Skip to main content
SpringerLink
Log in

HPLC and GC analyses of in vitro-grown leaves of the cancer bush Lessertia (Sutherlandia) frutescens L. reveal higher yields of bioactive compounds

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Lessertia frutescens L., commonly known as cancer-bush, is a medicinally reputed plant species indigenous to southern Africa. Field leaf extracts of this species are known to exhibit many curative properties. However, little is known about the bioactive compounds that are present in in vitro leaf extracts and seed extracts. The objective of this study was to verify the presence of and quantify l-canavanine, gamma amino butyric acid (GABA), arginine and d-pinitol in the seeds, field leaves and in vitro leaves of L. frutescens using gas and liquid chromatography. Methanolic extracts of in vitro leaves, field leaves and seeds were used. MRM chromatograms were recorded for l-canavanine and arginine using tandem mass spectrometry. GC chromatograms were recorded for GABA and d-pinitol using gas chromatography. d-Pinitol was found to be most abundant and was 14.75 and 18.17 mg/g in in vitro and field leaf extracts respectively, followed by GABA (7.29 and 3.48 mg/g), arginine (7.08 and 0.35 mg/g) and l-canavanine (0.55 and 0.08 mg/g). In the seed extracts, GABA content was found to be the highest (1.69 mg/g) followed by l-canavanine (0.37 mg/g), then d-pinitol (0.25 mg/g), and arginine (0.02 mg/g). In vitro leaves had higher quantities of all compounds, except for d-pinitol. This study therefore highlights the potential of bulking in vitro leaves for the extraction of the medicinal compounds, l-canavanine and GABA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Bell EA (2001) Ecological biochemistry and its development. Phytochemistry 56:223–227

    Article  PubMed  CAS  Google Scholar 

  • Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839–851

    Article  CAS  Google Scholar 

  • Chadwick WA, Roux S, van de Venter M, Louw J, Oelofsen W (2007) Antidiabetic effects of Sutherlandia frutescens in Wistar rats fed a diabetogenic diet. J Ethnopharmacol 109:121–127

    Article  PubMed  Google Scholar 

  • Cheng L, Xu Y, Grotewold E, Jin Z, Wu F, Fu C, Zhao D (2007) Characterization of anthocyanidin synthase (ANS) gene and anthocyanidin in rare medicinal plant-Saussurea medusa. Plant Cell Tissue Organ Cult 89:63–73

    Article  CAS  Google Scholar 

  • Colling J, Stander MA, Makunga NP (2010) Nitrogen supply and abiotic stress influence canavanine synthesis and the productivity of in vitro regenerated Sutherlandia frutescens microshoots. J Plant Physiol 167:1521–1524

    Article  PubMed  CAS  Google Scholar 

  • Dias LLC, Santa-Catarina C, Ribeiro DM, Barros RS, Floh EIS, Otoni WC (2009) Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor. Plant Cell Tissue Organ Cult 99:199–208

    Article  CAS  Google Scholar 

  • Ekanayake S, Skog K, Asp NG (2007) Canavanine content in sword beans (Canavalia gladiata): analysis and effect of processing. Food Chem Toxicol 45:797–803

    Article  PubMed  CAS  Google Scholar 

  • Fait A, Fromm H, Walter D, Galili G, Fernie AR (2007) Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci 13:14–19

    Article  PubMed  Google Scholar 

  • Fu X, Li XC, Smillie TJ, Carvalho P, Mabusela W, Syce J, Johnson Q, Folk W, Avery MA, Khan IA (2008) Cycloartane glycosides from Sutherlandia frutescens. J Nat Prod 71:1749–1753

    Article  PubMed  CAS  Google Scholar 

  • Ghias-Ud-Din M, Smith AE, Phillips DV (1981) Separation of pinitol and some other cyclitols by high-performance liquid chromatography. J Chromatogr 211:295–298

    Article  CAS  Google Scholar 

  • Gomes CI, Obendorf RL, Horbowicz M (2005) Myo-inositol, d-chiro-inositol, and d-pinitol synthesis, transport, and galactoside formation in soybean explants. Crop Sci 45:1312–1319

    Article  CAS  Google Scholar 

  • Hartmann T (2007) From waste products to ecochemicals: fifty years research of plant secondary metabolism. Phytochemistry 68:2831–2846

    Article  PubMed  CAS  Google Scholar 

  • Hartnett SM, Oosthuizen V, van de Venter M (2005) Anti-HIV activities of organic and aqueous extracts of Sutherlandia frutescens and Lobostemon trigonus. J Ethnopharmacol 96:113–119

    Article  Google Scholar 

  • Horbowicz M, Obendorf RL (1994) Seed desiccation tolerance and storability: dependence on flatulence-producing oligosaccahrides and cyclitols—review and survey. Seed Sci Res 4:385–405

    Article  CAS  Google Scholar 

  • Hrabák A, Bajor T, Temesi A (1994) Comparison of substrate and inhibitor specificity of arginase and nitric oxide (NO) synthase for arginine analogues and related compounds in murine and rat macrophages. Biochem Biophys Res Commun 198:206–212

    Article  PubMed  Google Scholar 

  • Jain R, Sinha A, Jain D, Kachhwaha S, Kothari, SL (2010) Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal. Plant Cell Tissue Organ Cult. doi:10.1007/s11240-010-9840-3

  • Kavanaugh D, Berge MA, Rosenthal GG (1990) A higher plant enzyme exhibiting broad acceptance of stereoisomers. Plant Physiol 94:67–70

    Article  PubMed  CAS  Google Scholar 

  • Kuo TM, Lowell CA, Nelsen TC (1997) Occurrence of pinitol in developing soybean seed tissues. Phytochemistry 45:29–35

    Article  CAS  Google Scholar 

  • Kuo YH, Rozan P, Lambein F, Frias J, Vidal-Valverde C (2004) Effects of different germination conditions on the contents of free protein and non-protein amino acids of commercial legumes. Food Chem 86:537–545

    Article  CAS  Google Scholar 

  • Li X, Atkinson RN, King SB (2001) Preparation and evaluation of new l-canavanine derivatives as nitric oxide synthase inhibitors. Tetrahedron 57:6557–6565

    Article  CAS  Google Scholar 

  • McManus MT, Bieleski RL, Caradus JR, Barker DJ (2000) Pinitol accumulation in mature leaves of white clover in response to a water deficit. Environ Exp Bot 43:11–18

    Article  CAS  Google Scholar 

  • Moshe D (1998) A biosystematic study of the genus Sutherlandia R.Br. (Fabaceae, Galgeae). M.Sc. Thesis (Botany), University of Johannesburg

  • Mostafa SE, Karam NS, Shibi RA, Alali FQ (2010) Micropropagation and production of arbutin in oriental strawberry tree (Arbutus andrachne L.). Plant Cell Tissue Organ Cult 103:111–121

    Article  Google Scholar 

  • Murakeözy EP, Smirnoff N, Nagy Z, Tuba Z (2002) Seasonal accumulation pattern of pinitol and other carbohydrates in Limonium gmelini subsp. hungarica. J Plant Physiol 159:485–490

    Article  Google Scholar 

  • Natelson S, Bratton GR (1984) Canavanine assay of some alfalfa varieties (Medicago sativa) by fluorescence: practical procedure for canavanine preparation. Microchem J 29:26–43

    Article  CAS  Google Scholar 

  • Ojewole JAO (2008) Anticonvulsant property of Sutherlandia frutescens R.BR. (variety incana E.MEY.) [Fabaceae] shoot aqueous extract. Brain Res Bull 75:126–132

    Article  PubMed  CAS  Google Scholar 

  • Pellati F, Benvenuti S (2008) Determination of ephedrine alkaloids in Ephedra natural products using HPLC on a pentafluorophenylpropyl stationary phase. Journal of Pharmaceut Biomed Anal 48:254–263

    Article  CAS  Google Scholar 

  • Phillipson JD (2007) Phytochemistry and pharmacognosy. Phytochemistry 68:2960–2972

    Article  PubMed  CAS  Google Scholar 

  • Prevoo D, Swart P, Swart AC (2008) The influence of Sutherlandia frutescens on adrenal steroidogenic cytochrome P450 enzymes. J Ethnopharmacol 118:118–126

    Article  PubMed  CAS  Google Scholar 

  • Rathore MS, Shekhawat NS (2009) Micropropagation of Pueraria tuberose (Roxb. Ex Willd.) and determination of puerarin content in different tissues. Plant Cell Tissue Organ Cult 99:327–334

    Article  CAS  Google Scholar 

  • Reid KA, Maes J, van Staden J, De Kimpe N, Mulholland DA, Verschaeve L (2006) Evaluation of the mutagenic and antimutagenic effects of South African plants. J Ethnopharmacol 106:44–50

    Article  PubMed  CAS  Google Scholar 

  • Shaik S, Dewir YH, Singh N, Nicholas A (2010) Micropropagation and bioreactor studies of the medicinally important plant Lessertia (Sutherlandia) frutescens L. S Afr J Bot 76:180–186

    Article  Google Scholar 

  • Shelp BJ, Bown AW, McLean MD (1999) Metabolism and functions of gamma-aminobutyric acid. Trends Plant Sci 4:446–452

    Article  PubMed  Google Scholar 

  • Shqueir AA, Brown DL, Klasing KC (1989) Canavanine content and toxicity of Sesbania leaf meal for growing chicks. Anim Feed Sci Technol 25:137–147

    Article  CAS  Google Scholar 

  • Sridhar KR, Seena S (2006) Nutritional and antinutritional significane of four unconventional legumes of the genus Canavalia—a comparative study. Food Chem 99:267–288

    Article  CAS  Google Scholar 

  • Stander BA, Marais S, Steynberg TJ, Theron D, Joubert F, Albrecht C, Joubert AM (2007) Influence of Sutherlandia frutescens extracts on cell numbers, morphology and gene expression in MCF-7 cells. J Ethnopharmacol 112:312–318

    Article  PubMed  CAS  Google Scholar 

  • Swaffer DS, Ang CY, Desai PB, Rosenthal GA (1994) Inhibition of the growth of human pancreatic cancer cells by the arginine antimetabolite l-canavanine. Cancer Res 54:6045–6048

    Google Scholar 

  • Tai J, Cheung S, Chan E, Hasman D (2004) In vitro culture studies of Sutherlandia frutescens on human tumor cell lines. J Ethnopharmacol 93:9–19

    Article  PubMed  Google Scholar 

  • Udedibie ABI, Carlini CR (1998) Questions and answers to edibility problem of the Canavalia ensiformis seeds—a review. Anim Feed Sci Technol 74:95–106

    Article  CAS  Google Scholar 

  • van Wyk BE, Albrecht C (2008) A review of the taxonomy, ethnobotany, chemistry and pharmacology of Sutherlandia frutescens (Fabaceae). J Ethnopharmacol 119:620–629

    Article  PubMed  Google Scholar 

  • Vermani K, Garg S (2002) Herbal medicines for sexually transmitted diseases and AIDS. J Ethnopharmacol 80:46–66

    Article  Google Scholar 

  • Wahid A, Gelani S, Ashraf M, Foolad MR (2007) Heat tolerance in plants: an overview. Environ Exp Bot 61:199–223

    Article  Google Scholar 

  • Wink M (2003) Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 64:3–19

    Article  PubMed  CAS  Google Scholar 

  • Xaba PM, Notten A (2003) Sutherlandia frutescens. South African National Biodiversity Institute (SANBI). Available online http://www.plantzafrica.com/plantqrs/sutherfrut.htm. Accessed 12 Feb 2007

  • Yazaki K (2006) ABC transporters involved in the transport of plant secondary metabolites. FEBS Lett 580:1183–1191

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Research funds were made available by the University of KwaZulu-Natal and the National Research Foundation.

Author information

Authors and Affiliations

  1. School of Biological and Conservation Sciences, University of KwaZulu-Natal, Westville Campus, Private bag X54001, Durban, 4000, South Africa

    Shakira Shaik, Nisha Singh & Ashley Nicholas

Authors
  1. Shakira Shaik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nisha Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ashley Nicholas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nisha Singh.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shaik, S., Singh, N. & Nicholas, A. HPLC and GC analyses of in vitro-grown leaves of the cancer bush Lessertia (Sutherlandia) frutescens L. reveal higher yields of bioactive compounds. Plant Cell Tiss Organ Cult 105, 431–438 (2011). https://doi.org/10.1007/s11240-010-9884-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-010-9884-4

Keywords

Search

Navigation