Keywords

Liver diseases, Egyptian victims; Hepatocellular carcinoma HCC, Egyptian plants, Spices, 13 Potent anti-hepG2.

INTRODUCTION

At present, enthusiasm remains high among scientists involved in the discovery of new potential anticancer agents from plants since the approval of taxol; the first novel antimicrotubule agent approved for clinical use in the treatment of ovarian, breast, lung, head and neck cancers (Goodman & Walsh 2001). In this concern, many medical reports showed that the major cause of death in most developing and 3^rd world countries are cardiovascular diseases and cancer with a latency of many years before clinical diagnosis (Gescher et al 2001).

In Egypt, liver diseases are the most agonizing endemic health problem especially in the rural governorates. About 20-25% of populations are suffering from hepatitis C virus, liver cirrhosis and /or hepatocellular carcinoma HCC (Frank et al 2000, Aboul-Enein et al 2012, Amara et al 2008, Khafagi et al 2001, Husein et al 2001, El-Sadawi et al 1996, Mostafa et al 2002). Unfortunately, incidence of hepatic diseases in Egypt is escalating and progressively affecting younger persons.

This encouraged us to screening the Egyptian wild and cultivated plants as well as traditionally consumed spices for their activity against hepatocellular carcinoma.

MATERIALS AND METHODS

Plant Material

Wild plants and weeds were collected from Zagazig farms, irrigation canals and desert vicinity of Sharkia governorate area (60 kilometers west to Sues Canal, Egypt) in March-April 2010-2011. The collected samples were kindly identified by Dr. Abd-Elhalim Abdel-Mogly, Prof. of Taxonomy, Flora Department, Agricultural Research Institute, Ministry of Agriculture, Cairo, Egypt. The fruits, spices and seeds were purchased from the local central markets and herbalists. Voucher specimens are kept in the Pharmacognosy Department, Faculty of Pharmacy, University of Zagazig, Egypt.

Preparation of Extracts

The whole plant, fresh fruits, seeds or spices (50 g each) were washed thoroughly with fresh water - to remove dust and insecticides remainders-then extracted by cold percolation using ethyl alcohol 70% (500 ml x 2). The combined extract then concentrated under reduced pressure using vacuum rotatory evaporator at a temperature not exceeding 45°C. The resulting aqueous residues were lyophilized into powder and submitted and /or kept in freezer at -20 °C for biological assay.

Biological Study (Hansen et al 1989)

Cell Culture

Human hepatocarcinoma cell line (HepG2), purchased from ATCC, USA, were used to evaluate the cytotoxic effect of the tested extracts. Cells were routinely cultured in DMEM (Dulbeco’s Modified Eagle’s Medium) which was supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulphate, and 250 ng/ml amphotericin B. Cells were maintained at sub-confluence at 37ºC in humidified air containing 5% CO₂. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37°C. Cells were used when confluence had reached 75%. Tested extracts were dissolved in dimethyl sulphoxide (DMSO), and then diluted thousand times in the assay. All cell culture material was obtained from Cambrex Bio Science (Copenhagen, Denmark). All chemicals were from Sigma/Aldrich, USA, except mentioned. All experiments were repeated three times, unless mentioned.

Cytotoxicity

Cytotoxicity was measured against HepG2 cells using the MTT Cell Viability Assay. MTT (3-[4,5-dimethylthiazole-2-yl]- 2,5-diphenyltetrazolium bromide) assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium rings of the yellow MTT and form a dark blue insoluble formazan crystals which is largely impermeable to cell membranes, resulting in its accumulation within healthy cells. Solubilization of the cells results in the liberation of crystals, which are then solubilized. The number of viable cells is directly proportional to the level of soluble formazan dark blue color. The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm.

Procedure

Cells (0.5X105 cells/well), in serumfree media, were plated in a flat bottom 96- well micro plate, and treated with 20μl of different concentrations of each tested extracts for 48 h at 37ºC, in a humidified 5% CO₂ atmosphere. After incubation, media were removed and 40 μl MTT solution/well were added and Incubated for an additional 4 h. MTT crystals were solubilized by adding 180 μl of acidified isopropanol / well and plate was shacked at room temperature, followed by photometric determination of the absorbance at 570 nm using micro plate ELISA reader. Triplicate repeats were performed for each concentration and the average was calculated. Data were expressed as the percentage of relative viability compared with the untreated cells compared with the vehicle control, with cytotoxicity indicated by <100% relative viability.

Calculation

Percentage of relative viability was calculated using the following equation:

[Absorbance of treated cells/ Absorbance of control cells)] X 100.

Then the half maximal inhibitory concentration (IC₅₀) was calculated from the equation of the dose response curve and the standard used was Doxorubicin (IC₅₀ 21.4 μg/ml).The results are presented in tables 1- 3.

Table 1: Strongly active anti-HepG2 extracts (< 20 µg/ml )

Table 2: Moderately active anti-HepG2 extracts (between 20-50µg/ml)

Table 3: Weakly active anti-HepG2 (IC₅₀ > 50µg/ml)

RESULTS AND DISCUSSION

Plants from the local farms or wild flora and traditional spices, which are daily used by the rural citizens and most of the other cities were collected and properly identified. The plant samples were cold extracted lyophilized and the resulting residues were submitted to in-vitro bioassay.

The obtained results expressed strong-(less than IC₅₀ 20 μg/ml , table 1) -to moderate activities( IC₅₀ 21 -50 μg/ml, table 2) according to the previous protocols of the American National Cancer Institute NCI (Boyd 1997 ). The most interesting of all tested samples are : the unorganized oleogum- resin asafoetida (Ferula foetida), the seeds of Erysimum corinthium, Ammi visnaga and Nigella sativa (black seed) with IC₅₀ 3.2, 2.5, 9.6 and 10.5 μg/ml, respectively. These promising results (IC₅₀ 3.2 and 2.5 μg/ml, respectively) require further investigation. In this concern, previous researchers confirmed that the cytotoxic or anti-carcinogenic activity of many brassicaceae plants are most probably ascribed to their contents of the allyl isothiocyanates and sulphurated compounds (Al-Gendy et al 2010, Nastruzzi et al 1996,). They also revealed that the hydrolysis products of some brassica glycosides showed a marked in vitro cytotoxicity against human erythroleukaemic K562 cells (Schonberg et al 1953). Considering the visnaga (IC₅₀ 16.2 μg/ml), although, the chemical constituents are well known in the middle east many years ago (Mahran et al 1954) yet none of its chemical contents have been tested or claimed to be active against HCC.

Among the other strong active extracts comes the black seed (Nigella sativa) although, to-our-knowledge, no previous reports on testing the seed extract or the oil as anti-HCC cells, yet in our study it showed very promising result with IC₅₀ 10.5 μg/ml. Previous report on the black seed indicated that, it only induces liver protection, while the results obtained by other authors indicated that both TQ and DIM were cytotoxic for several cell lines with very poor effect (IC₅₀'s 78 to 393 μg/ml) (Badary 1999, Haq et al 1999, Nagi et al 1999, Hassan et al 1998, Worthen et al 1998). Also, from spices tested in our study with remarkable strong activity are curcuma, long pepper and nutmeg with IC₅₀ 11.6, 10.5, 18.4 μg/ml, respectively. This is the first report on the activity of long pepper and nutmeg against HCC (Chan et al 1998, Jacob et al 2012, Sohn et al 2007).

Most interestingly, the results observed from three herbal extracts with strong unexpected results against HCC, these are the Egyptian tomato herb (Solanum lycopersicum), Hibiscus trionum and Hibiscus rosa-sinensis with IC₅₀ 9.6, 12.43 and 13.90 μg/ml, respectively. Concerning the herb of tomato which is generally used as animal feed, previous reports on its chemistry (Ripperger & Himmeireich 1994, Kumari et al 1985) indicated the presence of steroidal alkaloids, flavonoids and coumarins without any mention on cytotoxicity. In addition, previous chemical studies on Hibiscus plants showed high anthocyanin contents together with several flavonoidal compounds (Ateya et al 2012, Abdel Latif 2010). This is the first report on testing these Egyptian agricultural waste plants as active against HCC.

Concerning the vegetables in this study, although the Brassica and Cruciferous vegetables are well known -for long time- to contain the isothiocyanate sulforaphane well known inhibitors of carcinogen-induced mammary tumorigenesis (Marion 1997), unfortunately the Egyptian vegetables showed weak to very weak activities against HCC. In Egypt, rural areas consume one or more locally grown vegetables daily, a cultural dietary habit which could have greatly helped in reducing the incidence of hepatic diseases. An exception of these results was the aqueous extract of Broccoli which exhibited strong activity with IC₅₀ 16.2 μg/ml (table 1). A previously impressive report showed that, rats fed a broccoli diet showed a significant increase in colon and hepatic ubiquinone -reductase (Keck et al 2003).

ACKNOWLEDGEMENT

This work was kindly fully supported by the grant No. 2413 / 4 from the vice president sector for graduate studies and researches, University of Zagazig. Authors appreciate the help of Dr. Khaled Mahmoud, Cell Culture Laboratory, National Research Center, Cairo, Egypt for In-vitro assay.

REFERENCES

1. Aboul-Enein AM, Abu El-Ela F, Shalaby EA and El-Shemy HA. Traditional medicinal plants research in Egypt: Studies of antioxidant and anticancer activities. J. Med. Plants Res., vol.6 (5), pp.689- 703(2012).
2. A Latif S: Pharmacognostical Study on Certain Medicinal Plants and/ or Natural Products with Reported Antifertility Action, Ph.D. Thesis, Pharmacognosy Department, Faculty of Pharmacy, Cairo University (2010).
3. Al-Gendy A, El-Gindi O, Hafez AlS and Ateya AM. Glucosinolates, volatile constituents and biological activities of Erysimum corinthium Boiss. (Brassicaceae); Food Chemistry, 118, 519-524(2010).
4. Amara A, El-Masry M, Bogdady H. Plant crude extracts could be the solution: Extracts showing in vivo antitumorigenic activity. J. Pharm. Sci., 21(2): 159-171(2008).
5. Ateya AM, El Sayed Z I. and Fekry M.,Chemical Constituents, Cytotoxicity, Anti-oxidant, Hypoglycemic and Antihypertensive Activities of Egyptian Hibiscus trionum. Australian Journal of Basic and Applied Sciences, 6 (3): 756- 766(2012).
6. Ayoub NA. A trimethoxyellagic acid glucuronide from Conocarpus erectus leaves: Isolation, characterization and assay of antioxidant capacity. Pharm Biol, 48, 328-332(2010).
7. Badary OA. Thymoquinone attenuates ifosfamide-induced Fanconi syndrome in rats and enhances its antitumor activity in mice, J. Ethnopharmacol, 67(2), 135- 142(1999).
8. Boyd MR. The NCI in-vitro anti-cancer drug discovery screen, concept, Implementation and operation (1985 -1995). In: Teicher BA, ed., Anti-cancer Drug Development Guide, Preclinical screening, Clinical Trials and Approval. Totowa NJ: Humana Press, pp 23-42, (1997).
9. Chan MM, Huang HI, Fenton MR, Fong D. In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti- inflammatory properties, Biochem. Pharmacol, 55: 1555-62(1998).
10. El-Sadawi MS, Mostafa N S and Ateya AM. Molluscicidal Activity of Azolla pinnata; Zagazig J. Pharm. Sci., vol.5, No.2, pp 7- 12(1996).
11. Frank C, Mohamed MK, Strickland GT, Lavanchy D,Arthur RP, Magder LS, El- Khoby T, Abdel-Wahab Y, Ali Ohn ES, Anwar W, Sallam I., The role of parentral antischistosomal therapy in the spread of hepatitis C virus in Egypt. The Lancet (Pub. Med) Mar 11; 355(9207): 887-91. (2000).
12. Gescher A J, Sharma RA and Steward WP. Cancer chemoprevention by dietary constituents: a tale of failure and promise. The Lancet Oncology, vol. 2, pp 371-379, June (2001).
13. Goodman J, Walsh, V. The Story of Taxol: Nature and Politics in the Pursuit of an Anti- Cancer Drug. Cambridge University Press. pp 17- 51. (2001).
14. Hansen M B, Nielsen S E and Berg K. Re- examination and further development of a precise and rapid dye method for measuring cell growth/cell kill, J. Immunol. Methods, 119, 203-10(1989).
15. Haq Afrozul; Lobo, Peter I.; Al-Tufail, Mohammad; Rama, Nona R., Immunomodulatory effect of Nigella sativa proteins fractionated by ion exchange chromatography; Al- Int. J. Immuno pharmacol., 21(4), 283-295(1999).
16. Hassan, Mona M., Mohamed, Mervat E., Evaluation of the hepatoprotective effect of some drugs in paracetamol-induced liver cell toxicity in the rat, Alexandria J. Pharm.Sci., 12(2), 103-106(1998).
17. Husein AI, Ali-Shtayeh MS, Zatar NA, Jondi W. Antioxidant and anticancer activities of six Palestinian plants Used in Traditional Medicine (IYC-2011) 45. Medicinal Chem., 45: 450-461(2001).
18. Jacob S JP, Finub JS and Narayanan A."Synthesis of silver nanoparticles using Piper longum leaf extracts and its cytotoxic activity against Hep-2 cell line", Colloids and surface B: Biointerfaces, 91, p: 212-214 (2012).
19. Keck A-S, Qiao Q and Jeffery E Food matrix effects on bioactivity of Broccoli- derived sulforaphane in liver and colon of F344 rats. Journal of Agricultural and Food Chemistry, 51, 3320-3327(2003).
20. Khafagi I, Dewedar A, Kord M, Mohammed E. Identification and antibiotic sensitivity of bacteria occasionally isolated from differentiated and undifferentiated cultures of Sinai medicinal plants. Egyptian J. Biol., 3: 67-78(2001).
21. Kumari G, Rao J and Rao N, Flavonol 3-O- methylethers from Solanum pubescens Heyne”. J. Nat. Prod.48, 149-150 (1985).
22. Mahran GH, El alfyTS and Saber AH. A phytochemical study of the Afghanian asafoetida; Bull. Fac. Pharm. Cairo Univ., vol. I. 156(1954).
23. Marion N. Broccoli sprouts as inducers of carcinogen-detoxifying enzyme systems: Clinical, dietary, and policy implications, Proc. Natl. Acad. Sci. (USA), vol. 94, pp. 11149–11151(1997).
24. Mostafa NS, Ateya AM, Abou-Hashem MM, Koura SK and Desouki A Y. Study of the Molluscicidal Activity of Certain Wild Egyptian Plants Against the Snails intermediate hosts of Schistosoma and Fasciola. Med. J. Zagazig University, VIII (5), 443-452 (2002).
25. Nagi M N, Alam B, Osama A.; Al- Shabanah,Othman A.; Al-Sawaf, Hussein A.; Al-Bekairi, Abdullah M , Thymoquinone protects against carbon tetrachloride hepatotoxicity in mice via an antioxidant mechanism. , Int. Biochem. Mol. Biol. 47(1), 153-159(1999).
26. Nastruzzi C, Cortesi R, Esposito E, Menegatti E, Leoni O, Iori R . In-vitro cytotoxic activity of some glucosinolate- derived products generated by myrosinase hydrolysis. Journal of Agricultural and Food Chemistry, 44, 1014-1021(1996).
27. Qin Tian, PhD, Xiu-Hua Wang, MS, Wei Wang, PhD, Chuang-Nian Zhang, PhD, Ping Wang, MS, Zhi Yuan, PhD. Self-assembly and liver targeting of sulfated chitosan nanoparticles functionalized with glycyrrhetinic acid. Volume 8, Issue 6, Pages 870–879 (August 2012).
28. Ripperger H, Himmeireich U, Glyco- steroidal alkaloids in Solanum anguivi lam, Phytochemistry, 37(6), p1725 (1994).
29. Sohn J H, Han KL, Choo JH and Hwang JK. Macelignan protects HepG2 cells against tert-butylhydroxide-induced oxidative damage. Biofactors, 29(1), 1-10 (2007).
30. Worthen, D R; Ghosheh, O A, Crooks PA. The in- vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L. Anticancer Res. 18(3A), 1527-1532(1998).
31. Zhang C, Wang W, Lbiu T, Wu Y, Guo H, Wang P, Tian Q, Wang Y, Yuan Z. Doxorubicin-loaded glycyrrhetinic acid- modified alginate nanoparticles for liver tumor chemotherapy. Biomaterials, volume 33, issue 7, pages 2187–2196 (March 2012).