Phytochemical and Biological Studies of Helichrysum acutatum DC

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INTRODUCTION
Plants are a storehouse for different therapeutic molecules and have played a significant and crucial role in modern drug discovery and development. The search for safe, effective, and affordable health care has made many trusts traditional medicines. People believe these medicines are relatively safe for use from a natural source. Also, the search for new molecular compounds in treating and managing diseases has resulted in drug leads from natural products. Natural products account for more than 50% of modern drugs used clinically, with some possessing the ability to inhibit cancer cells. 1 Oxidative stress has been implicated in degenerative diseases such as Parkinson's, Alzheimer's, and cancer. Oxidative stress occurs when there is a shift in the production and removal of reactive oxygen species (ROS), favouring the production rather than the deduction. The National Cancer Institute (NCI) 2 defined ROS as unstable molecules containing oxygen that quickly react with other molecules in the cell. They are free radicals and could be referred to as oxygen radicals. Examples include peroxide, superoxide, hydroxyl radicals, and singlet oxygen species. 3 The build-up of ROS in the cell may cause damage to the DNA, RNA, proteins, and even cell death. The body's imbalance of antioxidants and free radicals leads to oxidative stress. Antioxidants react with free radicals in the body and terminate their chain reaction.
There are approximately 600 species in the genus Helichrysum (Asteraceae). Compounds including chalcones, diterpenes, phloroglucinol, and its derivatives have been isolated from the aerial parts and roots of some Helichrysum species. 4 Traditionally, some species have found use in treating diarrhoea, wounds, colds, coughs, and respiratory tract infections. 5 Essential oils from Helichrysum species have been used, with the most widely used essential oil being from Helichrysum italicum (Roth) G. Don. 6 Helichrysum acutatum DC. is a perennial woody herb with a flowering stem that grows in grasslands; Hillard (1983) 7 classified it morphologically into group 21. Other plants in this group are H. dasymallum Hilliard and H. oreophylum Klatt. Although H. acutatum DC is widely sold in the muthi market, there is no literature on its ethnopharmacology relevance. The locals in the market provided anecdotal information on its use in traditional medicine as an enema for newborns. Bohlmann and Abraham (1979) 8 investigated the plant's roots and isolated fourteen compounds, including chalcones and diterpenes. In the current study, the roots of Helichrysum acutatum, widely sold at the muthi market in Durban, were investigated to establish a rationale for its traditional use by the locals as no biological studies have previously been done on the plant.

Plant material
Plant material (root) of H. acutatum was purchased from Berea muthi market. The taxonomist in the School of Life Sciences, UKZN, authenticated the sample, and a voucher specimen (18271 01 900600) was deposited in the ward herbarium. The plant was air-dried and then crushed with a metal mortar and pestle to a smaller fragment for extraction.

General experimental procedures
Infra-red spectra were obtained using Perkin Elmer Spectrum 100 FT-IR spectrometer with universal ATR sampling. NMR spectra ( 1 H, 13 C, and 2D) were recorded on Bruker Avance III 400Hz spectrometer, using deuterated chloroform, methanol or DMSO at room temperature, with TMS as the internal standard. Column chromatography was carried out using Merck silica gel 60 (0.040-00063mm) as a stationary phase and solvents of different polarities as a mobile phase. The separated fractions were examined using Merck 20 cm × 20 cm silica gel 60 F254 aluminium sheets for TLC. The TLC plates were first visualised under UV (254 and 366 nm) before spraying with 10% H 2 SO 4 in methanol (MeOH) solution, followed by heating for the second visualisation. High-resolution mass spectra (HR-MS) were recorded on the Waters Micromass LCT Premier TOF-MS instrument. All reagents were of analytical grade and were sourced from either Merck (Darmstadt, Germany) or Sigma (St. Louis, USA) chemical companies.

Extraction and isolation
The roots of H. acutatum (1.3 kg) were oven-dried, crushed, and extracted sequentially with organic solvents of varying polarities, starting with the least polar to the most polar solvent in the order; DCM, EtOAc, and MeOH with the aid of a mechanical shaker. Each solvent was filtered and concentrated under reduced pressure using a rotary evaporator to give 5.4g of DCM, 4.5 g of EtOAc, and 20 g of MeOH extracts.
The DCM extract was subjected to separation on a silica gel column, with hexane and EtOAc as solvents using gradient elution to give 50 fractions of 100 mL monitored on TLC. Fraction 18 gave compound 1 (20 mg) as white flakes. The MeOH extract was purified on a packed silica gel column using hexane: EtOAc as the mobile phase, like the DCM extract. Fraction 20 gave compound 1, and fractions 40-45 were purified by washing with MeOH to give compound 2 (5 mg), an offwhite powder. The EtOAc extract was fractionated on silica gel and eluted sequentially with hexane and EtOAc, starting from 100% hexane that was stepped by 10% to 100% EtOAc. A total of 40 fractions of 100 mL were obtained. Fractions 26-28 gave compound 3 (15 mg), a orange powder.

Antioxidant activity
The DPPH radical scavenging ability and the ferric reducing antioxidant power (FRAP) were determined using established methods. 9,10 Butylated hydroxytoluene (BHT) and ascorbic acid served as positive controls. All experiments were carried out in triplicate.

Antibacterial susceptibility test
Three Gram-positive indicator bacteria, Bacillus subtilis ATCC 6653, methicillin-resistant Staphylococcus aureus ATCC 43000 and Mycobacterium smegmatis mc 2 155 and four Gram-negative indicator bacteria, beta-lactam-resistant Escherichia coli ATCC 35218, multidrugresistant Pseudomonas aeruginosa ATCC 27853, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae ATCC 700603 and the quorum sensing indicator Chromobacterium violaceum, were employed to evaluate the antibacterial activity. Three crude extracts of H. acutatum and two pure compounds were subjected to antibacterial screening using the agar well diffusion method. 11 The test samples were dissolved in MeOH to a final concentration of 20 mg/mL for the crude extracts and 10 mg/mL for the compounds.

Cell culture
Cryopreserved cells were rapidly thawed in the incubator and centrifuged. The cell pellet was recovered and propagated to 100% confluency in a 25 mL tissue culture flask with the addition of CCM (complete culture medium), which consists of 500 mL DMEM, supplemented with 1% L-glutamine, 1% penicillin-streptomycinfungizone and 10% FCS, in a humid environment (5% CO 2 , 37 0 C). The media was removed, and the cells were washed thrice with PBS. Trypsin was added to the flasks with Caco-2 and HepG2 cells to remove those that had adhered to the flasks. The flask with Hek-293 was agitated to remove the cells that had attached to it. The cells were resuspended in 2 mL for CCM and counted using the trypan blue method.

Sample preparation
Stock solutions of crude extracts were prepared by dissolving in DMSO and diluted with CCM to a concentration of 10 mg/ml, and eight different concentrations (0-5000 µg/mL) for the MTT assay were prepared from the stock. The final concentration of DMSO in each stock was less than 0.5%.

MTT assay
The viability of Caco-2, HepG2, and Hek-293 cells after exposure to varying concentrations of test samples for 24 h was evaluated using the MTT (tetrazolium salt reduction) assay. 2 × 10 5 cells in CCM were seeded in 96-well microplates and incubated at 37 0 C in 5% CO 2 overnight for adherence of cells to the plate. The medium was removed, and 100 µL of test samples prepared in CCM at varying concentrations were added to each well. The wells receiving only media served as the control. Treatment was done in triplicate for each test sample at 24 h. After 24 h, the medium was removed, 20 µL of MTT salt in CCM at a concentration of 5 mg/mL, and 100 µL PBS was added and incubated for 4 h at 37 0 C. After that, the MTT solution was removed, and the formazan crystals formed solubilised with 100 µL DMSO. The cell growth inhibition by tested samples was measured using a Bio-Tek µQuant plate spectrophotometer (Winooski, Vermont, United States) at 570 nm. Results were presented as percentage cell viability.

Analysis of mitochondrial membrane potential (MMP)
An increase in depolarization of the mitochondrial membrane with a subsequent decrease in mitochondrial membrane potential could activate pro-apoptotic factors. JC-10, a water-soluble dye, was used to probe the mitochondrial membrane potential in this assay. Cells were seeded at 2 × 10 4 into each well and allowed to adhere to the plate. IC 80 and IC 50 concentrations were prepared from the stock and added to each well. After 24 h, treatment media was removed and stored for other assays. Each well received 25 µL of JC-10 dye and 50 µL PBS before incubation at 37 0 C for 1 h in the dark. After 60 min of incubation, the JC-10/PBS solution was removed, and 80 µL PBS was added before the plates were read.

ATP quantification
The intracellular ATP levels were monitored using a CellTiter-Glo ® reagent (Promega) prepared according to the manufacturer's instructions. After taking the reading of the plate used for the mitochondrial membrane potential assay, 50 µL of PBS was added to each well, followed by 25 µL of CellTitre-Glo ® reagent. Next, a luminescent reading was done on a Modulus TM microplate luminometer (Turner Bio-Systems, California, USA).

LDH release assay
LDH is a stable cytoplasmic enzyme found in the plasma of living cells. Release of LDH from the cytoplasm to the surrounding cell culture due to loss of the plasma-wall integrity can be used to quantify cell viability and necrosis in-vitro. 50 µL of the treatment media from the mitochondrial membrane potential assay was pipetted into a 96-well plate. Thereafter, 25 µL of the assay buffer (iodonitrotetrazolium (INT) chloride, nicotinamide adenine dinucleotide (NAD) sodium salt, and lithium lactate) was added. Plates were incubated at room temperature in the dark for 30 min. The reaction was stopped by adding a 12.5 µL stop solution (acetic acid). LDH was quantified by measuring the absorbance at 500 nm using a Biotek µQuant spectrophotometer (Winooski, Vermont, United States).

Statistical analysis
Data were exported to Microsoft Excel for analysis and processed on GraphPad Prism v5.0 (GraphPad Software Inc., San Diego, California, United States). All data were normalised to the untreated control, and the student's t-test was used to determine statistically significant differences (P < 0.05). All data were expressed as mean ± SD (n = 3).

Identification of isolated compounds
Three compounds were isolated and elucidated from the roots of H. actutatum (Figure 1). These include a sterol, a sterol glycoside, and a phenolic acid. The DCM and MeOH extract of the root yielded stigmasterol (compound 1). The spectral data compared well to that published in the literature for this compound. 12 The MeOH extract produced stigmasterol glucoside (compound 2), as confirmed by literature. 13 The EtOAc extract yielded caffeic acid (compound 3

Antibacterial activity
The plant extracts were tested at 0.5 and 1 mg against three Grampositive bacteria and four Gram-negative bacteria, while the two phytocompounds were tested at 0.25 and 0.5 mg due to the limited mass of phytocompounds. No antimicrobial activity was observed against all three Gram-positive and all four Gram-negative indicator organisms with extracts and isolated phytocompounds (Table S1). 16

Antioxidant activity
The roots' crude DCM, EtOAc, and MeOH extracts were subjected to antioxidant testing using DPPH ( Figure S1) and FRAP ( Figure S2). The radical scavenging ability was in the order of BHT > EtOAc > ascorbic acid > MeOH > DCM. For the FRAP assay, the activity of the reference standards swapped and the order of reducing potential was ascorbic acid > EtOAc > BHT > MeOH > DCM.    Cytotoxicity testing

MTT assay
The different crude extracts were exposed to the tumour cell lines (Caco-2 and HepG2) and the normal human kidney cell line, Hek-293, for 24 h, using the MTT assay to evaluate their cytotoxicity ( Figure S3). Treatment with 50. 12

LDH release assay
The quantification of LDH released was used to determine cytotoxicity and necrosis. IC 80 and IC 50 concentrations of the DCM extract ( Figure  S4) increased LDH released by 1. 27

Mitochondrial membrane potential (MMP) assay
Depolarising the mitochondria led to the release of pro-apoptotic proteins, probed using JC-10 dye. Exposure of the different cell lines to the IC 80, and IC 50 values of the DCM extract ( Figure S5)

ATP assay
The amount of intracellular energy (ATP) was used to quantify cell viability and mitochondrial function. ATP was increased by 1.14-fold in Caco-2 after exposure to the IC 80 value of the DCM extract ( Figure  S6) but decreased by 1.72-fold (P < 0.05) with the IC 50 treatment. For the same extract, a 1.20 and 1.67-fold decrease was observed in HepG2 with the IC 80 and IC 50 therapies, respectively, while a 3.25 and 1.79-fold increase (P < 0.05) was observed in Hek-293 cell lines. Treatment with IC 80 and IC 50 concentrations of the EtOAc extract increased ATP by 1.22-fold and decreased it by1.39-fold (P < 0.05) in Caco-2, respectively. In HepG2, a 1. 18

DISCUSSION
The phytochemical investigation of the roots of H. acutatum led to the isolation of a sterol (stigmasterol), 12 a sterol glucoside (stigmasterol glucoside) 13 and a phenolic compound (caffeic acid). 15 Diterpenes, chalcones and phloroglucinol have been reported from the roots and aerial parts of H. acutatum, but these three compounds have not previously been isolated from the plant. 7 Caffeic acid is naturally present in many fruits, and this compound and its derivatives have been known to possess antioxidant, anticancer and antibacterial activities. These activities are mainly attributed to the free phenolic acid that has high bioavailability and good water solubility, the position of the OHs in the catechol moiety and the double bond in the carbonic chain. 17 This study showed H. acutatum to have relatively good antioxidant activity compared to the standards. The potent antioxidant activity demonstrated by the EtOAc extract could be due to the presence of caffeic acid, which has been reported to be an excellent free radical scavenger. 18  Mitochondria have been known to play an essential role in maintaining cell health and could be used to monitor cell viability. 27 A decrease in the mitochondrial membrane potential has been reported to be the first step in apoptosis, and apoptosis has been reported to require an increase in energy. 28 In cytotoxicity evaluations, cells either undergo an apoptotic or necrotic death. 29 In necrosis, cells swell, lose membrane integrity, and release their intracellular content into the external environment. LDH, a soluble enzyme found in the cell cytoplasm, is released when the cell membrane is compromised. The amount of LDH released can be used to quantify cell death.
At the IC 80 concentrations, all three extracts showed an increase in LDH release, a decrease in the mitochondrial membrane potential and increased ATP levels in Caco-2 cell lines (Table 1). While at the IC 50 concentrations, increased LDH release, decreased mitochondrial membrane potential, and decreased ATP levels were observed for the same cell lines (Table 1). Generation of ATP takes place in the mitochondria, a disruption in mitochondrial membrane potential could lead to depletion of intracellular ATP, and this was the case after treatment of Caco-2 tumour cell lines with IC 50 concentrations of the tested extract. These changes are concentration-dependent, and depletion of cellular ATP has been shown to switch cell death from apoptosis to necrosis. Apoptosis is a programmed cell death that requires energy (ATP), while necrosis is accidental cell death that does not require energy. 29 The effect of concentration on the mode of cell death was observed when MCF-7 was treated with the aqueous extract of Lepidium sativum Linn. extracts: apoptosis was induced in the cell when treated with 25 and 50% of the extract, but at a higher concentration of 75%, necrosis was induced. 30 At both concentrations, the plasma membrane was disrupted, leading to the release of LDH in Caco-2 cell lines. The high increase in LDH release observed for some extracts indicates the extent of membrane damage, while those with slight increases in the release of LDH indicate minimal membrane damage (Table 1).
Exposure of HepG2 cell lines to the IC 80 and IC 50 concentrations of the different extracts caused no depolarisation in mitochondrial membrane potential, nor was the plasma membrane disrupted, but a depletion in ATP levels was observed. The cytotoxic activity of the extract towards HepG2 was through depletion of intracellular energy without effect on the plasma membrane.
The results show that the plant can initiate apoptotic and necrotic cell death, depending on the concentration and the cell lines. Some plant extracts that have decreased mitochondrial membrane potential with subsequent cytochrome C release for apoptotic pathway initiation are Murraya koenigii (L.) Spreng, Annona reticulate L., Moringa oleifera Lam., Hibiscus sabdariffa L., Lablab purpureus (L.), and Euphorbia hirta Linn. 31

CONCLUSION
Three compounds were successfully isolated from the roots of H. acutatum, and this study is the first report of these compounds from the plant. The findings show H. acutatum extracts to have better antioxidant activity than antibacterial and anticancer activity for the tested microbes and cell lines. This study has established a toxicity profile and scientific basis for using H. acutatum as an antioxidant in traditional medicine and confirms its safety for human consumption. The study also highlights the lack of antibacterial activity of the species compared to others in the genus, which are well known for their use in traditional medicine in treating different infectious diseases.