Untitled Document
 
 
 
 
Untitled Document
Home
Current issue
Past issues
Topic collections
Search
e-journal Editor page

Growth Inhibitory Activity of Garcinia Hanburyi extracts on Cholangiocarcinoma Cell Lines.

ฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์ของสารสกัดจากต้นรงทองต่อเซลล์มะเร็งท่อน้ำดี

Wongwarut Boonyanugomol (วงศ์วรุตม์ บุญญานุโกมล) 1, Chariya Hahnvajanawong (จริยา หาญวจนวงศ์) 2, Vichai Reutrakul (วิชัย ริ้วตระกูล) 3, Natthinee Anantachoke (ณัฏฐินี อนันตโชค) 4




ของประเทศไทย การรักษาผู้ป่วยด้วยวิธีต่างๆยังให้ผลที่ไม่ดีนัก แนวทางเลือกอีกทางหนึ่งในการรักษาคือการค้นหายาตัวใหม่ๆจากสารสกัดธรรมชาติที่มีฤทธิ์ยับยั้งการเติบโตของเซลล์มะเร็งท่อน้ำดี

วัตถุประสงค์: เพื่อตรวจหาฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์ของสารสกัดหยาบและส่วนแยกจากเปลือกต้นและผลของต้นรงทองต่อเซลล์มะเร็งท่อน้ำดีของคน

วิธีการ: การตรวจหาฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์โดยใช้ Sulforhodamine B assay โดยรายงานผลเป็นค่าเฉลี่ยของค่า 50% inhibitory concentration (IC50) จากการทดลอง 3 ครั้ง

ผลการศึกษา: การศึกษาฤทธิ์ในการยับยั้งการเพิ่มจำนวนของเซลล์ของสารสกัดหยาบจากเปลือกต้นและผลของต้นรงทอง 4 ชนิด ต่อเซลล์มะเร็งท่อน้ำดี 5 ชนิด พบว่าสารสกัดเอธิลแอซีเตตจากเปลือกต้น (VR12874) และผล (VR11626) มีฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์มะเร็งท่อน้ำดี 5 ชนิด โดยมีค่าความเข้มข้นของสารสกัดที่สามารถยับยั้งเซลล์มะเร็งได้ 50% (IC50) ในช่วง 1.84±0.10 ถึง 2.49±0.03 mg/ml และ 1.69±0.04 ถึง 4.41±0.10 mg/ml ตามลำดับ ในขณะที่สารสกัดเมธานอลจากเปลือกต้น (VR12875) และผล (VR11627) ไม่แสดงฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์ จากผลการทดลองดังกล่าวได้นำ 8 ส่วนแยกที่ได้จาก VR12874 มาตรวจสอบฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์มะเร็งท่อน้ำดี 5 ชนิด พบว่า 5 ส่วนแยกคือ VR12877, VR12878, VR12881, VR12882 และ VR12883 มีฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์ในระดับที่สูง ในขณะที่ VR12876 และ VR12879 มีฤทธิ์ในระดับที่ต่ำ และ VR12880 ไม่แสดงฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์

วิจารณ์และสรุป: ผลการศึกษาครั้งนี้สรุปว่า สารสกัดเอธิลแอซีเตตจากเปลือกต้นและผลของต้นรงทองมีฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์มะเร็งท่อน้ำดีทั้ง 5 ชนิด ในขณะที่สารสกัดเมธานอลไม่มีฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์ จากผลที่ได้แสดงว่าสารสกัดเอธิลแอซีเตตน่าจะมีสารสำคัญซึ่งมีฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์ ความแตกต่างในการแสดงฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์ในแต่ละส่วนแยกอาจขึ้นกับชนิดหรือปริมาณของสารสำคัญที่อยู่ในแต่ละส่วนแยกนั้น

คำสำคัญ: ต้นรงทอง, ฤทธิ์ยับยั้งการเพิ่มจำนวนของเซลล์, เซลล์มะเร็งท่อน้ำดี

 

Background: Cholangiocarcinoma (CCA) is the highest incident of primary liver cancer in the northeastern  Thailand. All of the treatment approaches remain poor. One attractive strategy is the discovery of new drugs from medicinal plants which have a potential growth inhibitory activity on cholangiocarcinoma cells.

Objective: To examine the growth inhibitory activity of crude extracts and fractions from barks and fruits of Garcinia hanburyi on human cholangiocarcinoma cell lines.

Method: Growth inhibitory activity was assessed by sulforhodamine B assay. The mean IC50 values of three independent experiments were reported.

Results: The growth inhibitory activity of four extracts from barks and fruits of G. hanburyi on five human CCA cell lines was performed. It was found that the ethyl acetate extracts from barks (VR12874) and fruits (VR11626) of G. hanburyi exhibited strong growth inhibitory activity against five CCA cell lines with IC50 values range from 1.84±0.10 to 2.49±0.03 mg/ml and 1.69±0.04 to 4.41±0.10 mg/ml, respectively whereas the methanol extracts from barks (VR12875) and fruits (VR11627) showed no growth inhibitory activity on CCA cell lines tested. Subsequent to these results eight fractions from VR12874 were examined for their growth inhibitory activities against 5 human CCA cell lines. It was found that five fractions including  VR12877, VR12878, VR12881, VR12882 and VR12883 showed strong growth inhibitory activity whereas VR12876 and VR12879 exhibited low growth inhibitory activity and VR12880 showed no effect.

Discussion and conclusion: It could be concluded that the ethyl acetate extracts from barks and fruits of G. hanburyi showed strong growth inhibitory effect against 5 human CCA cell lines whereas the methanol extracts had no effect. These results indicated that the ethyl acetate extracts might contain active constituents. Variation in the growth inhibitory activities among fractions might  depend on the type or concentration of active constituents containing in each fractions.

Key words: Garcinia hanburyi, growth inhibitory activity, cholangiocarcinoma cell lines

 

Introduction

          Cholangiocarcinoma (CCA)  is defined as an adenocarcinoma arising from cholangiocytes or bile duct epithelial lining cells anywhere in the biliary tree, excluding the gallbladder and the ampulla of Vater1. It is a rare primary liver cancer in the Western countries and worldwide, but it is a major malignancy in the northeastern Thailand where liver fluke (Opisthorchis viverrini) infection is endemic2. The CCA is considered to be a multidrug and radioresistant tumor. The results of palliative chemotherapy and /or radiotherapy remain poor. Only in a minority of cases can be cured by surgery and the response rate is not satisfactory. Based on these reason, alternative treatments should be considered. One attractive strategy is the use of medicinal plants which serve as a major source of drug in both primary health care and clinical therapy for centuries.

Garcinia hanburyi Hook. f. (Guttiferae)  is a small to medium-sized tree found throughout Thailand. The latex is used as a dye and the folk medicine for potent purgative and infected wounds in Thai traditional medicine3. Previous phytochemical investigation on the latex, fruits and whole plant of G. hanburyi led to the identification of some cytotoxic caged xanthones.4-8 Many caged xanthones have been reported to exhibit cytotoxic effects in several mammalian cancer cell lines,5, 9-13 as well as anticancer and anti-tumor activities.14,15

           This study was undertaken to examine the growth inhibitory activity of crude extracts from the barks and fruits of G. hanburyi on human cholangiocarcinoma cell lines.

 

Materials and methods

          Human cancer cell lines

          Five human  CCA cell lines used in this study including KKU-100 (poorly-differentiated adenocarcinoma), KKU-M139 (squamous carcinoma), KKU-M156 (moderately-differentiated adenocarcinoma), KKU-M213 (adenosquamous carcinoma) and KKU-M214 (moderately-differentiated adenocarcinoma) were established in the Department of Pathology, Faculty of Medicine, Khon Kaen University. All cell lines were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS), and 100 U/ml penicillin, 100 mg/ml streptomycin.  All cell lines were maintained at 37 °C in a 5% CO2 humidified incubator and were subcultured weekly.

 

Plant extracts

          The ethyl acetate extract (VR12874), methanol extract (VR12875) and eight fractions from ethyl acetate extracts (VR12876-VR12883) from barks and an ethyl acetate extract (VR11626) and methanolic extract (VR11627) from fruits of G. hanburyi were kindly provided by Professor Vichai Reutrakul, Department of Chemistry, Faculty of Science, Mahidol University, Thailand. Stock solution of the extracts were made in DMSO and diluted in culture media to the desired final concentration.

          In vitro growth inhibitory assay

        The sulforhodamine B (SRB) assay was used in this study to estimate cell number indirectly by staining total cellular protein with the SRB.  The protocol was based on that originally described by Skehan et al. (1990)20 with slight modification. Briefly, cells at the exponential growth phase were detached with 0.25% trypsin-EDTA (Sigma) to make single-cell suspensions.  The viable cells were counted by trypan blue exclusion in a haemocytometer and diluted with medium to give final concentrations about 0.5 to 1×105 cells/ml.  Each cell suspension was seeded in 96-well microtiter plates in the volume of 190 ml/well and allowed to grow overnight. The cells were then treated with plant extracts by adding 10 ml/well of each concentration in triplicate to obtain final concentration of  0.8, 4 and 20 mg/ml. The cells with same final concentration of DMSO were used as the solvent-control wells.  The plates were incubated for  72 h at 37 °C in a 5% CO2 humidified incubator.  At the end of drug treatment, the cells were then fixed with cold trichloroacetic acid (TCA), and washed 5 times with distilled water.  The TCA-fixed cells were stained with SRB.  The bound dye was solubilized with 10 mM Tris buffer (pH 10).  The absorbance (OD) of each well (triplicate for each concentration) was measured by using ELISA plate reader (ELX-800; BIO-TEK INSTRUMENTS, INC.) at 510 nm.  The intensity of color developed in each well was corresponded to the cell number. Percentage of cell survival will be calculated by using equation below. IC50 value was expressed as concentration of extract in microgram per milliliter that caused a 50% growth inhibition comparing with controls.

 

 

 

Statistical analysis

The statistical analysis was carried out by mean± SE.

 

Results

          Effect of G. hanburyi extracts on cell growth

          Of 4 crude extracts and 8 fractions from barks and fruits of G. hanburyi, the growth inhibitory activities were examined against five human CCA cell lines. After 72 h, cell viability was determined with SRB assay and the results are shown in Table1. The treatment of all cell lines with VR12874 markedly decreased cell viability with IC50 values ranging from 1.84±0.10 to 2.49±0.03 mg/ml (Table1), whereas the VR12875 showed no growth inhibitory activity on all cell lines with IC50 values >20 mg/ml. The extract VR11626 also showed strong growth inhibitory activity against all cell lines with IC50 values ranging from 1.69±0.04 to 4.41±0.10 mg/ml while the VR11627 showed no growth inhibitory activity on CCA cell lines tested (Table1).

          Subsequent to these results 8 fractions from the VR12874 were also examined for their growth inhibitory activities against 5 human CCA cell lines. It was found that VR12877 and VR12881 were the two most effective extracts against these CCA cell lines with IC50 values ranging from 1.26±0.30 to 2.33±0.03 mg/ml.The VR12878, VR12882 and VR12883 also showed strong growth inhibitory activity against all cell lines with IC50 values ranging from 0.77±0.01 to 7.91±0.02 mg/ml. The low growth inhibitory activities were observed when VR12876 and VR12879 were tested against these cell lines with IC50 values ranging from 15.64±0.06 to 18.25±0.77 mg/ml (Table1).

 

Table1 The IC50 values of G. hanburyi crude extracts and fractions on 5 human CCA cell lines. The results are expressed as mean±SE from 3 independent experiments.

 

NR= None reactive

 

Discussion and conclusion

          The search for anticancer agents from natural sources has been successful worldwide. Active constituents have been isolated and are nowadays used to treat human tumors. The ethnopharmacological knowledge is helpful in the searching for plants with potential cytotoxic activity.

 

          In the present study we analyzed the in vitro effect of G. hanburyi extracts on five CCA cell lines. Our results demonstrate that the ethyl acetate extracts from bark (VR12874) and fruit (VR11626) of G. hanburyi had marked growth inhibitory effects on the five human CCA cell lines whereas no effect was observed in methanol extract from bark (VR12875) and fruit (VR11627). In addition, seven fractions from VR12874 also showed strong to low growth inhibitory activities against five human CCA cell lines. These results indicated that the ethyl acetate extracts might contain active constituents. Variation in the growth inhibitory activities among fractions might be depending on the type or concentration of active constituents containing in each fraction. According to the previous reports several active components have been isolated from G. hanburyi for example desoxymorellin16, isomorellin17, isomorellinol5, gambogic acid10,18, and forbesione19. In addition, some cytotoxic caged xanthones isolated from resin, fruits and whole plant of G. hanburyi have been reported4-8. Many caged xanthones have been shown to exhibit cytotoxic activities in several mammalian cancer cell lines including human gastric cancer BGC-823, human lung carcinoma SPC-A1, human gastric carcinoma MGC-803 and HeLa cells9,11,13,15, as well as anticancer and anti-tumor activities14,15. In the present study the crude extracts and fractions of G. hanburyi were used. The active components in these extracts have already been characterized. We are now in the process of studying the exact mechanism of the growth inhibitory activity and ability to induce apoptosis of these active components on human CCA cell lines. More research on the application of G. hanburyi in cancer treatment is warranted.

 

Acknowledgement

       We gratefully acknowledge Associate Professor Banchob Sripa, Department of Pathology, Faculty of Medicine, Khon Kaen University, for providing CCA cell lines. This research was granted by the Center for Innovation in Chemistry : Postgraduate Education and Research Program in Chemistry (PERCH-CIC). We also thank Professor Vichai Reutrakul for his supply of plant extracts.

References

1. Uttaravichien T, Bhudhisawasdi V, Pairojkul C, Pugkhem A.  Intrahepatic cholangiocarcinoma in Thailand.  J Hepatobiliary Pancreat. Surg. 1999; 6: 128-35.

2. Bunyaratavej S, Meenakanit V, Tantachamrun T, Srinawat P, Susilavorn P, Chongchitnan N. Nationwide survey of major liver diseases in Thailand  analysis of 3305 biopsies as to year-end 1978. J Med Assoc Thai 1981; 64: 432-9.

3. Saralamp P, Chuakul W, Temsiririrkkul R, Clayton T. Medicinal plants in Thailand. Volume 1, Bangkok: Amarin Printing and Publishing, 1996: 97.

4.Asano J, Chiba K, Tada M, Yoshii T. Cytotoxic xanthones from Garcinia hanburyi. Phytochemistry 1996; 41: 815-20.

5. Lin LJ, Lin LZ, Pezzuto JM, Cordell GA. Isogambogic acid and isomorellinol from Garcinia hanburyi. Mag Reson Chem 1993; 31: 340-7.

6. Lu GB, Yang XX, Huang QS. Isolation and structure of neoigambogic acid from Gamboge (Garcinia hanburyi). Yao Xue Xue Bao 1984; 19: 636-9.

7. Lu GB, Fang J. Determination of gambogic acid and neogambogic acid in Tenghuang (Garcinia hanburyi) by HPLC. Zhogcaoyao 1988; 19: 298-300.

8. Sukpondma Y, Rukachaisirikul V, Phongpaichit S. Antibacterial caged-tetraprenylated xanthones from the fruits of Garcinia hanburyi. Chem Pharm Bull 2005; 53: 850-2.

 9. Asano J, Chiba K, Tada M, Yoshii T. Cytotoxic xanthones from Garcinia hanburyi. Phytochemistry 1996; 41: 815-20.

10. Ollis WD, Ramsay MVJ, Sutherland IO. The constitution of gambogic acid. Tetrahedron 1965; 21: 1453-70.

11. Zhao L, Guo QL, You QD, Wu ZQ, Gu HY. Gambogic acid induces apoptosis and regulates expressions of bax and Bcl-2 protein in human gastric carcinoma MGC-803 cells. Biol Pharm Bull 2004; 2: 106-10.

12. Han QB, Cheung S, Tai J, Qiao CF, Song JZ, Xu HX. Stability and cytotoxicity of gambogic acid and its derivative, gambogoic acid. Biol Pharm Bull 2005; 28: 2335-7.

13. Liu W, Guo QL, You QD, Zhao L, Gu HY, Yuan ST. Anticancer effect and apoptosis induction of gambogic acid in human gastric cancer line BGC-823. World J Gastroenterol 2005; 11: 3655-9.

14. Reutrakul V, Santisuk T, Noessner G, Schmidt J, Nickel B, Klenner T, Hose S. Novel xanthone compounds, their preparation and use medicament. Eup Pat Appl 2004; 20: 312-7.

15. Wu ZQ, Guo QL, You QD, Zhao L, Gu  HY. Gambogic acid inhibits proliferation of human lung carcinoma SPC-A1 cells in vivo and in vitro and represses telomerase activity and telomerase reverse transcriptase mRNA expression in the cells. Biol Pharm Bull 2004; 27: 1769-74.

16. Bhat HB, Nair PM, Venkataraman K. The colouring matters of Garcinia morella. Part V. Isolation of desoxymorellin and dihydroisomorellin. Ind J Chem 1964; 2: 405-9.

17. Kartha G, Ramachandran GN, Bhat HB, Nair PM, Raghavan VKV, Venkataraman K. The constitution of morellin. Tetrahedron Lett 1963; 4: 459-72.

18. Karanjgaonkar CG, Nair PM, Venkataman K. Morellic, isomorellic and gambogic acid. Tetrahedron Lett  1966; 7: 687-91.

19. Leong YW, Harrison LJ, Bennett GJ, Tan HTW. Forbesione, a modified xanthone from Garcinia forbesii. J Chem Res 1996; 392-3.

20. Skehan P, Storeng R, Scudiero D.  New colorimetric Cytotoxicity Assay for Anticancer-drug Screening. J Natl Cancer Inst 1990, 82: 1107-12.

 

Untitled Document
Article Location

Untitled Document
Article Option
       Abstract
       Fulltext
       PDF File
Untitled Document
 
ทำหน้าที่ ดึง Collection ที่เกี่ยวข้อง แสดง บทความ ตามที่ีมีใน collection ที่มีใน list Untitled Document
Another articles
in this topic collection

Cancer Chemoprevention from Dietary Phytochemical (เคมีป้องกันมะเร็ง :กลไกการป้องกันของยาและสารจากธรรมชาติ)
 
Role of Natural Products on Cancer Prevention and Treatment (บทบาทของผลิตภัณฑ์ธรรมชาติในการป้องกันและรักษามะเร็ง)
 
Prescription-Event Monitoring: New Systematic Approach of Adverse Drug Reaction Monitoring to New Drugs (Prescription-Event Monitoring: ระบบการติดตามอาการไม่พึงประสงค์จากการใช้ยาใหม่ )
 
The use of Digoxin in Pediatrics (การใช้ยาดิจ๊อกซินในเด็ก)
 
<More>
Untitled Document
 
This article is under
this collection.

Pharmacology
 
 
 
 
Srinagarind Medical Journal,Faculty of Medicine, Khon Kaen University. Copy Right © All Rights Reserved.
 
 
 
 

 


Warning: Unknown: Your script possibly relies on a session side-effect which existed until PHP 4.2.3. Please be advised that the session extension does not consider global variables as a source of data, unless register_globals is enabled. You can disable this functionality and this warning by setting session.bug_compat_42 or session.bug_compat_warn to off, respectively in Unknown on line 0