Development of a natural product library using hyphenated techniques and evaluation of its constituents as potential anti-cancer agents

Abstract

Worldwide, cancer is among the most common public health issues with an estimated death toll in 2020 of close to 10 million individuals. Although cancer treatments are continuously improving as the use of personalised medicinal treatments have shown great potential, the incident rate of cancer is predicted to exponentially increase over the next 10 years. Cancer treatment is still a reductionist approach since cancer is a variety disease at the cellular level, which makes treatment even more challenging. The rising incidence of cancer worldwide and the reductionist approach in treatments emphasises the need for new and more efficient cancer treatments. The use of natural products in medicinal practices is one of mankind’s oldest traditions. Since 1982, The FDA has approved over 809 natural products for the treatment and management of communicable and noncommunicable diseases like HIV, bacterial infections and cancers. Natural products still offer novel and chemically diverse compounds that display greater drug-likeness behaviour than synthetic compounds. In the past, natural products have been exceptionally successful in drug discovery which proves their potential for novel therapeutic agents against variety diseases like cancer. South Africa’s biodiversity is potentially one of the world’s richest and untapped sources for novel natural products. In 2018, it was estimated that South Africa contained over 67 000 animal and 20 401 plant species. Additionally, South Africa has one of the richest multicultural heritages in the world that has established an impressive knowledge on the use of indigenous medicinal plants.

The work completed in this study has created a trusted South African natural product library that is ready for modern HTS against various diseases. In this project, the robust procedures developed for the standardisation of plant extracts and fractions were miniaturised from the high-throughput methods of pre-existing natural product library platforms, primarily the NCI NPNPD project. A validation study that used a paclitaxel spiked extract of Brassica oleracea (cabbage) was also conducted to ensure the integrity of plant samples after the plant preparative methods. A second extract of Brassica oleracea was used as a negative control and did not contain paclitaxel. The UPLC-PDA-HRMS analysis of the spiked fractions indicated that paclitaxel was concentrated in fraction 6 and was quantified at 0.1409 mg/mL in the final standardised fraction. The recovery of the spiked paclitaxel was calculated to be 6.23 %, which indicated that the preparation of the spiked extract for fractionation was ineffective. The spiked extract and respective fractions were screened against the human lung cancer cell line A549 where the presence of other interfering compounds resulted in a low cell proliferation inhibition for fraction 6 of 31.43 %. Irrespective of the low activity, it was determined that the plant preparative methods did not degrade potential bioactive compounds nor induce false positives as later in the project the presence of a few extremely bioactive fractions were observed.

Additionally, from a subset of selected medicinal plant species in the South African natural product library at University of Pretoria was evaluated for potential anti-cancer compounds. For plant species selection an ethnopharmacological preferential scoring system was developed that preferentially scored the South African medicinal plants most likely to succeed based on their traditional usage. From a final list of the top 100 preferentially scored medicinal plant species, 34 (representing 21 plant families) were standardised and high-throughput screened against the human cancer cell lines A549, MCF7 and Caco2. The ultrasonic bath extraction method developed produced sufficient high-quality extracts for downstream standardisation from ± 7.0 g of plant material. Of the 34 plant extracts produced, only one required a second extraction. The automated plant extract fractionation method developed by the NCI NPNPD project was used to fractionate 34 plant extracts on C8 SPE cartridges using a single needle Gilson GX-241 ASPEC® liquid handler. Each plant extract was fractionated into 7 unique fractions of decreasing polarity which resulted in the total production of 272 unique fractions. The average mass recovery after fractionation was 46.75 % but a slight revision in the preparation of the plant extracts before fractionation will improve the recovery. The standardisation of the plant extracts and respective fractions followed a multi-step process using a Hamilton Microlab® STARlet™ automated liquid handler. The STARlet™ automated liquid handler successfully standardised more than 97 % of the plant extracts and fractions at 5 mg/mL in DMSO. For the few plant extracts and fractions that had insufficient mass to be standardised, their respective concentrations in 100 µL DMSO were recorded appropriately. All the standardised extracts and fractions were stored in airtight screw capped pre-barcoded polypropylene vials at -20 °C under dry atmospheric conditions inside a Hamilton Verso® Q20 robotic freezer. The unique internal barcoding format and database structure developed in this project was able to successfully catalogue and correlate all the information relating to the preparation, standardisation and storage of each standardised extract and fraction.

It was noted that only 3 standardised fractions showed excellent cell proliferation inhibition above 80 % against the cell line A549 at 50 μg/mL. It was noted that the 3 standardised fractions with potent activity were from fraction numbers 3 and/or 4. At the screening concentration 25 µg/mL, only 7 and 3 standardised fractions showed above 90 % cell proliferation inhibition against the cell lines MCF7 and Caco2, respectively. The potent fractions were concentrated in the fractions number 2 and 4. The benefit of fractionating the plant extracts on a C8 SPE cartridge pre-HTS was demonstrated as most active fractions demonstrated better activity than their respective crude extracts. It was demonstrated that in a time and cost-efficient manner, the HTS of a small subset of standardised fractionated plant extracts from the South African natural product library has rapidly identified numerous “hits” for the investigation of potentially novel anti-cancer compounds.

Using the in vitro HTS results of the South African natural product library, a single hit fraction from Mikania natalensis was prioritised for post-HTS investigation. At the time of investigation there was no reported phytochemical analysis or biological activity on M. natalensis which made it attractive for potentially novel anti-cancer compounds. The dereplication of the hit fraction used modern UPLC-PDA-HRMS analysis and online databases such as ChemSpider, PubChem, Reaxys and the Dictionary of Natural Products which quickly lead to the tentative identification of a single unique chromatographic peak as mikanin 3-O-sulphate, named M3S. The easy upscaling of the plant preparative methods allowed for the rapid isolation of the biologically active compound. Approximately 18.26 g of dried leaf material from M. natalensis was used to produce 2.30 g of an upscaled MeOH/DCM crude extract. Only 0.745 g of the extract was used in upscaled extract fractionation which produced 24.66 mg of the hit fraction for isolation purposes. The 24.66 mg of the hit fraction was prepared in 1.0 mL ACN/H2O and injected onto the semi-preparative HPLC-PDA-MS. By using mass-directed isolation on the HPLC-PDA-MS, 0.98 mg of the tentatively identified compound M3S was isolated. The identity of M3S was further verified by structural confirmation using both 1D (1H and 13C) and 2D (HSQC and HMBC) NMR experiments in combination with ACDLabs™ Spectrus Processor. The isolated M3S was rescreened and the IC50 concentrations against the cancer cell lines MCF7 and Caco2 were 146.2 µM and 65.36 µM, respectively. The presence dihydromikanolide in the leaf extract of M. natalensis was also confirmed by SCXRD analysis of a single crystal from the dried fraction 5 of the upscaled extract fractionation of M. natalensis. The absolute configuration of dihydromikanolide was reported to be less uncertain than current literature. This was the first report of dihydromikanolide and M3S, that has shown encouraging anti-cancer activity against the cancer cell lines MCF7 and Caco2, present in the leaf extract of M. natalensis.