile to well-characterize its expanded activities.Although research was focused on the therapeutic effects of this mushroom,little information is available about its antitumor property in vivo.The object of this study is to evaluate the antitumor activity of dry matter of culture broth(DMCB)ofAntrodia cam phoratain submerged culture using H22-bearingmice.Meanwhile,the antitumor activity ofDMCB was compared with that of sporoderm-broken spores ofGanoder m a lucidum(SSGL)using the H22-bearingmice.

Materials andM ethods

Reagents and cell lines

H22 is a hepatocarcinoma of the mouse.Mice bearing the ascitic-type hepatocarcinoma H22 were purchased from Shanghai Institute of Pharmaceutical Industry, China.This model comes from a chemically induced hepatocarcinoma[5].DMCB ofAntrodia cam phoratain submerged culture was culture by the method of Ao et al[6].SSGL were purchased from Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences.All the other chemicals used were either of analytical grade or of the highest purity commercially available.

An imals

Male K M mice(CLA grade,4-6 weeks,weight 20±2 g)were purchased from the ShanghaiBK Experimental Animal Center and acclimatized under controlled temperature,20±2℃;humidity,60% ±5%;12 h light and 12 h dark cycle conditions for 2 week before the start of the experiments.They were allowed to feed standard laboratory diet and water.The exper imental procedures are in compliance with the National Institutes of Health Guide for Care and Use ofLaboratoryAn imals. Evaluation of inhibitory activity afterDMCB exposed to the H22-bearingmiceThe mice bearing the ascetic-type hepatocarcinoma H22 were sacrificed 7 days after tumor implant.The skin was sterilized and the ascitic fluid was drawn through the muscles of the abdominal wall using a sterile syringe,and then diluted to a concentration of 2.5×107 cells/mL in PBS solution.Each mouse was injected 0.2 mL tumor cell suspensionsprepared above in the right flank subcutaneously.Twentyfour hours after inoculation,mice were divided randomly into five groups,with 10 mice in each group.Group 1,which served as model group,received vehicle (0.5%CMC-Na in distilled water)only.Group 2 were given standard drug 5-Fu(25 mg/kg b.w.,i.p.)daily.Group 3 were given SSGL (500 mg/kg b.w.), while groups 4 and 5 received DMCB ofAntrodia camphorata(500 and 1000 mg/kg b.w.,respectively). The vehicle or test drugs were administered orally for 15 days or until death of the mouse.The mice weights were recorded before each drug administration.On the fifteenth day,the mice were sacrificed and tumorswere peeled off carefully and weighed after washing off any remaining blood with PBS.

L ife extension of DMCB on the H22-bearing m ice

Fiftymice were inoculated with tumor cells prepared as above in the right flank subcutaneously.Twenty-four hours later,the mice were divided into five groups randomly as the same with the above.Group 1,which served asmodel group,received vehicle(0.5%CMCNa in distilled water)only.Group 2 were given standard drug 5-Fu(25 mg/kg b.w.,i.p.)daily.Group 3 were given SSGL(500 mg/kg b.w.),while groups 4 and 5 received DMCB ofAntrodia cam phorata(500 and 1000 mg/kg b.w.,respectively).The survival time of animalswas monitored and recorded daily.The test was continued for 60 days and those that lived more than 60 dayswere defaulted as60 days.Prolonged survival rate(%)was calculated as[(median survival days of experimental group-median survival days of control group)/median survival days of control group] ×100%.

Histopathological study

The tumorswere removed from the animals and the tissueswere fixed in 10%for malin for at least 24 h.Then the paraffin sections were prepared(automatic tissue processor,Autotechnique)and cut into 5μm thick sections in a rotary microtome(Lieca 2135,Germany). The sectionswere then stained with hematoxylin and e-osin(H&E)dye and were studied using light microscope(Olympus BX60,Japan)for histopathological changes.

Statisticalmethods

All experimental data were expressed asmean value± standard deviation.Experimental data on the growth of H22 in mice were analyzed using one-way analysis of variance(ANOVA)and the significant differences between two groups were assessed by T-test.Data were considered statistically significant if P valueswere 0.05 or lower.Using SPSS Statistical Software,survival time of each groups of mice transplanted with H22 hepatocarcinoma was compared using the Kaplan-Meiermethod and the significant differences between two groups were assessed by Log Rank test.

Results

Effects on the growth of H22 transplanted into m ice

Effects ofDMCB ofAntrodia cam phoratain submerged culture on tumor-bearingmice were shown in Table 1. DMCB ofAntrodia cam phoratacould significantly inhibit the growth of H22 cells transplanted in mice.Dosages of 500 and 1000 mg/kg produced tumor inhibition index of 42.0%and 46.4%,respectively,which were significantly different from the model group(P＜0.05). Although the capacity for 5-Fu to inhibit tumor growth is greater,the mice of this group suffered from debilitation and weight loss(Table 2).During the period of administration,the mice of the SSGL and DMCB group were in better physical condition and showed weight gain.DMCB ofAntrodia cam phoratawas associated with a continuous gain of bodyweight ofmice,with the increase clearly different from the model group,and significantly different compared to the 5-Fu group(P＜0.05).DMCB ofAntrodia cam phoratahad no obvious effect on the weight of spleen and thymus,with the spleen index and thymus index not significantly different than those in the model group,although they were much higher than 5-Fu group(Table 2).

Table 1 Effect ofAntrodia cam phoratain submerged culture on tumor-bearing m ice

Table 2 Effect ofAntrodia cam phoratain submerged culture on body weight,thymus index and spleen index(n=10,x ±s)

Effects on the survival of m ice with transplanted H22 hepatocarcinoma

Table 3 shows that DMCB ofAntrodia cam phoratasignificantly prolonged the survival time ofmice transplanted with H22 hepatocarcinoma and had long-term survivor.The group that received 1000 mg/kg ofDMCB had their prolonged survivalrate (%) increased by 29.4%,which was comparable to SSGL treated group (23.5%).5-Fu also prolonged the survival time of mice transplanted with the H22 hepatocarcinoma,although no mice from this group were long-ter m survivors,and animals from this group suffered from debilitation(Table 3).

Table 3 Effect ofAntrodia cam phoratain submerged culture on survival t ime of m ice

Note:SSGL:Sporoderm-broken spores ofGanoder ma lucidum;DMCB:Drymatter of culture broth.△Two mice died before the experiment finished;＊ Compared to model group,P＜0.05.

Histopathaologicalobservation of tumor tissue from H22-bearing m ice

Histopathaological observation showed that,in the model group,tumor cellswere arranged tightly,with a large nucleus and clearly apparent nucleolus(Fig.1A).By contrast,the tumor tissues of 5-Fu treated mice showed obvious necrosis and pycnosis(Fig.1B).In the SSGL group(500 mg/kg)and DMCB group(1000 mg/kg), tumor cellswere arranged loosely,with denaturalization and putrescence both very obvious(Fig.1C,D).

Fig.1 Histopathaological observation of tumor tissues from H22-bear ing m ice

D iscussion

Antrodia cam phorata,a traditional Chinese herb found locally in Taiwan,has been widely used to treat cancer and inflammation.Previous studies revealed that an activated steroid acid ofAntrodia cam phoratafruiting bodies,zhankuic acid,exhibited a significant cytotoxic effect against P338 murine leukemia in vitro[7].Furthermore,Hseuet al.have reported that aqueous extract ofAntrodiacam phoratamycelia possesses cytotoxic effect against HL-60 leukemia cells[8].The maleic and succinic acid derivatives of the mycelium ofAntrodia cam phoratahave also shown appreciable cytotoxicity against LLC cells[9].Fruiting bodies ofAntrodia camphoratahas been reported to possess antitumor activity against hepatic H22 tumor on mice by activating the immune response of the host[10].In this study,we report the inhibitory effect of dry matter of culture broth ofAntrodia cam phoratain submerged culture on mice Hepatoma 22in vivo.

The ascitic-type liver cancer H22 comes from a hepatocarcinoma of mouse.This model was established as an induced hepatocarcinoma in Russia.In 1962,Yanget al.(Shanghai Drugs Research Institute of the Chinese Medical Academy of Science)subcutaneously implanted H22 hepatocarcinoma from C3HA mice into K M mice,with the substantial hepatocarcinoma developing into ascitic-type liver cancer.Thereafter,the ascitictype liver cancer H22 grew in the abdominal cavity of K M mice.H22 could grow in many mouse strains with100%incidence of growth and high invasiveness and metastasis.The facility and stability of the H22 tumor model made it one of most common translated tumor models employed in tumor and drugs research in China[11].In this research,DMCB ofAntrodia cam phorata(1000 mg/kg)could significantly inhibit H22 hepatocarcinoma growth,and prolong the survival time ofmice transplanted with H22 hepatocarcinomawhile not showing any toxicity and side effect.

Antrodia cam phoratahas been reported to contain many bioacitive components,such as polysaccharides,triterpenoids and phenyl compounds[12].Further studies are needed to demonstrate the constituents likely to be responsible for the in vivo antitumor activities ofAntrodia cam phoratain submerged culture.In conclusion,DMCB ofAntrodia cam phoratahas anticancer properties in a murine model of H22 hepatocarcinoma.This observation suggests that further development and exploration ofDMCB may enable it to become an effective clinical agent.The isolation and testing of constituents likely to be responsible for the antitumor activities ofAntrodia cam phoratain submerged culture is under progress in our lab.Acknowledgement This work was supported by the grant from National High-Tech Program of China (No.2007AA021506)and the program forNew Century Excellent Talents inUniversity ofChina(No.NCET-07-0380).

1 Lu Z M,TaoWY,Zou XL,et al.Protective effects of mycelia ofAntrodia camphorataandA r m illariella tabescensin submerged culture against ethanol-induced hepatic toxicity in rats.J Ethnophar m,2007,110:160-164.

2 Chang CY,Lue MY,Pan T M.Determination of adenosine, cordycepin and ergosterol contents in cultivatedAntrodia cam phorataby HPLC method.J Food D rug Anal,2005,13: 338-342.

3 LuMK,ChengJJ,LaiWL,et al.Adenosine as an active component ofAntrodia cinnam omeathat prevents rat PC12 cells from serum deprivation-induced apoptosis through the activation of adenosine A2A receptors.Life Sci,2006,79:252-258.

4 Wu H,Pan CL,Yao YC,et al.Proteomic analysis of the effect ofAntrodia cam phorataextract on human lung cancer A549 cell.Proteom ics,2006,6:826-835.

5 Wang JF,LingMY,Chu HY.The establishment and biological character of ascitic fluid type liver cancer(H22-Fo/L) in mice.J Pathol China,1991,20:65.

6 Ao ZH,Fu HZ,Zou XL.Large scale Zhangzhi fungus deep fermenting process.2003,CN 1456661-2003-11-19.

7 Chen CH,Yang S W,Shen YC.New steroid acids fromAntrodia cinnamom ea,a fungal parasite of Cinnamomum micranthum.J Nat Prod,1995,58:1655-1661.

8 Hseu YC,ChangWC,Hseu YT,et al.Protection of oxidative damage by aqueous extract fromAntrodia cam phoratamycelia in nor mal human erythrocytes.Life Sci,2002,71:469-482.

9 Nakamura N,Hirakawa A,Gao JJ,et al.Five new maleic and succinic acid derivatives from the mycelium ofAntrodia camphorataand their cytotoxic effects on LLC tumor cell line.J Nat Prod,2004,67:46-48.

10 Meng FY.The antineoplastic property ofAntrodia cam phorataand its effect on the immune function of the tumor bearing mouse.Master thesis,Harbin Medical University,China, 2005.

11 Yang YX,Zhu L,He X,et al.Antitumor activity of mitoxantrone-nanosphere against murine liver tumor H22.J Sichuan UnivM ed,2004,35:68-70.

12 Song TY,Yen GC.Antioxidant properties ofAntrodia camphoratain submerged culture.J Agri Food Chem,2002,50: 3322-3327.

Inhibitory Effect of DryMatter of Culture Broth of Antrodia cam phoratain Submerged Culture on M ice Hepatoma 22

XU Hong-yu1,KONG Xiao-ming1,AO Zong-hua2,LU Zhen-ming2,XU Zheng-hong1,2＊

1Laboratory of Phar m aceutical Engineering,School of M edicine and Phar maceutics,Jiangnan University;2The Key Laboratory of Industrial B iotechnology,M inistry of Education,Jiangnan University,W uxi 214122,China

The inhibitory effect of dry matter of culture broth(DMCB)ofAntrodia cam phoratain submerged culture were evaluated with the mice transplanted Hepatoma 22in vivo.The results showed thatDMCB(500 or 1000 mg/kg b. w.)could inhibit tumor growth,with the inhibition rates of 42%and 47%,respectively.Meanwhile,the survival time of the H22-bearingmice from DMCB treated group(1000 mg/kg b.w.)was prolonged,when compared with that ofmodel group.The histopathology of tumors from the various groups indicated that the tumor cells of untreated mice grew vigorously,whereas the tumor cells from DMCB treated groups had clear nucleus pycnosis and necrosis areas.Antrodia camphoratain submerged culture possess potent antitumor acitivityin vivo.

Antrodia cam phorata;anti-tumor;Hepatoma 22;submerged culture

July 23,2008;Accepted October 27,2008 Foundation Item:This work was supported by the National High-Tech Program ofChina(No.2007AA021506)and the program forNew Century Excellent Talents in University of China(No.NCET-07-0380).

R285;Q946.91

A

1001-6880(2010)03-0411-05

＊Corresponding author Tel:86-510-85918206;E-mail:zhenghxu@163. com