CYTOTOXIC EFFECT AND PHYTOCHEMICAL STUDY OF PETROLEUM ETHER EXTRACT OF TILIA CORDATA MILL

Gehan F. Abdel Raoof^1*, Hala M. Mohammed²

¹Pharmacognosy Department, National Research Centre, Dokki, 12622, Giza, Egypt.

²Home Economics department (Nutrition & Food Science), Faculty of Agricultue, Cairo University, Egypt.

DOI: https://doi.org/10.22270/ujpr.v4i4.292

ABSTRACT

The aim of this research was to investigate the chemical composition of petroleum ether extract of Tilia cordata aerial parts as well as to evaluate its cytotoxic activity. Gas chromatography and gas chromatography–mass spectrometry (GC-MS) were used to analyze the unsaponifiable matter and fatty acid methyl esters.  Moreover, the cytotoxicity was examined against human hepatoma HepG2 cell line and breast adenocarcinoma MCF cell line. The result showed that thirteen compounds were identified in the fatty acid methyl esters fraction representing 93.71% of the total identified peak area. The major compounds were Octadecanoic acid methyl ester (36.26%) and eicosanoic acid methyl ester (29.42%), whereas nineteen compounds in the unsaponifiable fraction were identified representing 90.56 % of the total beak area. The major compounds were 1- Nonene (30.44%), 1-Hexadecene (24.83%) and phytol (10.40%). Moreover, petroleum ether extract showed a potent cytotoxic effect against human hepatoma HepG2 cell line and a moderate cytotoxic effect on breast adenocarcinoma MCF7 human tumor cell line. So the current research aims to be the first step toward the use of petroleum ether extract of Tilia cordata aerial parts as a potent cytotoxic drug.

Keywords: Aerial parts, chemical composition, cytotoxicity, petroleum ether extract, Tilia cordata.

INTRODUCTION

Tilia cordata belongs to family Tiliaceae, it is used in folk medicine for many purposes, its flowers are widely used for the treatment of fever and anxiety. It contains flavonoids, volatile oils and tannins¹. The flower of Tilia cordata reported to have a potent antioxidant activity². The aerial parts of Tilia cordata showed antioxidant and anti-tyrosinase activities³. Moreover, the aerial parts contain various phytoconstituents such as; coumarins, triterpenes, flavonoids, tannins, saponins and carbohydrates³. In addition, our recent research showed that aerial parts of Tilia cordata showed a powerful anti-inflammatory, antinociceptive and nephroprotective activities⁴. Moreover, kaempferol 3-O-rutinoside, quercetin 3-O-β-galactoside, kaempferol 3-O-rutinoside, quercetin, vitexin and kaempferol were isolated and identified from aerial parts of Tilia cordata⁴.

The current research aims to find the correlation between the lipoidal matter of petroleum ether extract of Tilia cordata aerial parts and their effect on some human cell line carcinoma. So this research clarified the chemical composition of petroleum ether extract of Tilia cordata aerial parts as well as evaluated its cytotoxic activity. 

So current study aims to be the first step toward the use of petroleum ether extract of Tilia cordata aerial parts as a potent cytotoxic drug with the aim of producing a natural drug.

MATERIALS AND METHODS

Plant material

Tilia cordata aerial parts were collected from the Agricultural Research Centre, Giza, Egypt, in March 2017. The plant was identified by Dr. Mohammed El-Gebaly, Department of Botany, National research centre (NRC).

Preparation of the lipoidal matter

The powder of the air-dried aerial parts of Tilia cordata (800g) was exhaustively extracted with light petroleum (60–80°C) in a continuous extraction apparatus (Soxhlet). The extract was evaporated under vacuum to yield 28g of dry residue, representing 3.5% of the air-dried aerial parts.

Investigation of the lipoidal matter

Saponification of the petroleum ether extract

The petroleum ether extract (PtE) (1g) was subjected to saponification according to the method reported by Tsuda et al.⁵ Percentages of the unsaponifiable matter and the total fatty acid were found to be 38 and 60%, respectively.

Preparation of fatty acid methyl esters

Free fatty acids obtained by saponification were methylated according to the method reported by Finar 1967 ⁶.

GC/MS analysis

Both the unsaponifiable and the saponifiable fractions were studied to identify their contents using GC/MS analysis. The constituents were identified by comparison of their mass spectral fragmentation patterns with those of the available database libraries, Wiley (Wiley International, Colorado, USA) and NIST (Nat. Inst. St Technol., Colorado, USA), and/or published data^7,8. Quantitative determination was carried out on the basis of the peak area integration.

Cytotoxicty assay procedures

Tumor cell lines

Human hepatocellular liver carcinoma (HepG2) and human breast carcinoma (MCF-7) cell lines were obtained in frozen state under liquid nitrogen (-180ºC) from the American Type Culture Collection. The tumor cell lines were maintained by serial sub-culturing in the National Cancer Institute, Cairo, Egypt.

Culture media

The cells were suspended in RPMI 1640 medium (Sigma Aldrich) supplemented with 10% fetal calf serum (SIGMA, USA) in presence 1% antibiotic antimycotic mixture (10.000 U/ml K-penicillin, 10.000 μg/ml streptomycin sulphate and 25 μg/ml amphotericin B) and 1% L-glutamine (all purchased from Lonza, Belgium). 

Assay method for cytotoxic activity

The cytotoxicity against Hep-G2 and MCF-7 cells were tested in the National Cancer Institute, according to the SRB (Sulforhodamine  B) assay by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method, Adriamycin® (Doxorubicin) 10 mg vials (Pharmacia, Sweden) was used as the reference drug. The method was described in⁹.

RESULTS AND DISCUSSION

The results showed that nineteen compounds in the unsaponifiable fraction were identified representing 90.56 % of the total peak area. The major compounds were 1- Nonene (30.44%), 1-Hexadecene (24.83%) and phytol (10.40%) (Table1). Moreover, thirteen compounds were identified in the fatty acid methyl esters fraction representing 93.71% of the total identified peak area. The major compounds were Octadecanoic acid methyl ester (36.26%) and Eicosanoic acid methyl ester (29.42%) (Table 2).

The cytotoxic activity

There are many researches that showed the cytotoxic effect of hydrocarbons and triterpenoids against many human tumor cell lines^10,11 . The current research aims to find the correlation between the lipoidal matter of petroleum ether extract of Tilia cordata aerial parts and their effect on some human cell line carcinoma. The cytotoxicity of petroleum ether extract of Tilia cordata aerial parts was evaluated against, HepG2 and MCF7 cell lines using Doxorubicin as reference drug. The results showed that the extract had cytotoxic activity against the tested cell lines (IC₅₀ (µg/ml) =5.42 and 9.67), respectively, while Doxorubicin showed activity with IC₅₀ (µg/ml) =3.62 and 3.34, respectively. So this result showed that petroleum ether extract of Tilia cordata aerial parts had a potent cytotoxic effect against HepG2  and moderate activity against  MCF7 (Table 3).

CONCLUSION

This work was carried out to investigate the chemical composition of petroleum ether extract of Tilia cordata aerial parts as well as to evaluate its cytotoxicity against human hepatoma HepG2 cell line and breast adenocarcinoma MCF7 cell line. The result revealed that petroleum ether extract showed a potent cytotoxic effect against human hepatoma HepG2 cell line  and a moderate cytotoxic effect on breast adenocarcinoma MCF7 human tumor cell line. In conclusion, this study aims to be the first step toward the use of petroleum ether extract of Tilia cordata aerial parts as anticancer agent upon further clinical studies.

ACKNOWLEDGEMENT

We acknowledge to National Research Centre and Faculty of Agriculture, Cairo University for using laboratory instruments in doing research.

CONFLICT OF INTEREST

No conflict of interest associated with this work.

REFERENCES

1. Bradley P. ed. British Herbal Compendium1992; 1:142–144.
2. Vinha AF, Sérgio VPB, Ana C, Marisa M. Comparison between the phytochemical and antioxidant properties of plants used in plant infusions for medicinal purposes. J Agric Sci 2013;5(11):11-19.
3. Rashed K, Medda R, Spano D, Pintus F. Evaluation of antioxidant, anti tyrosinase potentials and phytochemical composition of four Egyptian plants. Int Food Res J 2016; 23(1): 316-321.
4. Fawzy G, Younes K, Waked E, Mahmoud, H. Anti-inflammatory, antinociceptive and nephroprotective activities of Tilia cordata and isolation of bioactive compounds. Mater Environ Sci 2018; 9 (6):1908-1914.
5. Tsuda K, Sakai K, Tanabe K, Kishida Y. Isolation of 22-dehydrocholesterol from Hypnea japonica. J Am Chem Soc 1960; 82:1442–1443.
6. Finar IL. Organic chemistry. 5^th London, UK: Longmans Green and Co. Ltd; 1967; 1:212.
7. Adams RP. Identification of essential oils by ion trap mass spectroscopy. New York: Academic Press Inc.; 1995.
8. Jennings W, Shibamato T. Qualitative analysis of flavor and fragrance volatiles by glass capillary gas chromatography. New York: Academic Press; 1981.
9. Said A, Omer E A , El Gendy M A M , Fawzy G, Abd EL-Kader A E, Fouad R. Volatile constituents and cytotoxic activity of the fruits of pleiogynium timorense (Dc.) Leenh J Mater Environ Sci 2018; 9(8): 2274-2279.
10. Bishayee A, Ahmed S, Brankov N, Perloff M. Triterpenoids as potential agents for the chemoprevention and therapy of breast cancer. Front Biosci 2011; 16:980–990.

11. Khamsan S, Liawruangrath B, Liawruangrath Teerawutkulrag A, Pyne SG, Garson MJ. Antimalarial, anticancer, antimicrobial activities and chemical constituents of essential oil from the aerial parts of Cyperu skyllingia Rec Nat Prod 2011; 5:324–7.

Table 1: GC/MS analysis of USM from petroleum ether extract of Tilia cordata aerial parts

Table 2: GC/MS analysis of fatty acids of petroleum ether extract of Tilia cordata aerial parts identified as the methyl esters

Table 3: Cytotoxic activity of petroleum ether extract of Tilia cordata aerial parts

IC₅₀: the concentration that produces 50% inhibition