Determination of cardiac glycosides and total phenols in different generations of Securigera securidaca suspension culture

Document Type : Original paper


1 Department of Pharmacognosy, Faculty of Pharmacy and Medicinal Plants Research Center, Tehran University of Medical Sciences, Tehran, Iran.

2 Department of Pharmacognosy, Faculty of Pharmacy and Medicinal Plants Research Center, Tehran University of Medical Sciences, Tehran, Iran


Background and objectives: The seeds of Securigera securidaca (L.) Deg. & Dorf. (Fabaceae) are used as anti-diabetic remedy in Iranian folk medicine. The aim of the present study was to establish the callus and suspension culture of S. securidaca seeds for the first time and to determine the major secondary metabolites including cardiac glycosides and total phenols.
Methods: The culture of S. securidaca from seeds was initiated in hormone-supplemented MS medium containing 1 and 0.1 ppm 2, 4-D solution for solid and suspension cultures, respectively, sucrose and vitamins (B1, B2, B6, Folic acid, Biotin, Nicotinamide and Ca pantothenate) at 25 °C and 12 h photoperiods. The cardiac glycosides were determined based on the calibration curve of securidaside which was isolated from the seeds extract of S. securidaca. Total phenolic compounds of different generations of suspension culture were determined using Folin Ciocalteu reagent.
Results: Callus culture of S. securidaca was grown light cream to pale yellow in color and soft in texture while the cells of suspension culture grew cream to yellow with isolated cells and small aggregates. The production of cardiac glycosides in the 7th generation were more than the seeds extract (p<0.05) and from the 8th generation it reached to a steady state. The amount of phenolic contents were elevated and reached to its peak during the 8th generation but it was much lower than the seeds extract.
Conclusion: In vitro culture of S. securidaca showed possibility for production of cardiac glycosides more than the seeds extract.


[1] Recshinger KH. Securigera securidaca (Legominoseae). In: Recshinger KH, Ed. Flora Iranica. Graz: Akademische Druck-u, 1984.
[2]  Ghitasi I,  Nikbakht MR, Sadeghi H, Sabzali V, Sabzali S, Shahrani M. The hypoglycemic effects of a hydro-alcoholic extract from Securigera securidaca seeds on induced diabetic in male rats. J Shahrekord Univ Med Sci. 2007; 8(4): 68-73.
[3] Hosseinzade H, Ramezani M, Danaei AR. Antihyperglycaemic effect and acute toxicity of Securigera securidaca L. seed extract in mice. Phytother Res. 2002; 16(8): 745-747.
[4] Porchezhian E, Ansari SH.  Effect of Securigera securidaca on blood glucose levels of normal and alloxan-induced diabetic rats. Pharma Biol. 2001; 39(1): 62-64.
[5] Pouramir M, Shahaboddin ME, Moghadamnia AA, Parastouei K. To study the effects of Securigera securidaca (L.) seed against alloxan-induced hyperglycemia. J Med Plants Res. 2011; 5(14): 3188-3191.
[6] Roostazadeh A, Firoozrai M, Shabani M. Effect of aqueous seed extract of Securigera Securidaca on erythrocytes catalase activity in type 1 diabetic rats. Qom Univ Med Sci J. 2008; 1(4): 9-14. 
[7] Zahedi-Asl S, Marahel H, Zare B. Study on the effects of chloroformic extract of Securigera Securidaca on serum glucose level and liver glycogen content of mice. J Kerman Univ Med Sci. 2005; 12(1): 32-38.
[8] Ali AA, Mohamed MH, Kamel MS, Fouad MA, Spring O. Studies on Securigera securidaca (L.) Deg. et Dorfl. (Fabaceae) seeds, an antidiabetic egyptian folk medicine. Pharmazie. 1998; 53(10): 710-715.
[9] Garjani AR, Fatahiazad F, Zakher A, Allaf Akbari N, Azarmie YA. The effect of total extract of Securigera securidaca L. seeds on serum lipid profiles, antioxidant status, and vascular function in hypercholesterolemic rats. J Ethnopharmacol. 2009; 126(3): 525-532.
[10] Mard SA, Bahari Z, Eshaghi N, Farbood Y. Antiulcerogenic effect of Securigera securidaca L. seed extract on various experimental gastric ulcer models in rats. Pakistan J Biol Sci. 2008; 11(23): 2619-2623.
[11] Komissarenko AN, Kovalev VN. Hydroxy coumarins and flavones of Securigera securidaca. Chem Nat Compd. 1987; 23(2): 252.
[12] Zamula VV, Maksyutina NP, Kolesnikov DG. Cardenolides of Securigera securidaca. Khim Prir Soedin. 1965; 1(3): 153-156.
[13] Vanisree M, Lee CY, Lo SF, Nalawade SM, Lin CY, Tsay HS. Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Bot Bull Acad Sinica. 2004; 45(1): 1-22.
[14] Solich P, Sedliakova V, Karlicek R. Spectrophotometric determination of cardiac glycosides by flow-injection analysis. Anal Chim Acta. 1992; 269(2): 199-203.
[15] Tofighi Z. Phytochemical investigation of Securigera securidaca (L.) Deg. & Dorf. Ph.D. thesis. Facaulty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, 2013.
[16] Singleton VL, Orthofer R. Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent.  Method Enzymol. 1999; 299(1): 152-178.
[17] Mraz M, Opletal L, Sovova M, Drasar P, Havel M. Inhibition of Na+, K+-ATPase by the glycosides from Coronilla varia. Planta Med. 1992; 58(5): 467-468.
[18] Viqar Uddin A, Anwer B. Spectroscopic data of steroid glycosides: cardenolides and pregnans. USA: Springer, 2006.
[19] Eisenbeib M, Kreis W, Reinhard E. Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures. Plant Physiol Bioch. 1999; 37(1): 13-23.
[20] Hagimori M, Matsumoto T, Obi Y. Studies on the production of Digitalis cardenolides by plant tissue culture. III Effects of nutrients on digitoxin formation by shoot-forming cultures of Digitalis purpurea L. grown in liquid media. Plant Cell Physiol. 1982; 23(7): 1205-1211.
[21] Ohlsson AB, Bjork L, Gatenbeck S. Effect of light on cardenolide production by Digitalis lanata tissue cultures. Phytochemistry. 1983; 22(11): 2447-2450.
[22] Sahin G, Verma SK, Gurel E. Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey. Plant Physiol Bioch. 2013; 73: 139-143.
[23] El-Beltagi HS, Ahmed OK, El-Desouky W. Effect of low doses γ-irradiation on oxidative stress and secondary metabolites production of rosemary (Rosmarinus officinalis L.) callus culture. Radiat Phys Chem. 2011; 80(9): 968-976.
[24] Abohatem M, Zouine J, El Hadrami I. Low concentrations of BAP and high rate of subcultures improve the establishment and multiplication of somatic embryos in date palm suspension cultures by limiting oxidative browning associated with high levels of total phenols and peroxidase activities. Sci Hortic-Amsterdam. 2011; 130(1): 344-348.
[25] Lim FL, Yamb MF, Asmawi MZ, Chan LK. Elicitation of orthosiphon stamineus cell suspension culture for enhancement of phenolic compounds biosynthesis and antioxidant activity. Ind Crop Prod. 2013; 50: 436-442.
[26] Cai Z, Kastell A, Speiser C, Smetanska I. Enhanced resveratrol production in Vitis vinifera cell suspension cultures by heavy metals without loss of cell viability. Appl Biochem Biotech. 2013; 171(2): 330-340.
[27] Tofighi Z, Asgharian P, Goodarzi S, Hadjiakhoondi  A, Ostad SN, Yassa N. Potent cytotoxic flavonoids from Iranian Securigera securidaca. Med Chem Res. 2014; 23(4): 1718-1724.