Enhanced Accumulation of Betulinic Acid in Transgenic Hairy Roots of Senna obtusifolia Growing in the Sprinkle Bioreactor and Evaluation of Their Biological Properties in Various Biological Models

Abstract

Betulinic acid, which is found in transgenic roots of Senna obtusifolia (L.) H.S.Irwin & Barneby, is a pentacyclic triterpene with distinctive pharmacological activities. In this study, we report the differences in the content of betulinic acid and selected anthraquinones in transgenic S. obtusifolia hairy roots with overexpression of the PgSS1 gene (SOPSS2 line) and in transformed hairy roots without this genetic construct (SOA41 line). Both hairy root lines grew in 10 L sprinkle bioreactor. Additionally, the extracts obtained from this plant material were used for biological tests. Our results demonstrated that the SOPSS2 hairy root cultures from the bioreactor showed an increase in the content of betulinic acid (38.125 mg/g DW), compared to the SOA41 hairy root line (4.213 mg/g DW). Biological studies have shown a cytotoxic and antiproliferative effect on U‐87MG glioblastoma cells, and altering the level of apoptotic proteins (Bax, p53, Puma and Noxa). Antimicrobial properties were demonstrated for both tested extracts, with a stronger effect of SOPSS2 extract. Moreover, both extracts showed moderate antiviral properties on norovirus surrogates.

Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots

“…Hairy roots were grown in the sprinkle bioreactor as previously described [ 17 ]. In this study, we attempted to increase the content of betulinic acid by applying MeJA on a hairy root culture.…”

“…Preparation of plant extracts for analyses was carried out according to the protocol presented previously [ 17 ]. Briefly, the extracts of transgenic hairy roots grown in a sprinkle bioreactor with MeJA were prepared as follows: A total of 20 g of dry root mass was extracted for 15 min with 80% ( v / v ) aqueous methanol (1000 mL) at 35 °C.…”

“…In the following step, the cells were incubated with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at 37 °C for 1.5 h. After this time, MTT was carefully removed and DMSO was added to each well and vortexed at low speed for 5 min to fully dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference at 630 nm using a Bio-Tek Synergy HT Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA), according to our previous studies [ 17 ].…”

“…Hao et al [ 16 ] showed that methyl jasmonate (MeJa), in combination with salicylic acid (SA), can significantly affect the accumulation of tanshinone and the expression of biosynthetic genes in hairy roots, overexpressing the geranylgeranyl diphosphate synthase overexpression line ( SmGGPPS ) in Salvia miltiorrhiza. The results of our previous work have shown that a combination of many strategies, such as the genetic manipulation and cultivation of hairy roots in a bioreactor, can lead to a significant increase in valuable secondary metabolites in Senna obtusifolia hairy root cultures [ 17 ]. This valuable plant is widely used in traditional Chinese medicine to prevent and treat diabetes, hyperlipidemia, hypertension, photophobia, headache, dizziness, liver and Alzheimer’s disease [ 18 , 19 ].…”

“…Additionally, an extract from these roots grown in the bioreactor has been shown to have a cytotoxic and antiproliferative effect on U87MG glioblastoma cells and to alter the level of selected apoptotic proteins. Antimicrobial and moderate antiviral properties have also been demonstrated for this extract [ 17 ].…”