Sains Malaysiana 43(2)(2014): 185–194

In vitro Evaluation of Biological Activities of Citrus Leaf Extracts

(Penilaian in vitro Aktiviti Biologi Ekstrak Daun Limau)

MUHAMMAD ADNAN¹, ATIKA UMER², IMTIAZ AHMAD¹, KHIZAR HAYAT³

& SAMINA N. SHAKEEL*¹

¹Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan

²National Institute of Health, Islamabad, Pakistan

³Department of Chemistry, COMSATS Institute of Information Technology, Abbotabad,

22060, Pakistan

Received: 6 January 2013/Accepted: 18 May 2013

ABSTRACT

Leaves extracts of three different citrus species namely Citrus sinensis (Malta), Citrus paradisi (Grape fruit) and Citrus jambhiri (Khatti) were evaluated for their antioxidant, cytotoxic, antitumor, antimicrobial activities and finally the mineral contents were determined. Four types of extraction solvents (100, 80 and 50% methanol and distilled water heated at 50^oC) were used for extraction of antioxidant compounds. Extracts yield increased with the elevated levels of aqueous component of organic solvent and our data showed the highest yield in aqueous extracts. All extraction regimes showed in vitro antioxidant activity. Citrus paradisi showed the highest total flavonoid content (TFC), total phenolic content (TPC), reducing power and 1,1-Diphenyl-2- picrylhydrazil free radical scavenging activity in all combinations of solvents followed by Citrus sinensis and Citrus jambhiri. Antioxidant activity was also increased with higher aqueous component of organic solvents in each case. While Citrus sinensis (in 100% methanolic solvent) and Citrus paradisi (in 50% methanolic extract) showed significant cytotoxicity (LD₅₀ values <1000). Antitumor activity was observed in all extracts, however Citrus sinensis and Citrus paradisi (in 50% aqueous methanolic) extracts had the highest antitumor activity from the selected citrus species whereas no antimicrobial activity was observed at higher concentrations (4000 μgmL_-1) against specified strains. We found high calcium contents in all three citrus species tested by atomic absorption method. The results showed that the leaves of selected Citrus species possess significant antioxidant, antitumor and cytotoxic activities. Citrus leaves extracts can be potentially helpful in antioxidant protection in food as well as in human body against lipid peroxidation and free radicals damage. It can further be evaluated after in vivo studies using animal models or identifications of high throughput methods for enhanced biological activities.

Keywords: Biological activities; citrus; in vitro evaluation; mineral micronutrients

ABSTRAK

Ekstrak daun tiga spesies limau berbeza iaitu Citrus sinensis (Malta), Citrus paradisi (Grape fruit) dan Citrus jambhiri (Khatti) telah dinilai untuk aktiviti antioksidan, sitotoksik, antitumor dan agen antimikrob serta menentukan kandungan mineral mereka. Empat jenis pelarut pengekstrakan (100, 80 dan 50% metanol dan air suling yang dipanaskan pada 50^oC) telah digunakan untuk pengekstrakan sebatian antioksidan. Hasil ekstrak meningkat dengan peningkatan tahap komponen akueus pelarut organik dan data kami menunjukkan hasil tertinggi dalam ekstrak akueus. Semua rejim pengekstrakan menunjukkan aktiviti antioksidan in vitro. Citrus paradisi menunjukkan kandungan flavonoid keseluruhan (TFC), kandungan fenolik keseluruhan (TPC), kuasa penurunan dan aktiviti skaveng radikal bebas 1,1-difenil-2-pikrilhidrazil tertinggi dalam semua kombinasi pelarut diikuti oleh Citrus sinensis dan Citrus jambhiri. Aktiviti antioksidan juga ditingkatkan dengan peningkatan komponen akueus pelarut organik untuk setiap kes. Manakala Citrus sinensis (dalam pelarut metanol 100%) dan Citrus paradisi (dalam ekstrak metanol 50%) menunjukkan kesitotoksikan yang signifikan (nilai LD₅₀ < 1000). Aktiviti antitumor telah diperhatikan dalam semua ekstrak. Walau bagaimanapun, ekstrak Citrus sinensis dan Citrus paradisi (dalam akueus metanol 50%) didapati mempunyai aktiviti antitumor tertinggi daripada spesies limau terpilih manakala tiada aktiviti antimikrob telah diperhatikan pada kepekatan yang lebih tinggi (4000 μgmL_-1) terhadap strain khusus. Kami mendapati kandungan kalsium adalah tinggi dalam kesemua spesies limau yang diuji oleh kaedah penyerapan atom. Hasil menunjukkan bahawa daun spesies limau terpilih memiliki aktiviti antioksidan, antitumor dan sitotoksik yang signifikan. Ekstrak daun limau berpotensi membantu dalam perlindungan antioksidan dalam makanan serta dalam badan manusia terhadap peroksidasi lipid dan kerosakan oleh radikal bebas. Ia boleh dinilai selanjutnya berikutan kajian in vivo menggunakan model haiwan atau pengecaman kaedah berdaya pemprosesan tinggi untuk aktiviti biologi tertingkat.

Kata kunci: Aktiviti biologi; limau; mikronutrien mineral; penilaian in vitro

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*Corresponding author; email: snq28@yahoo.com