There is a global demand for natural ingredients from plant sources with multifunctional properties for application in the food and nutraceutical industries. In 2018, the market for herbal dietary supplements in the United States increased by 9.4% and the value of this market reached an estimated 8.842 billion dollars across all market channels, marking the strongest US sale growth of herbal supplements since 1998 [1
]. Similarly, there is a growing demand for foods and medicines that are known for their customary usage by Indigenous communities. These interests range from general desires for prevention from acute and chronic diseases, maintenance of good health, well-being, immune system strengthening and energy sustenance, to seeking out traditional foods/native plants that provide natural antioxidants and antimicrobials. There are many plant bioactive compounds of particular interest such as saponins, polyphenols including tannins and alkaloids, which are known to assist in providing the above benefits to human health and well-being.
(common name: Gumby Gumby) is a shrub tree, native to Australia. This species belongs to the genus Pittosporum and the family Pittosporaceae, consisting of approximately 200 species in nine genera [2
]. P. angustifolium
has been found mainly in inland Australia, New Zealand and many other parts of the world [2
]. Different botanical tissues of P. angustifolium
have been traditionally used as Indigenous bush medicine across inland Australia for hundreds of years to enhance general health and well-being. The infusions from leaves were used to treat cold and coughs and to induce lactation [3
]. Decoction made from the fruits was taken orally or applied to treat skin problems such as eczema and pruritus [2
]. In addition, P. angustifolium
has been traditionally used for treatment of rheumatoid arthritis and other inflammatory conditions [5
]. Recently, Madikizela and McGaw [6
] summarized information on traditional medicinal applications of the genus Pittosporum for treatment of a wide range of infections such as inflammatory, spasmodic, malarial and microbial infections (e.g., narcotics, chronic bronchitis, leprous infection, rheumatic, bruises, sciatica, chest infection and certain skin diseases). Interestingly, all parts of the Pittosporum plants, including leaf, bark, root, flower, fruit pulp, seed and even wood, have been reported to show potential medicinal applications in many countries such as Australia, China, India and South Africa [6
The increasing interest in drug discovery from native medicinal plants has led to many studies on extraction, identification and quantification of bioactive compounds in different species of Pittosporum genus, P. angustifolium
]. Several bioactive compounds have been identified in the crude extracts of P. angustifolium
such as triterpenoid saponins in leaves and seeds [7
], phenolic acids and flavonoids in leaves (Figure 1
], tannins and essential oils in leaves and fruits [11
]. Among them, triterpenoid saponins, essential oils and non-tannin polyphenols are reported as main bioactive compounds in the Pittosporum genus [6
], whereas tannins and alkaloids are minor compounds. There seems to be no alkaloids present in the leaves and fruits of P. angustifolium
Despite the wealth of available literature on bioactive compounds and their associated medicinal properties, there is still a gap in knowledge regarding potential effects of P. angustifolium bioactive compounds due to different growing conditions (wild vs. cultivated), diverse botanical tissues (leaf vs. stem) and geographical locations. Therefore, the present study aimed (i) to determine proximate composition, minerals and trace elements, bioactive compounds, antioxidant capacity and antimicrobial activity of Australian grown P. angustifolium as an initial measure of their nutritional value and bioactive potential and (ii) to assess the impact of different plant parts, growing conditions and geographic locations on bioactive compounds and associated bioactivities.
2. Materials and Methods
2.1. Plant Material
Approximately 5 kg of P. angustifolium (1 year-old) whole branch (length ≤ 30 cm; stem diameter < 5 mm) collected in 2018 from the field (Clermont, Queensland (QLD), Australia) were provided by Gumby Gumby Australia, Ltd. (Clermont, Australia). The cultivated sample was divided into 3 different parts: leaves, stems and the whole branch (without separation as used for traditional medicinal applications). The ratio between leaf and stem was 65–70:30–35 (w/w). The samples were freeze-dried at −50 °C for 48 h (CSK Climateck, CSK Scientific, Brisbane, Australia) and blended into a fine powder using a Waring Laboratory Blender (Australian Scientific, Australia).
The wild harvested sample, collected in 2018 from subtropical forest, included P. angustifolium whole branch from QLD (provided by Gumby Gumby Australia, Ltd.) and leaves from South Australia (SA) (supplied by Bush Food Australia, Ltd., Wilmington, Australia). After harvesting, the samples were air-dried indoors and blended into a fine powder (ca. 3 kg) as with the cultivated samples. The final (powdered) samples had a moisture content ≤5% and were stored at −35 °C for further analysis.
The following abbreviations were used to label the samples: QLD–Cul–WB, QLD–Cul–Leaf, QLD–Cul–Stem (cultivated P. angustifolium whole branch, leaf and stem samples collected in QLD); QLD–Wild–WB (P. angustifolium whole branch collected from the wild in QLD) and SA–Wild–Leaf (P. angustifolium leaves collected from the wild in SA).
Polyphenol and carotenoid standards (HPLC grade), including (+/−) -catechin, gallic acid, rutin, isoquercetin, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, lutein, zeaxanthin and trans-beta-carotene, were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), oleanolic acid and 1,4-dithiothreitol (DTT) were also from Sigma-Aldrich.
Pteroylmonoglutamic acid (PteGlu), tetrahydrofolate (H4folate), 5-methyltetrahydrofolate (5-CH3-H4folate), 5-formyltetrahydrofolate (5-CHO-H4folate), and their corresponding labeled isotopes were sourced from Merck Eprova (Schaffhausen, Switzerland).
Cultures of Staphylococcus aureus (strain 6571) and Escherichia coli (strain 9001) were obtained from the National Collection of Type Cultures (NCTC, Health Protection Agency Center for Infection, London, UK). Candida albicans (strain 90028) was sourced from the American Type Culture Collection (ATCC, In Vitro Technologies Pty, Ltd., Noble Park, Australia). Plate count agar and potato dextrose agar media (Thermo Fisher Scientific, Scoresby, Australia) were used to test antibacterial and antifungicidal activity.
2.3.1. Proximate Analysis
Proximate analysis was performed at Symbio Alliance Laboratories (Eight Mile Plains, Australia), a National Association of Testing Authorities (NATA) accredited laboratory that complies with ISO/IEC 17,025:2005. The following NATA accredited in-house or standard AOAC methods were used: protein (AOAC method 990.03, [15
]); total fat (AOAC method 991.36, [16
]); saturated, mono-unsaturated, poly-unsaturated and trans fatty acids by gas chromatography with flame-ionization detector (in-house method CFH068.2); dry matter (AOAC method 934.01, [17
]); ash content (AOAC method 942.05, [17
]); total sugar, total dietary fiber and available carbohydrate by high performance liquid chromatography equipped with refractive index detector (in-house methods CFH001.1, CF057 and CF029.1). Proximate analysis was performed in duplicate including measurement of uncertainty.
2.3.2. Mineral and Trace Element Analysis
Analysis of minerals (Ca, K, Mg, Na and P) was performed using inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 700, Agilent Technologies, Tokyo, Japan) after hot-block digestion. Analysis of trace elements was performed using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700) after microwave digestion. The analysis was carried out at the Forensic and Scientific Services, Queensland, a NATA accredited laboratory. Details of the method have been described previously by Akter et al. [18
2.3.3. Analysis of Polyphenols
Polyphenols were analyzed using a Waters AcquityTM
UPLC-PDA System (Waters, Rydalmere, Australia) with detailed chromatographic conditions summarized in Supplementary Table S1
. Peak identities were confirmed using a Thermo high resolution Q Exactive mass spectrometer equipped with electrospray ionization (ESI) source and a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific Pty, Ltd., Scoresby, Australia). A full MS scan in negative ion mode was acquired from m/z
120 to 1000 at a resolving power of 70,000 full-width at half maximum. For the compounds of interest, a MS/MS scan range of m/z
100–1000 was selected, with normalized collision energy (NCE) at 35 V. The compound identification was based on comparing retention time, UV-Vis spectra, mass spectra and fragmentation patterns with those obtained from available standards and/or literature. Polyphenols were quantified at 320 nm, using external calibration curves of different polyphenol standards as stated in Section 2.2
2.3.4. Analysis of Carotenoids
Carotenoids were analyzed using a Waters AcquityTM
UPLC-PDA system (Supplementary Table S1
). Detected carotenoid compounds were identified using the same UHPLC-MS/MS system as described for the polyphenol analysis (Section 2.3.3
) but employing an atmospheric pressure chemical ionization (APCI) operated in positive mode. A full MS scan (m/z
80–1200) was acquired. For the compounds of interest, a MS/MS scan range of m/z
80–650 was selected, with NCE at 20 V. Carotenoids were quantified at 450 nm, using external calibration curves of all-trans beta carotene, lutein and zeaxanthin. Concentrations of carotenoid standards were determined spectrophotometrically (Cintra 303, GBC Scientific Equipment, Braeside, Australia) using the specific molar absorption coefficients of carotenoids in solutions [23
Folates were analyzed by stable isotope dilution assay (SIDA) and UHPLC-PDA-MS/MS (Striegel et al. [24
]). Briefly, 100 mg of powdered sample was extracted with MES buffer (pH 5). Labeled isotopic compounds (IS), including [13
folate and [13
folate, were added at appropriated concentrations. A Shimadzu UHPLC-ESI-MS/MS system (Shimadzu Corp., Kyoto, Japan) equipped with a Shimadzu 8060 triple quadrupole mass spectrometer was employed (Supplementary Table S1
). Multiple reaction monitoring (MRM) in positive mode was optimized to quantify individual folate vitamers and their corresponding labeled isotopes. External calibration curves for quantification of folate vitamers were constructed based on the ratios of peak areas of analytes vs. IS.
Ascorbic acid (L-AA) extraction and analysis followed Campos et al. [25
], with slight modifications. Briefly, 200 mg powdered sample was extracted with 3% meta-phosphoric acid containing 8% acetic acid and 1-mM EDTA. The reduction of dehydroascorbic acid (DHAA), which was also present in the extracts/samples, to L-AA was performed following the method of Spinola et al. [26
]. Vitamin C (L-AA + DHAA) was determined using a Waters UPLC-PDA system (Supplementary Table S1
) and an external calibration curve of L-AA at 245 nm was used for quantification.
Non-Folate B Vitamins
Analysis of vitamins B1, B2, B3, B5, B6, B7 and B12 was conducted at Symbio Alliance Laboratories, using NATA accredited in-house HPLC-PDA methods (CFH363, CFH364 and CFH366). The analysis were performed in duplicate including measurement of uncertainty.
2.3.6. Total Phenolic Content
The free and bound polyphenol extracts were used for total phenolic content (TPC) measurement employing the Folin–Ciocâlteu reagent [27
]. A microplate absorbance reader (Sunrise, Tecan, Maennedorf, Switzerland) was used at 700 nm. TPC was expressed as mg of gallic acid equivalents (GAE) per g of sample, using an external gallic acid standard curve (0–105 mg/L).
2.3.7. DPPH Radical-Scavenging Capacity
The DPPH free radical-scavenging assay [28
] was used to determine the radical-scavenging capacity of the samples. The scavenging capacity was measured on a microplate absorbance reader (Sunrise, Tecan) at 517 nm. Ascorbic acid (0–0.1 mg/mL) was added for comparison. The % DPPH scavenging capacity was calculated using the following equation:
% DPPH = [(A0 − A1)/A0] × 100;
are the absorbance values of the control and the test samples, respectively.
2.3.8. Total (Condensed) Tannins
Extraction of tannins followed the method described by Karamac et al. [29
] with slight modifications. Briefly, approximately 1 g powdered sample was extracted with acetone in a sonication bath (70 °C, 15 min). After cooling, the supernatant was retained by centrifugation at 3900 rpm for 10 min. The extraction was repeated 3 times and the supernatants were combined and concentrated at 40 °C in a miVac sample Duo concentrator (Genevac, Inc, Gardiner, NY, USA). The dried extract was redissolved in methanol and subjected to the Vanillin-HCl assay (Price et al. [30
]), using a Sunrise microplate reader at 500 nm. (+/−)-Catechin (0–1.5 g/L) was used to prepare an external calibration curve. Total tannin content (TTC) was expressed as mg of catechin equivalents (CaE) per 100 g of sample.
2.3.9. Total Saponins
Extraction and quantification of saponins followed the spectrophotometric method described previously [31
] with modifications. Approximately 1 g powdered sample was extracted with 80% methanol at rt while shaking on an orbital shaker (RP1812, Paton Scientific, Victor Harbor, Australia) at 100 rpm overnight. The supernatant was collected after centrifugation (3900 rpm, 10 min), while the residue was re-extracted twice with 80% methanol (for 1 h). The supernatants were combined and evaporated until dryness at 40 °C in a miVac sample Duo concentrator. The dried extract was redissolved in water and successively extracted with diethyl-ether to remove the pigments, followed by extraction of saponins with saturated n-butanol. The n-butanol extracts were combined and dried under reduced pressure using a rotary evaporator (Buchi Rotavapor R-100, BÜCHI Labortechnik AG, Flawil, Switzerland). The dried extract was redissolved in aqueous methanol 80% (v/v
) and subjected to the Vanillin-H2
], using a Sunrise microplate reader at 540 nm. Oleanolic acid (0–0.5 g/L) was used to prepare an external calibration curve. Total saponins were expressed as mg of oleanolic acid equivalents (OE) per 100 g of sample.
2.3.10. Antimicrobial Activity
Powdered samples (1 g) were extracted 3 times with water or methanol in a sonication bath (30 min, rt). The supernatants were combined after centrifugation and evaporated at 60 °C and 40 °C for water and methanolic extracts, respectively, using a miVac sample Duo concentrator. Aqueous methanol 20% (v/v
) was used to freshly reconstitute the extract precipitates. Well diffusion assay followed the method described previously by Phan et al. [21
] was applied to test the antimicrobial activity against Staphylococcus aureus
, a Gram-positive bacteria; Escherichia coli
, a Gram-negative bacteria and Candida albicans
, a fungi. Penicillin and streptomycin (1 g) (Gibco, Life Technologies, Scoresby, Australia) and 10 μg fluconazole (Sigma-Aldrich) were used as antibacterial and antifungal controls, respectively. Aqueous methanol 20% (v/v
) was also included in the assay to evaluate the effect of solvent on microbial growth. The agar plates were incubated at 37 °C for 24 h or 48 h (depending on growth), and the inhibition zones formed around the wells were recorded. The results were expressed as strong (>13 mm), moderate (6–12 mm), weak (≥5 mm) or no inhibitory activity (<5 mm) [33
2.3.11. Statistical Analysis
A one-way analysis of variance (ANOVA), using Minitab 17 for Windows (Minitab, Inc., State College, PA, USA), was employed to test the variances of measurements. A p-value of 0.05 or less was considered as statistically significant. Pearson’s correlation coefficient analysis was also applied to test correlations between bioactive compounds and bioactivities.
The present study, to the best of our knowledge, reports for the first time the proximate composition, minerals and trace elements, vitamins and carotenoids in P. angustifolium leaves and stems collected from the wild or cultivated. P. angustifolium leaves could be identified as a rich source of saponins and polyphenols, whereas carotenoids, tannins and vitamins (B and C) were present at lower levels. The results indicate that multiple factors, such as growing condition, geographic location and different botanical tissues, can have a significant effect on the bioactive compounds in P. angustifolium and subsequently its bioactivity. However, further studies with more samples (total number and replicates), different seasons and growing locations, are strongly recommended to substantiate the results of the present study. The finding of the Pearson’s correlation suggests that only polyphenols have a significant correlation with the DPPH radical-scavenging capacity, whereas the antifungal activity against C. albicans was positively correlated with both polyphenols and saponins. This study further confirms the relationship between (phyto) chemicals and biologic properties in P. angustifolium, suggesting potential applications of this Australian indigenous plant as a functional (food) ingredient and/or a natural fungicide.