{"id":60470,"date":"2024-09-30T11:08:42","date_gmt":"2024-09-30T11:08:42","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=60470"},"modified":"2024-10-09T18:19:25","modified_gmt":"2024-10-09T18:19:25","slug":"evaluation-of-the-secondary-metabolites-and-bioactivity-of-south-african-bulbine-natalensis-under-simultaneous-elevated-carbon-dioxide-and-temperatures","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/evaluation-of-the-secondary-metabolites-and-bioactivity-of-south-african-bulbine-natalensis-under-simultaneous-elevated-carbon-dioxide-and-temperatures\/","title":{"rendered":"Evaluation of the Secondary Metabolites and Bioactivity of South African Bulbine Natalensis Under Simultaneous Elevated Carbon Dioxide and Temperatures"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Increased atmospheric CO2 <\/sub>drives\nthe increase in temperature, which could have adverse effects on plant growth\nand productivity.1<\/sup> The latest recorded concentration of atmospheric\nCO2 <\/sub>was just above 410 parts per million (ppm).2<\/sup> Temperatures,\nparticularly in South Africa, are predicted to increase between 3 and 6 \u02daC over\nthe next seven decades, with some parts of the country experiencing summer\ntemperatures above 45 \u02daC.3<\/sup> Environmental changes like this could\nnegatively impact the bioactivity of medicinal plants. Bulbine natalensis<\/em> is a broad-leaved succulent medicinal plant\nindigenous to South Africa, and is commonly used for the treating skin ailments\nsuch as wounds, scars and ringworms, blood related disorders, diabetes and aids\nin fertility issues.4<\/sup> These properties owe to the presence of\nsecondary metabolite compounds, which are derivatives of primary metabolites.\nThey play an important physiological role in all plants, such as their\ninvolvement as defence mechanisms against both abiotic and biotic environmental\nfactors.5, 6<\/sup> Secondary metabolites are also responsible for their\npharmaceutical properties, hence why medicinal plants have been used for the\ntreatments of a variety of illnesses and diseases.6, 7<\/sup> The\nphyto-pharmacological properties of B.\nnatalensis<\/em> has been reported in previous studies, however, the focus was\nmainly on the leaves, with limited studies reported on the underground stems\nand roots.8, 9<\/sup> In addition, there is an increasing trend in the\nresearch on climate change impacts on medicinal plants, which can provide\nvaluable information on how these plants would adapt to the evolving climate,\nand how their medicinal properties would be affected as a result. Currently,\nthe inclusion of abiotic stress impacts in medicinal plant research in South\nAfrica is limited. Hence the aim of this study was to evaluate the secondary metabolic\nresponses of B. natalensis<\/em> and their\neffect on the species\u2019 antioxidant and antibacterial activities under\nsimultaneous exposure to elevated CO2<\/sub> and high temperatures. <\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Experimental design<\/strong><\/p>\n\n\n\n

Matured\nB. natalensis<\/em> plants (n = 85) were\npurchased from the Witkoppen Nursery in Randburg, Gauteng Province in February\n(summer). All of the plants were left to acclimatize for one month, and\nmaintained in the rooftop greenhouse at the University of the Witwatersrand,\nJohannesburg. The plants were then transferred into three climate simulation\nchambers (Conviron\u00ae), each with different simulations as follows: control\nchamber (400 ppm CO\u2082\nand 27\/25 \u00b0C day\/night temperatures- ambient) and experiment chambers (600 ppm\nand 800 ppm, each paired with temperature ranges of 40\/30 \u00b0C and 45\/35 \u00b0C\nday\/night temperatures respectively). In addition, all plants were exposed to\nthese conditions for up to 192 hours (eight days), to simulate the predicted\naverage duration of a heat wave in South Africa. Five pots of B. natalensis<\/em> were harvested at 48-hour\nintervals: 48, 96, 144, and 192 hours respectively. Following each harvest, the\nplant parts were taken to the laboratory, where they were rinsed with distilled\nwater, and placed in a hot-air drier for up to four days at set at 40 \u02daC. The\ndried samples were ground into fine powder using an electric grinder. The\npowdered samples were then stored at room temperature in a dark cabinet\nawaiting extraction.<\/p>\n\n\n\n

Preparation of methanol extracts<\/strong><\/p>\n\n\n\n

Powdered\nsamples of B. natalensis<\/em> plant parts (3\ng) were each added into autoclave bottles containing 25 ml of 80% methanol\n(MeOH). Once mixed, the extracts were sonicated for 25 minutes in a water bath set\nat 50 \u02daC, in which 20 ml of 80% acetone was added. Following sonication, the\nextracts were centrifuged for 10 minutes at 3500 rotations per minute (rpm).10,\n11<\/sup><\/p>\n\n\n\n

Qualitative screening of phytocompounds<\/strong><\/p>\n\n\n\n

Preliminary screening of phytochemical\ncompound groups were investigated using several test methods. 12-17<\/sup>\nThe phytocompounds of interest for this study were phenolics, saponins,\nvolatile oils, terpenoids, steroids, tannins, flavonoids, glycosides, coumarins\nand phlobatannins. <\/p>\n\n\n\n

Quantification of phytochemical content<\/strong><\/p>\n\n\n\n

The quantification of the phytochemical content\nwas determined using ultraviolet spectrophotometry technique.<\/p>\n\n\n\n

Total phenolic content (TPC) <\/strong><\/p>\n\n\n\n

A Folin-Ciocalteu solution (750 \u03bcL- 1:10)\nmixed with a volume of 2.5 ml saturated sodium carbonate (Na2<\/sub>CO3<\/sub>)\nand 0.3 ml of the plant extracts were prepared in volumetric flasks, followed\nby 7 ml of distilled water to dilute the mixtures.11<\/sup> All vials were\nincubated in a dark cabinet for a period of two hours at ambient temperature, and\nthe absorbance of the mixtures were recorded at a wavelength of 765 nm,\nfollowing incubation. A gallic acid calibration curve equation (y = 0.0495 \u2013\n0.0259, r2<\/sup>= 0.9994) was used to determine the total phenolic content\nof all extracts.<\/p>\n\n\n\n

Total flavonoid content (TFC) <\/strong><\/p>\n\n\n\n

Methanol extracts (300 \u03bcL) of B. natalensis<\/em> were added into 10 ml\nvolumetric flasks, followed by 4 ml of distilled water and 300 \u03bcL of sodium\nnitrate (NaNO3<\/sub>) solution. The mixtures in the flasks were left to\nsettle for five minutes before aluminium chloride solution (3 ml) was added,\nfollowed by another six minute waiting period before adding 1 M of sodium\nchloride (NaOH- 2 ml). All mixtures were then made up to 10 ml with distilled\nwater.10, 11<\/sup> Each flask was vortexed for 15 minutes before recording\nthe absorbance at a wavelength of 510 nm.18<\/sup> The total flavonoid\ncontent of the extracts were then quantified using the quercetin calibration\ncurve equation, y = 0.2388 \u2013 0.0019, r2<\/sup> = 0.9997.<\/p>\n\n\n\n

Total tannin content (TTC) <\/strong><\/p>\n\n\n\n

B.\nnatalensis<\/em> extracts (100 \u03bcL)\nwere mixed with 7.5 ml of distilled water and 0.5 ml of Folin-Ciocalteu reagent\nin glass vials. A solution of 35% Na2<\/sub>CO3 <\/sub>was then added into\neach vial before filling them up to 10 ml with distilled water. The mixtures\nwere vortexed and incubated for 30 minutes at ambient temperature, and then the\nabsorbance of the extracts were recorded at a wavelength of 725 nm. The gallic\nacid calibration curve equation (y = 0.046x \u2013 0.0264, r2<\/sup> = 0.9833)\nwas used to quantify the total tannin content of the extracts.19<\/sup> <\/p>\n\n\n\n

Total proanthocyanidin content (TPAC)<\/strong><\/p>\n\n\n\n

Methanol extracts (500 \u03bcL) mixed with 3 ml\nof 4% vanillin-methanol solution and 1.5 ml of HCl were added into vials, and\nthen vortexed before incubating for 15 minutes at ambient temperature. A\nwavelength of 500 nm was used to record the absorbance of the extracts, and the\nstandard catechin calibration curve equation (y = 0.9554x + 0.0003, r2<\/sup>\n= 0.9927) was used to calculate the total proanthocyanidin content.20<\/sup><\/p>\n\n\n\n

Ultra-violet spectrophotometry assessment of the in-vitro antioxidant activity<\/strong><\/p>\n\n\n\n

DPPH radical quenching assay<\/strong><\/p>\n\n\n\n

The 2, 2 diphenylpicryhydrazyl antioxidant assay was\nfollowed for this experiment, with some alterations to the adopted methodology.21<\/sup>\nA stock solution of DPPH was prepared from 50 mg of DPPH powder and 80%\nmethanol (100 ml), from which a work solution (1:5) was made using the stock\nsolution and 80% methanol. Reaction mixtures consisting of 10-50 \u03bcL of the\nmethanol extracts were mixed with 0.7 ml of the work solution and filled up to\n1 ml using 80% methanol. The solutions were incubated in a cabinet for 45\nminutes to avoid any interference with light before measuring the absorbance at\na wavelength of 517 nm. A blank solution (80% methanol) as well as a control\nsolution (work solution and 80% methanol) were used, and a % inhibition was\nobtained using the equation: scavenging (%) = [(Absample \u2013 Abblank)\/Abcontrol] \u00d7 100.<\/p>\n\n\n\n

Hydrogen peroxide scavenging assay<\/strong><\/p>\n\n\n\n

The\nassessment of the hydrogen peroxide scavenging potential of B. natalensis<\/em> under elevated CO2<\/sub>\nand temperatures was performed for this study.22<\/sup> A hydrogen peroxide\nsolution (40 mM) containing phosphate buffer (pH 7.4) and 30% hydrogen peroxide\nwas prepared. The extracts (10-50 \u03bcL) were mixed with 0.6 ml of hydrogen\nperoxide solution (40 mM), and then incubated for 10 minutes in a dark cabinet.\nThe absorbance of the solutions were measured at a wavelength of 230 nm. Blank\n(phosphate buffer) and positive control (phosphate buffer and 40 mM hydrogen\nperoxide) solutions were used for the analysis of the scavenging activity using\nthe equation % scavenged H2<\/sub>O2 <\/sub>= [(Ac <\/sub>\u2013 As<\/sub>)\/Ac<\/sub>]\n\u00d7 100, where Ac<\/sub> represents the\npositive control solution, and As<\/sub> represents the plant extract\nsolution mixed with hydrogen peroxide.<\/p>\n\n\n\n

Iron chelating assay<\/strong><\/p>\n\n\n\n

Few\nalterations were made to the method used for the investigation of the iron\nchelating activity of B. natalensis<\/em>.23<\/sup>\nSolution mixtures were prepared using plant extracts (10-50 \u03bcL) and a solution\nof 2 mM ferric (II) chloride (50 \u03bcL). A volume of 0.2 ml of ferrozine (5 mM)\nwas then added to initiate a reaction. The solution mixtures were vortexed and\nleft to stand for 10 minutes at room temperature. A blank solution consisting\nof 80% methanol and a positive control containing ferric (II) chloride and\nferrozine were used as part of the analysis. The absorbance of the solutions\nwere measured at 562 nm, and the % chelation was obtained using the equation: %\nchelation = [(A0<\/sub> \u2013 A1<\/sub>)\/A0<\/sub>] \u00d7\n100, with A0<\/sub> being the absorbance value of the positive control\nsolution and A1<\/sub> representing the extract samples. <\/p>\n\n\n\n

Qualitative antibacterial activity assay<\/strong><\/p>\n\n\n\n

Culture preparations <\/strong><\/p>\n\n\n\n

Gram-positive\nStaphylococcus aureus<\/em> (ATCC 25923)\nand Gram-negative Escherichia coli <\/em>(ATCC\n25922) were grown in Baird-Parker and Muller-Hinton agar, respectively. Both\nmicrobial cultures and agar were purchased from Thermo-Fisher Laboratory\nSpecialties (Pty) Ltd. <\/p>\n\n\n\n

Agar-well diffusion assay<\/strong><\/p>\n\n\n\n

A\nslightly modified method for the agar-well diffusion assay was followed to\nassess the qualitative antibacterial activity of B. natalensis.<\/em>24, 25<\/sup> Culture plates were placed in an\nincubator for 24 hours at 37 \u02daC to allow the microbes to grow successfully.\nFollowing the incubation, a sterile borer was used to make wells (six\nmillimetres in diameter) in the agar, and then the plant extracts, as well as a\nnegative control (dimethyl sulfoxide) were added into the wells. The agar\nplates were refrigerated for one hour for sufficient diffusion of the extracts\nand the negative control into the agar. The plates were incubated once again\nfor another 24 hours in order to determine the growth inhibitory activity of\nthe extracts. A zone of inhibition, which is a value indicating the extract\u2019s\nantibacterial activity, was obtained using Vernier callipers and measured in\nmillimetres.<\/p>\n\n\n\n

Data analysis<\/strong><\/p>\n\n\n\n

Three\nreplicates were performed for the quantitative phytochemical, antioxidant, and\nantibacterial activity analyses, and all values were expressed as mean \u00b1\nstandard error (SE). A one-way analysis of variance (ANOVA) and Tukey HSD\npost-hoc tests were performed to determine the overall significance across all\nplant parts (p<\/em> < 0.05) and where\nthe difference lies amongst the plant parts using R Studio\u00ae statistical\nsoftware. <\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Phytochemical analysis<\/strong><\/p>\n\n\n\n

Qualitative screening of phytocompounds<\/p>\n\n\n\n

Leaves <\/strong><\/p>\n\n\n\n

Eight out of 10 compound groups were detected in the control leaf extract of B. natalensis<\/em>, from which phenolics, tannins, and coumarins had the highest presence. In selective elevated CO2 <\/sub>and high temperature treatments, coumarins, tannins, steroids and phenolics showed a higher presence of the respective compounds, although there was no difference when compared to the control with regards to phenolics, tannins and coumarins. About 70% of the phytochemical compounds were present in all treatments, including the control, whereas saponins were only present at 800ppm & 40\/30 \u02daC after the 48-hour harvest.<\/p>\n\n\n\n\n\n
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Figure 1: Qualitative screening of phytocompounds in B. natalensis<\/em> leaves under simultaneous elevated CO2<\/sub> and temperatures.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Underground stems <\/strong><\/p>\n\n\n\n

Four compound groups, namely saponins, steroids, terpenoids and glycosides, were noted in the control underground stem extract. Phlobatannins were the only phytochemical group to show a high presence at 600ppm & 45\/35 \u02daC (192 hours), 800ppm & 40\/30 \u02daC (144 hours and 192 hours) and 800ppm & 45\/35 \u02daC (48 hours) treatments, respectively. Saponins and terpenoids were the only compounds to maintain consistency in all treatments. Contrastingly, both coumarins and volatile oils were undetected in the underground stems.<\/p>\n\n\n\n\n\n
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Figure 2: Qualitative screening of phytocompounds in B. natalensis<\/em> underground stems under simultaneous elevated CO2<\/sub> and temperatures.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Roots <\/strong><\/p>\n\n\n\n

In the control root extract of B. natalensis<\/em>, half of the tested phytochemical groups were present. Similar to the underground stems, only one group was reported to have a higher presence. However, in the case of the roots, it was saponins, in three separate treatments: 600ppm & 45\/35 \u02daC (48 hours), 800ppm & 40\/30 \u02daC (192 hours) and 800ppm & 45\/35 \u02daC (48 hours and 96 hours) respectively. Five compound groups retained a consistent presence whereas coumarins and volatile oils were not detected.<\/p>\n\n\n\n\n\n
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Figure 3: Qualitative screening of phytocompounds in B. natalensis<\/em> roots under simultaneous elevated CO2<\/sub> and temperatures.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Quantitative analysis<\/strong><\/p>\n\n\n\n

Total phenolic content<\/strong><\/p>\n\n\n\n

The TPC of B. natalensis<\/em> leaves showed a progressive increase between the\ncontrol (867.0 \u00b1 64.50 mg\/100g GAE) and 96 hours (1883.1 \u00b1 102.43 mg\/100g GAE).\nA decrease was noted at 144 hours, which then slightly increased at 192 hours.\nThe control had a significantly lower (p<\/em>\n< 0.05) TPC than the leaves harvested under 600ppm & 40\/30 \u02daC (Figure\n4A). The concentration of total phenolics in the underground stems decreased\nfrom the control (1909.2 \u00b1 4.80 mg\/100g GAE) up to 144 hours (p<\/em> < 0.05), followed by an increase,\nwith the highest recorded concentration under 600ppm & 40\/30 \u02daC at 192\nhours (1833.3 \u00b1 35.16 mg\/100g GAE). The highest TPC from the root samples under\nelevated conditions was reported at 48 hours (3718.9 \u00b1 42.49 mg\/100g GAE),\nwhich was almost quadruple the concentration of the control (1002.8 \u00b1 24.7\nmg\/100g GAE). In decreasing order follows the 144-hour, 96-hour and 192-hour\nharvest periods, and lastly, the control (p<\/em>\n< 0.05). The roots had greater TPC than the leaves and underground stems\nunder 600ppm & 40\/30 \u02daC.<\/p>\n\n\n\n

The greatest significance was noted at 144\nhours, with a TPC of 1960.8 \u00b1 6.74 mg\/100g GAE (p<\/em> < 0.05) in the leaves harvested under 600ppm & 45\/35 \u02daC,\nwhich was followed by 192 hours, 48 hours, 96 hours, and the control (Figure\n4B). The TPC in the underground stems had doubled at 48 hours (3910.4 \u00b1 6.73\nmg\/100g GAE) from the control. The highest content was recorded at 144 hours\n(4725.3 \u00b1 23.33 mg\/100g GAE; p<\/em> <\n0.05), following a decrease at 96 hours. The TPC then decreased from 144 hours\nto 192 hours. The same pattern was observed from the TPC in the leaves. The\ngreatest decrease in TPC in the roots was reported at 96 hours (1896.9 \u00b1 11.66\nmg\/100g GAE), which was three times lower in concentration compared to 48 hours\n(3668.0 \u00b1 26.94 mg\/100g GAE). The TPC slightly decreased at 192 hours following\na gradual increase between 96 and 144 hours. Overall, the control was\nsignificantly different to all samples harvested under elevated conditions (p<\/em> < 0.05). All three plant parts had\ngreater concentrations under 600ppm & 45\/35 \u02daC compared to their respective\ncontrols.<\/p>\n\n\n\n

A concentration of 2314.4 \u00b1 17.75 mg\/100g\nGAE was the highest recorded at 48 hours from the leaves harvested under 800pm\n& 40\/30 \u02daC (p<\/em> < 0.05), which\ndecreased at 96 hours (Figure 4C). The TPC increased again at 144 hours before\ndecreasing at 192 hours. The greatest significance was recorded after 96 hours,\nwith a TPC of 6294.3 \u00b1 26.94 mg\/100g GAE (p<\/em>\n< 0.05) from the underground stems. This was followed by 48 hours, 144\nhours, 192 hours, and the control, which had the lowest TPC by comparison. In\nthe roots, the TPC had doubled from the control to 48 hours, with a\nconcentration of 2442.4 \u00b1 151.63 mg\/100g GAE, which then decreased from 96\nhours to 144 hours. The highest TPC came from the roots harvested at 192 hours\n(3890.2 \u00b1 84.38 mg\/100g GAE; p<\/em> <\n0.05). The underground stems had higher TPC between 48 and 144 hours, compared\nto the leaves and roots. The roots contained a comparatively higher\nconcentration of total phenolics, followed by the underground stems and the\nleaves at 192 hours.<\/p>\n\n\n\n

The TPC in the leaves saw a noticeable increase from the control to 144 hours (1312.9 \u00b1 32.04 mg\/100g GAE; p<\/em> < 0.05), and then decreased at 192 hours (Figure 4D). A huge increase in total phenolics in the underground stems was reported at 48 hours (5499.7 \u00b1 6.73 mg\/100g GAE) with the next highest value at 144 hours, followed by 192 hours, 96 hours and the control (p<\/em> < 0.05). The TPC in the roots under 800ppm & 45\/35 \u02daC, at first, increased from 48 to 96 hours (2422.2 \u00b1 23.33 mg\/100g GAE; p<\/em> < 0.05), then decreased from 144 hours to 192 hours. The underground stems possessed greater TPC than the leaves and roots in all harvest periods under 800ppm & 45\/35 \u02daC. Although the leaves and roots were the only plant parts to have higher TPC in each concurrent elevated CO2 <\/sub>and high temperature treatment in contrast to their controls, the underground stems had an overall higher TPC.<\/p>\n\n\n\n\n\n
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Figure 4: Total phenolic content of various B. natalensis<\/em> plant organs under simultaneous elevated CO2<\/sub> and high temperatures.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Total flavonoid content<\/strong><\/p>\n\n\n\n

An increase in the TFC was noticed at 48\nhours (358.9 \u00b1 51.17 mg\/100g QE) from the control (134.4 \u00b1 21.40 mg\/100g QE).\nThe concentration slightly decreased at 96 hours, and then increased again at\n144 hours, before doubling down at 192 hours (172.8 \u00b1 15.17 mg\/100g QE). An\noverall significant difference was noted for the leaves under 600ppm &\n40\/30 \u02daC against the control (p<\/em> <\n0.05; Figure 5A). The TFC in the underground stems at 192 hours was slightly\nhigher (265.0 \u00b1 3.33 mg\/100g QE) in comparison to the control (259.7 \u00b1 8.18\nmg\/100g QE). The three preceding harvest periods under 600ppm & 40\/30 \u02daC\nwere lower in TFC by comparison. Only the stems harvested at 48 and 144 hours\nwere significantly different to the control (p<\/em> < 0.05). The concentration of total flavonoids in the roots\nincreased between the control (238.3 \u00b1 43.40 mg\/100g QE) and 96 hours (897.5 \u00b1\n24.75 mg\/100g QE; p<\/em> < 0.05). At\n144 hours, the TFC took a great fall (285.6 \u00b1 5.37 mg\/100g QE; p<\/em> < 0.05) which then increased at 192\nhours. The leaves and roots alternated in terms of the TFC, with the leaves\nhaving a greater content at 48 and 144 hours, and the roots at 96 and 192 hours\nrespectively. They were also the only plant parts to contain higher TFC than\ntheir respective controls.<\/p>\n\n\n\n

The leaves had a slight decrease in TFC at\n48 hours (130.8 \u00b1 5.83 mg\/100g QE) from the control, before increasing at 96\nhours (268.3 \u00b1 23.12 mg\/100g QE; p<\/em>\n< 0.05; Figure 5B). A moderate decrease was noted at 144 hours (p<\/em> < 0.05), which was followed by\nanother increase at 192 hours. A similar trend was observed in the underground\nstems, where a fluctuation in the TFC was reported. A drop in concentration\nfrom the control to 48 hours was noted (p<\/em>\n< 0.05), which increased to its highest content at 96 hours (232.2 \u00b1 1.11\nmg\/100g QE). The TFC decreased at 144 hours before increasing, however\nindifferently, at 192 hours. The greatest difference in the TFC of the roots\nwas recorded at 144 hours, with a concentration of 423.9 \u00b1 3.13 mg\/100g QE, (p<\/em> < 0.05) followed by the control,\n192 hours, 48 hours, and 96 hours. Under 600ppm & 45\/35 \u02daC, the leaves\nshowed comparatively higher TFC at 96 and 192 hours. The underground stems and\nroots each had higher concentrations at 48 and 144 hours respectively.<\/p>\n\n\n\n

A significant increase in total flavonoids\nunder 800ppm & 40\/30 \u02daC at 48 hours was reported in the leaves, which\ngradually increased all the way to 192 hours, with the highest recorded\nconcentration of 501.3 \u00b1 40.29 mg\/100g QE in the leaves (Figure 5C). The\ncontrol was significantly different to the harvested samples under treatment (p<\/em> < 0.05). Compared to the leaves, B. natalensis<\/em> underground stems\ndisplayed an inverse pattern in TFC. The greatest concentration was reported at\n48 hours (607.8 \u00b1 12.75 mg\/100g QE), showing a great increase from the control.\nThe TFC then took a downturn from 96 hours to 192 hours (356.7 \u00b1 26.94 mg\/100g\nQE). The control had the lowest TFC compared to each harvested sample from this\ntreatment. Furthermore, a significant difference was reported between 48 and\n144 hours against the control (p<\/em> <\n0.05). The highest concentration of total flavonoids in the roots was reported\nat 96 hours (232.9 \u00b1 24.29 mg\/100g QE) followed by 48 hours, 192 hours and 144\nhours, under 800ppm & 40\/30 \u02daC. All the above mentioned concentrations had\nlower TFC than the control. In the last three harvest periods (96 to 192\nhours), the leaves contained greater TFC than the underground stems and roots.<\/p>\n\n\n\n

About 75% of the methanol leaf extracts of B. natalensis<\/em> were reported to have higher TFC under elevated CO2 <\/sub>and high temperatures (800ppm & 45\/35 \u02daC; p<\/em> < 0.05), in contrast to the control, namely at 48 hours, 96 hours, and the highest at 192 hours (462.2 \u00b1 15.76 ng\/100g QE; Figure 5D). The TFC in the underground stems showed a recurrent increase form the control to 96 hours (533.9 \u00b1 5.47 mg\/100g QE; p<\/em> < 0.05). The concentration moderately decreased at 144 hours and elevated again at 192 hours (p<\/em> < 0.05). The roots recorded the most significant increase in the concentration of total flavonoids at 48 hours (388.1 \u00b1 6.46 mg\/100g QE; p<\/em> < 0.05) followed by 192 hours, 144 hours and 96 hours. Similar to the underground stems, the control had the lowest TFC compared to the samples harvested under 800ppm & 45\/35 \u02daC. The plants exposed to 800ppm & 45\/35 \u02daC showed the underground stems to have the highest TFC between 96 and 192 hours, compared to the leaves and roots. The leaves, however, yielded a higher TFC at 48 hours.<\/p>\n\n\n\n\n\n
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Figure 5: <\/strong>Total flavonoid content of various B. natalensis<\/em> plant organs under simultaneous elevated CO2<\/sub> and high temperatures.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Total tannin content<\/strong><\/p>\n\n\n\n

The highest concentration of total tannins\nin the leaves was recorded at 48 hours under 600ppm & 40\/30 \u02daC (3228.62 \u00b1\n1.81 mg\/100g GAE; p<\/em> < 0.05). This\nwas followed by the control, with a slightly lower concentration (3008.3 \u00b1\n38.50 mg\/100g GAE), 192 hours, 144 hours, and 96 hours (Figure 6A). B. natalensis<\/em> underground stems showed an\nincreasing pattern in the TTC from the control (2890.0 \u00b1 53.10 mg\/100g GAE) to\n192 hours (6958.69 \u00b1 21.97 mg\/100g GAE). Each of the samples harvested under\nelevated conditions were significantly different against the control (p<\/em> < 0.05). Overall, the roots showed\nconsistency in TTC, with slight increases and decreases under elevated CO2\n<\/sub>and high temperatures. The concentration was highest at 96 hours (5480.43\n\u00b1 6.27 mg\/100g GAE; p<\/em> < 0.05),\nfollowed by 48 hours, 192 hours, 144 hours, and the control (1645.0 \u00b1 34.50\nmg\/100g GAE). The underground stems and roots were higher in TTC under elevated\nconditions compared to the leaves. Contrastingly, the leaves did have a greater\ncontent in the control.<\/p>\n\n\n\n

A steady increase was noted in the TTC in\nthe leaves between 48 and 144 hours, with the latter having the greatest\nconcentration (2808.33 \u00b1 9.06 mg\/100g GAE). The control, however, was\nsignificantly higher (p<\/em> < 0.05;\nFigure 6B) in TTC than every sample that was exposed to 600ppm & 45\/35 \u02daC.\nThe TTC in the underground stems continued to increase from the control to 192\nhours, where a concentration of 7199.64 \u00b1 17.28 mg\/100g GAE was recorded (p<\/em> < 0.05). The concentration of TTC\nin the roots nearly quadrupled in concentration at 48 hours (5946.02 \u00b1 4.79\nmg\/100g GAE; p<\/em> < 0.05) from the\ncontrol. Some consistency was noted between 96 and 192 hours, as the\nconcentrations slightly differed between 100 and 250 mg\/100g GAE (ANOVA;\nTukey). B. natalensis<\/em> underground\nstems had greater TTC in 75% of the harvest periods (96 to 192 hours) than the\nroots and leaves.<\/p>\n\n\n\n

A concentration of 2672.46 \u00b1 172.13\nmg\/100g GAE of total tannins was recorded after the 96-hour harvest of B. natalensis<\/em> leaves. All of the samples\nharvested under 800ppm & 40\/30 \u02daC had significantly lower TTC than the\ncontrol (p<\/em> < 0.05; Figure 6C). The\nTTC in the underground stems gradually declined from 48 hours (6647.10 \u00b1 7.89\nmg\/100g GAE) to 192 hours. The concentrations between 48 and 144 hours had at\nleast twice the concentration of the control (p<\/em> < 0.05). A similar pattern as in the underground stems was\nobserved from the TTC in the roots. The concentration steadily decreased from\n48 hours (5686.67 \u00b1 6.03 mg\/100g GAE) to 192 hours (p<\/em> < 0.05). Overall, B.\nnatalensis<\/em> underground stems and roots both had greater TTC compared to the\nleaves.<\/p>\n\n\n\n

The highest concentration recorded, compared to all other elevated CO2 <\/sub>and high temperature treatments was reported in the leaves at 48 hours, under 800ppm & 45\/35 \u02daC (4642.03 \u00b1 38.34 mg\/100g GAE; p<\/em> < 0.05; Figure 6D). The TTC decreased from 96 to 192 hours. Only the first two harvest periods showed greater tannin content than the control. Following each harvest period, there was an increase in the TTC in the underground stems (p<\/em> < 0.05), with the highest concentration recorded at 192 hours (8333.69 \u00b1 3.14 mg\/100g GAE). There were slight differences in the TTC in the roots, showing consistency in the concentrations throughout this treatment. The roots had higher content at 96 hours (3313.77 \u00b1 26.68 mg\/100g GAE) followed by 48 hours, 144 hours, and 192 hours. In comparison to the control, the roots under treatment had greater TTC (p<\/em> < 0.05). The underground stems contained greater concentrations of total tannins than the other plant organs.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 6: Total tannin content of various B. natalensis<\/em> plant organs under simultaneous elevated CO2<\/sub> and high temperatures.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Total proanthocyanidin content<\/strong><\/p>\n\n\n\n

The concentration of total\nproanthocyanidins in B. natalensis<\/em>\nleaves had increased from 48 hours to 96 hours (294.9 \u00b1 0.32 mg\/100g CE), which\nthen decreased in the last two harvest periods (Figure 7A). The control (362. 3\n\u00b1 9.50 mg\/100g CE) was significantly higher (p<\/em> < 0.05) in TPAC than the leaves under 600ppm & 40\/30 \u02daC. A\nsignificantly higher concentration of total proanthocyanidins in the\nunderground stems was reported at 192 hours (864.1 \u00b1 34.27 mg\/100g CE, p<\/em> < 0.05) in comparison to the\ncontrol (858.3 \u00b1 1.70 mg\/100g CE). The underground stems harvested between 48\nand 144 hours were lower in TPAC than the control. The TPAC in the roots at 48\nhours (462.2 \u00b1 0.23 mg\/100g CE) was the only harvest period that showed higher\ncontent than the control (373.3 \u00b1 9.50 mg\/100g CE) (p<\/em> < 0.05). Overall, the results showed that the underground\nplant parts contained greater TPAC than the leaves.<\/p>\n\n\n\n

A continual decrease in the concentration\nof total proanthocyanidins was observed in the leaves under 600ppm & 45\/35\n\u02daC from 48 hours to 192 hours (Figure 7B). The control was significantly higher\nin TPAC compared to the leaves exposed to elevated CO2 <\/sub>and high\ntemperatures (p<\/em> < 0.05). The TPAC\nincreased between 48 and 144 hours (940.4 \u00b1 2.48 mg\/100g CE) in the underground\nstems. The control was higher in content in contrast to 48 hours, 96 hours, and\n192 hours (p<\/em> < 0.05). The roots\ndisplayed greater concentrations of total proanthocyanidins at 192 hours (573.3\n\u00b1 0.85 mg\/100g CE; p<\/em> < 0.05),\nfollowed by the 48 hours, 96 hours, the control, and 144 hours. The leaves were\nlower in TPAC in this treatment compared to the species\u2019 underground plant\nparts.<\/p>\n\n\n\n

Although a concentration of 307.9 \u00b1 0.76\nmg\/100g CE was reported at 144 hours and was subsequently the highest TPAC in\nthe leaves under 800ppm & 40\/30 \u02daC, the control was still significantly\nhigher in content overall (p<\/em> <\n0.05; Figure 7C). The TPAC in the underground stems continued to increase from\n48 to 144 hours (776.1 \u00b1 0.78 mg\/100g CE) until it decreased at 192 hours, by\nalmost half the concentration of the previous harvest period. The control,\nhowever, remained the highest in TPAC by comparison (p<\/em> < 0.05). At least 75% of the harvested roots showed greater\nTPAC, at 192 hours (870.6 \u00b1 1.13 mg\/100g CE), 96 hours and 144 hours, than the\ncontrol (p<\/em> < 0.05). The\nsubterranean plant parts of B. natalensis<\/em>\ncontained higher concentrations of total proanthocyanidins than the leaves.<\/p>\n\n\n\n

A higher TPAC was reported at 48 hours (367.1 \u00b1 0.23 mg\/100g CE; p<\/em> < 0.05) with the control sample trailing closely behind, and 144 hours (Figure 7D). These were the only leaf extracts to have a concentration above 300 mg\/100g CE. There was a slight decrease in the TPAC in the underground stems between the control (p<\/em> < 0.05) and 48 hours (849.5 \u00b1 0.91 mg\/100g CE), which continued to 96 hours. It was from here where the concentration increased until 192 hours. The roots contained a greater TPAC at 144 hours (430.5 \u00b1 0.46 mg\/100g CE; p<\/em> < 0.05), which was by comparison, higher than the control. B. natalensis<\/em> underground stems displayed the highest concentration of total proanthocyanidins, followed by the roots and leaves, under 800ppm & 45\/35 \u02daC.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 7: Total proanthocyanidin content of various B. natalensis<\/em> plant organs under simultaneous elevated CO2<\/sub> and high temperatures.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Antioxidant activity (AA)<\/strong><\/p>\n\n\n\n

DPPH<\/strong><\/p>\n\n\n\n

B.\nnatalensis<\/em> leaves fairly\nreduced the production of DPPH radicals at 48, 144 and 192 hours, and a weaker\nactivity reported at 96 hours (Table 1). All the harvested leaves under 600ppm\n& 40\/30 \u02daC had slightly higher IC50 <\/sub>concentrations than the\ncontrol (3.88 \u00b1 0.16 mg\/ml). About half of the underground stems showed a good\nAA at 96 hours (3.00 \u00b1 0.32 mg\/ml) and 144 hours (3.49 \u00b1 0.31 mg\/ml)\nrespectively. The poorest activity was reported at 48 hours, 192 hours, and the\ncontrol, of which the latter displayed the weakest DPPH scavenging activity\n(35.86 \u00b1 0.29 mg\/ml). Most of the roots harvested under elevated CO2 <\/sub>and\nhigh temperatures had weaker scavenging activities, in comparison to the\ncontrol, which had excellent AA (0.36 \u00b1 0.02 mg\/ml). The leaves and underground\nstems each had better DPPH radical quenching at 48 and 192 hours, as well as 96\nand 144 hours respectively, compared to the roots. However, all three plant\nparts under 600ppm & 40\/30 \u02daC were significantly different to their respective\ncontrols (p<\/em> < 0.05).<\/p>\n\n\n\n

Three of the harvest periods (48, 96 and\n192 hours) had lower IC50 <\/sub>concentrations from the leaf extracts than\nthe control (p<\/em> < 0.05; Table 1).\nEach of the values indicated an excellent potential to reduce DPPH radicals,\nespecially at 48 hours (0.27 \u00b1 0.05 mg\/ml). All the underground stem extracts\nshowed significantly greater DPPH scavenging activity than the control (p<\/em> < 0.05), with values ranging\nbetween 0.24 to 0.67 mg\/ml. An adequate radical quenching activity was reported\nfrom the roots. Although a good AA was recorded at 48 and 96 hours\nrespectively, the control still exceeded their potential. Overall, the\nunderground stems, followed by the leaves, greatly outperformed the roots in\nreducing DPPH radicals under 600ppm & 45\/35 \u02daC. <\/p>\n\n\n\n

The leaves harvested at 96 and 192 hours\nunder 800ppm & 40\/30 \u02daC showed excellent AA (1.64 \u00b1 0.11 mg\/ml and 0.61 \u00b1\n0.09 mg\/ml respectively) compared to 48 and 144 hours, which displayed a good\nDPPH scavenging activity (Table 1). The control, however, was slightly weaker\nin activity than the leaves harvested under 800ppm & 40\/30 \u02daC (p<\/em> < 0.05). The IC50 <\/sub>values\nof the underground stems significantly decreased from the control to 144 hours\nwhere the greatest activity was recorded (0.76 \u00b1 0.09 mg\/ml). Similar to the\nleaves, all of the underground stems under this treatment scavenged DPPH\nradicals much better than the control (p<\/em>\n< 0.05). The strongest AA from the roots harvested under 800ppm & 40\/30\n\u02daC, was reported at 192 hours (0.51 \u00b1 0.05 mg\/ml) which was indifferently\nhigher in concentration than the control. A good activity was observed at 48\nand 144 hours, and the weakest activity was reported at 96 hours. <\/p>\n\n\n\n

The IC50 <\/sub>concentrations of the leaf extracts had decreased from 9.78 \u00b1 0.44 mg\/ml at 48 hours (good AA) to 1.30 \u00b1 0.12 mg\/ml at 96 hours (excellent AA) and the lowest concentration at 144 hours (0.49 \u00b1 0.04 mg\/ml; Table 1). Only the latter two harvest periods had a higher scavenging potential than the control (p<\/em> < 0.05). A positive activity was observed in the underground stems, with 50% of the harvest periods exhibiting an excellent radical quenching activity (96 hours \u2013 0.82 \u00b1 0.07 mg\/ml and 192 hours \u2013 1.58 \u00b1 0.17 mg\/ml, respectively) while the other half had a good AA (48 and 144 hours). The roots displayed a polarizing DPPH radical scavenging activity, with a good AA reported at 48 and 144 hours, and a weak AA noted at 96 and 192 hours. All the recorded IC50 <\/sub>concentrations of the roots under 800ppm & 45\/35 \u02daC were significantly higher than the control (p<\/em> < 0.05). The greatest radical scavenging activity was found to come from the underground stems, specifically at 48, 96 and 192 hours. <\/p>\n\n\n\n

Table 1: The DPPH scavenging potential of B. natalensis<\/em> exposed to simultaneous elevated CO2<\/sub> and temperatures.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment<\/strong><\/p>\n<\/td>\n

\n

Harvest period<\/strong><\/p>\n<\/td>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

Underground stems<\/strong><\/p>\n<\/td>\n

\n

Roots<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

3.87d<\/sup> \u00b1 0.16<\/p>\n<\/td>\n

\n

35.86a<\/sup> \u00b1 0.29<\/p>\n<\/td>\n

\n

0.36e<\/sup> \u00b1 0.02<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

600ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

5.50c<\/sup> \u00b1 0.27<\/p>\n<\/td>\n

\n

22.4b<\/sup> \u00b1 0.23<\/p>\n<\/td>\n

\n

11.41c<\/sup> \u00b1 0.32<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

13.74a<\/sup> \u00b1 0.32<\/p>\n<\/td>\n

\n

3.00c<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

8.65d<\/sup> \u00b1 0.17<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

9.11b<\/sup> \u00b1 0.20<\/p>\n<\/td>\n

\n

3.49c<\/sup> \u00b1 0.31<\/p>\n<\/td>\n

\n

38.15a<\/sup> \u00b1 0.40<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

6.18c<\/sup> \u00b1 0.21<\/p>\n<\/td>\n

\n

22.89b<\/sup> \u00b1 0.44<\/p>\n<\/td>\n

\n

13.49b<\/sup> \u00b1 0.49<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

3.87b<\/sup> \u00b1 0.16<\/p>\n<\/td>\n

\n

35.86a<\/sup> \u00b1 0.29<\/p>\n<\/td>\n

\n

0.36e<\/sup> \u00b1 0.02<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

600ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.27b<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.51b<\/sup> \u00b1 0.06<\/p>\n<\/td>\n

\n

2.89d<\/sup> \u00b1 0.42<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

1.77c<\/sup> \u00b1 0.13<\/p>\n<\/td>\n

\n

0.67b<\/sup> \u00b1 0.06<\/p>\n<\/td>\n

\n

7.09c<\/sup> \u00b1 0.35<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

25.42a<\/sup> \u00b1 0.06<\/p>\n<\/td>\n

\n

0.42b<\/sup> \u00b1 0.03<\/p>\n<\/td>\n

\n

16.5b<\/sup> \u00b1 0.37<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

1.91c<\/sup> \u00b1 0.29<\/p>\n<\/td>\n

\n

0.24b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

20.5a<\/sup> \u00b1 0.42<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

3.87a<\/sup> \u00b1 0.16<\/p>\n<\/td>\n

\n

35.86a<\/sup> \u00b1 0.29<\/p>\n<\/td>\n

\n

0.36d<\/sup> \u00b1 0.02<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

800ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

2.39b<\/sup> \u00b1 0.12<\/p>\n<\/td>\n

\n

11.95b<\/sup> \u00b1 0.41<\/p>\n<\/td>\n

\n

3.77b<\/sup> \u00b1 0.55<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

1.64c<\/sup> \u00b1 0.11<\/p>\n<\/td>\n

\n

3.13d<\/sup> \u00b1 0.44<\/p>\n<\/td>\n

\n

29.99a<\/sup> \u00b1 0.19<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

2.55b<\/sup> \u00b1 0.12<\/p>\n<\/td>\n

\n

0.76e<\/sup> \u00b1 0.09<\/p>\n<\/td>\n

\n

2.29c<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

0.61d<\/sup> \u00b1 0.09<\/p>\n<\/td>\n

\n

5.17c<\/sup> \u00b1 0.85<\/p>\n<\/td>\n

\n

0.51cd<\/sup> \u00b1 0.05<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

3.87c<\/sup> \u00b1 0.16<\/p>\n<\/td>\n

\n

35.86a<\/sup> \u00b1 0.29<\/p>\n<\/td>\n

\n

0.36e<\/sup> \u00b1 0.02<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

800ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

9.78b<\/sup> \u00b1 0.44<\/p>\n<\/td>\n

\n

2.86c<\/sup> \u00b1 0.44<\/p>\n<\/td>\n

\n

3.13d<\/sup> \u00b1 0.35<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

1.30d<\/sup> \u00b1 0.12<\/p>\n<\/td>\n

\n

0.82d<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

20.14b<\/sup> \u00b1 0.24<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

0.49d<\/sup> \u00b1 0.04<\/p>\n<\/td>\n

\n

7.24b<\/sup> \u00b1 0.22<\/p>\n<\/td>\n

\n

8.35c<\/sup> \u00b1 0.23<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

27.14a<\/sup> \u00b1 0.35<\/p>\n<\/td>\n

\n

1.58cd<\/sup> \u00b1 0.17<\/p>\n<\/td>\n

\n

24.55a<\/sup> \u00b1 0.20<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are expressed as mean (mg\/ml) \u00b1 SE. Mean values of different superscript letters in the same column indicates significance (p<\/em> < 0.05).<\/p>\n\n\n

Hydrogen peroxide<\/strong><\/p>\n\n\n\n

Of the three harvest periods where an\nexcellent H2<\/sub>O2<\/sub>reducing power was reported in\nthe leaves under 600ppm & 40\/30 \u02daC (IC50 <\/sub>values lower than 1\nmg\/ml) namely at 48, 96 and 192 hours, the latter two had the strongest\nactivity (0.19 \u00b1 0.04 mg\/ml and 0.18 \u00b1 0.04 mg\/ml respectively; Table 2). The\ncontrol, however, had the greatest scavenging power (p<\/em> < 0.05). A gradual decrease in the IC50 <\/sub>concentrations\nof the underground stems was observed from the control (0.61 \u00b1 0.05 mg\/ml) to\n144 hours (0.22 \u00b1 0.03 mg\/ml; p<\/em> <\n0.05), indicating greater AA compared to the stems harvested at 192 hours. The\nroots showed an excellent (96 and 192 hours) to good (48 and 144 hours) H2<\/sub>O2<\/sub>scavenging activity under 600ppm & 40\/30 \u02daC, however, none of these\nwere stronger at hydroxyl ion reduction than the control (0.24 \u00b1 0.04 mg\/ml).\nThe leaves and underground stems both had scavenged H2<\/sub>O2<\/sub>ions much better than the roots.<\/p>\n\n\n\n

Although each of the harvested leaves\nunder 600ppm & 45\/35 \u02daC had excellent activity, with the greatest H2<\/sub>O2<\/sub>quenching reported at 48 hours (0.46 \u00b1 0.14 mg\/ml), the control still\nshowed a slight but significantly stronger AA (Table 2). The underground stems\nhad a fairly good AA. The scavenging potential was better at 96 and 192 hours,\nwhere IC50 <\/sub>values were lower (0.36 \u00b1 0.02 mg\/ml and 1.17 mg\/ml\nrespectively). All the root extracts displayed excellent H2<\/sub>O2<\/sub>radical scavenging activity, with the IC50 <\/sub>values being lower\nthan 1 mg\/ml. The roots harvested after 144 hours had the strongest AA with an\nIC50 <\/sub>concentration of 0.15 \u00b1 0.03 mg\/ml. <\/p>\n\n\n\n

A slightly stronger AA in the leaves was\ndiscovered at 96 hours (0.25 \u00b1 0.03 mg\/ml), followed by 192 hours and 48 hours\nunder 800ppm 40\/30 \u02daC, of which both had higher IC50 <\/sub>concentrations\nthan the control (Table 2). A comparatively weaker, but good AA was reported at\n144 hours. The AA in the underground stems greatly improved from 48 hours to\n192 hours under elevated CO2 <\/sub>and high temperature conditions. This\nwas evident from the continual decrease in the IC50 <\/sub>concentrations,\nwith a better scavenging power recorded at 144 hours (0.41 \u00b1 0.02 mg\/ml) and\n192 hours (0.17 \u00b1 0.04 mg\/ml) respectively. An excellent H2<\/sub>O2<\/sub>\nradical scavenging activity was reported in all root extracts. The greatest\nactivity was noted at 96 hours (0.44 \u00b1 0.03 mg\/ml), which is approximately\ndouble the concentration of the control. All plant parts had excellent H2<\/sub>O2<\/sub>scavenging activity at 96 and 192 hours. The ANOVA results showed that\nonly the leaves showed an overall significant difference (p<\/em> < 0.05), whereas the underground stems and roots were\ninsignificant.<\/p>\n\n\n\n

The leaves had a great H2<\/sub>O2<\/sub> reducing power in all harvest periods from the 800ppm & 45\/35 \u02daC, with the IC50 <\/sub>concentrations being lower than 1 mg\/ml (Table 2). Although there was no significant difference between the concentrations recorded for each harvest period and the control, the latter had the lowest concentration. A much better scavenging activity from the underground stems was reported at 96 (0.10 \u00b1 0.02 mg\/ml) and 144 hours (0.15 \u00b1 0.01 mg\/ml) in comparison to the control (p<\/em> < 0.05), 48 hours and 192 hours, which the latter had a good activity. The roots showed a similar trend in the excellent scavenging potential against H2<\/sub>O2 <\/sub>radicals as leaves, however in contrast, the roots slightly reduced the production of radicals better than the leaves. Only two harvest periods had lower IC50 <\/sub>concentrations, particularly at 144 hours (0.18 \u00b1 0.04 mg\/ml) and 192 hours (0.21 \u00b1 0.03 mg\/ml) than the control. Both the underground stems and roots showed stronger H2<\/sub>O2<\/sub>scavenging activity than the leaves at 96 and 144 hours, as well as 48 and 192 hours respectively. <\/p>\n\n\n\n

Table 2: The hydrogen peroxide radical scavenging potential of B. natalensis<\/em> exposed to simultaneous elevated CO2<\/sub> and temperatures. <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment<\/strong><\/p>\n<\/td>\n

\n

Harvest period<\/strong><\/p>\n<\/td>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

Underground stems<\/strong><\/p>\n<\/td>\n

\n

Roots<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

0.29b<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.61b<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.24b<\/sup> \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

600ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.71s<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.38ab<\/sup> \u00b1 0.06<\/p>\n<\/td>\n

\n

3.24a<\/sup> \u00b1 0.30<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

0.19s<\/sup> \u00b1 0.04<\/p>\n<\/td>\n

\n

0.27b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

1.36ab<\/sup> \u00b1 0.10<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

2.31s<\/sup> \u00b1 0.32<\/p>\n<\/td>\n

\n

0.22b<\/sup> \u00b1 0.03<\/p>\n<\/td>\n

\n

4.64a<\/sup> \u00b1 0.65<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

0.18s<\/sup> \u00b1 0.04<\/p>\n<\/td>\n

\n

2.94a<\/sup> \u00b1 0.41<\/p>\n<\/td>\n

\n

1.94ab<\/sup> \u00b1 0.08<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

0.29b<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.61b<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.24a<\/sup> \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

600ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.46ab<\/sup> \u00b1 0.14<\/p>\n<\/td>\n

\n

7.06a<\/sup> \u00b1 1.08<\/p>\n<\/td>\n

\n

0.45a<\/sup> \u00b1 0.09<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

1.16a<\/sup> \u00b1 0.34<\/p>\n<\/td>\n

\n

0.36b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.51a<\/sup> \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

0.62ab<\/sup> \u00b1 0.06<\/p>\n<\/td>\n

\n

2.51b<\/sup> \u00b1 0.24<\/p>\n<\/td>\n

\n

0.15a<\/sup> \u00b1 0.03<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

0.83a<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

1.17b<\/sup> \u00b1 0.09<\/p>\n<\/td>\n

\n

0.82a<\/sup> \u00b1 0.08<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

0.29b<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.61a<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.24a<\/sup> \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

800ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.78b<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

2.67a<\/sup> \u00b1 1.92<\/p>\n<\/td>\n

\n

1.20a<\/sup> \u00b1 0.12<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

0.25b<\/sup> \u00b1 0.03<\/p>\n<\/td>\n

\n

0.68a<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.44a<\/sup> \u00b1 0.03<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

6.94a<\/sup> \u00b1 0.31<\/p>\n<\/td>\n

\n

0.41a<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.85a<\/sup> \u00b1 0.07<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

0.77b<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

0.17a<\/sup> \u00b1 0.04<\/p>\n<\/td>\n

\n

0.86a<\/sup> \u00b1 0.06<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

0.29a<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.61b<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.24a<\/sup> \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

800ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.73a<\/sup> \u00b1 0.10<\/p>\n<\/td>\n

\n

0.72a<\/sup> \u00b1 0.08<\/p>\n<\/td>\n

\n

0.56a<\/sup> \u00b1 0.09<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

0.35a<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

0.10a<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.59a<\/sup> \u00b1 0.06<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

0.83a<\/sup> \u00b1 0.08<\/p>\n<\/td>\n

\n

0.15a<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

0.18a<\/sup> \u00b1 0.04<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

0.57d<\/sup> \u00b1 0.04<\/p>\n<\/td>\n

\n

2.82a<\/sup> \u00b1 0.26<\/p>\n<\/td>\n

\n

0.21a<\/sup> \u00b1 0.03<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are expressed as mean (mg\/ml) \u00b1 SE. Mean values of different superscript letters in the same column indicates significance (p<\/em> < 0.05).<\/p>\n\n\n

Iron chelating activity<\/strong><\/p>\n\n\n\n

The leaves displayed a good iron chelating\nactivity under 600ppm & 40\/30 \u02daC, however, the control excellently quenched\nthe radicals in comparison (0.19 \u00b1 0.01 mg\/ml; Table 3). All the underground\nstem extracts yielded great iron quenching activity, with the 48 hour (0.69 \u00b1\n0.04 mg\/ml) and 144 hour (0.71 \u00b1 0.05 mg\/ml) harvest periods having\ncomparatively lower IC50 <\/sub>values than the control (0.89 \u00b1 0.02\nmg\/ml). The roots showed the best iron radical scavenging activity compared to\nthe underground stems and leaves. All of the IC50 <\/sub>values recorded\nunder 600ppm & 40\/30 \u02daC were lower than 1 mg\/ml. The control however, was\nsignificantly stronger (0.33 \u00b1 0.01 mg\/ml). <\/p>\n\n\n\n

The lowest IC50 <\/sub>concentrations\nfrom the methanolic leaf extracts were reported at 96 hours (0.40 \u00b1 0.09 mg\/ml)\nand 144 hours (0.41 \u00b1 0.09 mg\/ml) respectively (Table 3). Overall, the leaves\nshowed an excellent iron reducing activity. The underground stems showed the\nstrongest reducing power at 144 hours (0.07 \u00b1 0.02 mg\/ml), followed by the 48\nhours and the control. A good AA was reported at 96 and 192 hours. An overall\ngood AA was observed for the roots, showing significantly higher IC50 <\/sub>concentrations\nthan the control. Of the three plant parts, the leaves had stronger iron\nradical scavenging activity under 600ppm & 45\/35 \u02daC, followed by the\nunderground stems and the roots. <\/p>\n\n\n\n

The iron chelating activity of the leaves\nslowly decreased at each of the harvest periods, with the lowest IC50 <\/sub>concentration\nat 48 hours (0.36 \u00b1 0.02 mg\/ml) to the highest at 192 hours (1.63 \u00b1 0.18 mg\/ml;\nTable 3). At least 75% of the harvest periods showed greater iron chelating\npower at 48 hours (0.99 \u00b1 0.03 mg\/ml), 144 hours (1.99 \u00b1 0.07 mg\/ml) and 192\nhours (0.87 \u00b1 0.20 mg\/ml) respectively for the underground stems. The roots\nexhibited an excellent scavenging activity between 48 hours and 144 hours. The\ngreatest activity was reported at 96 hours (0.66 \u00b1 0.06 mg\/ml), which was\nmoderately stronger than the control. All the plant organs displayed excellent\niron chelating activity at 48 hours and 144 hours, with the harvested material\nfrom the latter period having a stronger antioxidant power. <\/p>\n\n\n\n

The leaves showed the greatest iron radical reducing power at 96 hours (0.14 \u00b1 0.02 mg\/ml), followed by the control, 144 hours, 192 hours and 48 hours. Half of the underground stem extracts had more powerful iron radical quenching than the control, particularly at 48 hours (0.07 \u00b1 0.01 mg\/ml) and 144 hours (0.07 \u00b1 0.02 mg\/ml). The IC50 <\/sub>concentrations recorded from the root extracts decreased continually, indicating a stronger AA with each harvest period. The roots harvested at 144 and 192 hours had the best scavenging activity (0.80 \u00b1 0.36 mg\/ml and 0.80 \u00b1 0.11 mg\/ml respectively). B. natalensis <\/em>was overall, significantly different (p < 0.05; ANOVA) in the metal chelating activity, however, the control was found to be significantly different to each of the harvest periods in several treatments (600ppm & 40\/30 \u02daC- leaves and underground stems and 600ppm & 45\/35 \u02daC- roots) respectively.<\/p>\n\n\n\n

Table 3: The iron chelating potential of B. natalensis<\/em> exposed to simultaneous elevated CO2<\/sub> and temperatures.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment<\/strong><\/p>\n<\/td>\n

\n

Harvest period<\/strong><\/p>\n<\/td>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

Underground stems<\/strong><\/p>\n<\/td>\n

\n

Roots<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.19b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

0.89b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.33b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong><\/p>\n

 <\/strong><\/p>\n

600ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

1.54a<\/sup> \u00b1 0.14<\/p>\n<\/td>\n

\n

0.69c<\/sup> \u00b1 0.04<\/p>\n<\/td>\n

\n

0.84a<\/sup> \u00b1 0.10<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

1.95a<\/sup> \u00b1 0.17<\/p>\n<\/td>\n

\n

1.07a<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

0.58ab<\/sup> \u00b1 0.02<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

2.62a<\/sup> \u00b1 0.37<\/p>\n<\/td>\n

\n

0.71c<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.74a<\/sup> \u00b1 0.03<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

2.39a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

1.19a<\/sup> \u00b1 0.05<\/p>\n<\/td>\n

\n

0.83a<\/sup> \u00b1 0.09<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.19b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

0.89c<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.33b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong>600ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.57a<\/sup> \u00b1 0.03<\/p>\n<\/td>\n

\n

0.80c<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

3.08a<\/sup> \u00b1 0.13<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

0.40ab<\/sup> \u00b1 0.09<\/p>\n<\/td>\n

\n

2.48b<\/sup> \u00b1 0.21<\/p>\n<\/td>\n

\n

3.42a<\/sup> \u00b1 0.43<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

0.41ab<\/sup> \u00b1 0.09<\/p>\n<\/td>\n

\n

0.07c<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

3.81a<\/sup> \u00b1 0.47<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

0.57a<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

4.96a<\/sup> \u00b1 0.46<\/p>\n<\/td>\n

\n

3.19a<\/sup> \u00b1 0.09<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.19b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

0.89b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.33b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong>800ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.36b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.99b<\/sup> \u00b1 0.03<\/p>\n<\/td>\n

\n

1.19b<\/sup> \u00b1 0.06<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

0.57b<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

2.18a<\/sup> \u00b1 0.38<\/p>\n<\/td>\n

\n

0.66b<\/sup> \u00b1 0.06<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

1.57a<\/sup> \u00b1 0.23<\/p>\n<\/td>\n

\n

1.99a<\/sup> \u00b1 0.07<\/p>\n<\/td>\n

\n

1.36b<\/sup> \u00b1 0.08<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

1.63a<\/sup> \u00b1 0.18<\/p>\n<\/td>\n

\n

0.87b<\/sup> \u00b1 0.20<\/p>\n<\/td>\n

\n

4.69a<\/sup> \u00b1 0.44<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.19b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

0.89b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.33b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/strong>800ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

0.57a<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.07b<\/sup> \u00b1 0.01<\/p>\n<\/td>\n

\n

2.10a<\/sup> \u00b1 0.30<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

0.14b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

3.62a<\/sup> \u00b1 0.16<\/p>\n<\/td>\n

\n

1.21ab<\/sup> \u00b1 0.08<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

0.50a<\/sup> \u00b1 0.03<\/p>\n<\/td>\n

\n

0.07b<\/sup> \u00b1 0.02<\/p>\n<\/td>\n

\n

0.80b<\/sup> \u00b1 0.36<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

0.55a<\/sup> \u00b1 0.04<\/p>\n<\/td>\n

\n

3.96a<\/sup> \u00b1 0.37<\/p>\n<\/td>\n

\n

0.80b<\/sup> \u00b1 0.11<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are expressed as mean (mg\/ml) \u00b1 SE. Mean values of different superscript letters in the same column indicates significance (p<\/em> < 0.05).<\/p>\n\n\n

Antibacterial activity <\/strong><\/p>\n\n\n\n

600ppm and 40\/30 \u02daC <\/strong><\/p>\n\n\n\n

The harvested leaves from this treatment all displayed an intermediate antibacterial activity against S. aureus<\/em>, with a range between 13 and 17 mm (Table 4). Surprisingly, the control sample did not show any inhibitory activity, indicating bacterial resistance. The underground stems also had the same activity as the leaves, but with slightly lower zones of inhibition (10-13 mm) under 600ppm & 40\/30 \u02daC, even with the control having a larger zone of inhibition (15.33 \u00b1 0.67 mm; Table 4). The roots also had the same intermediate activity as the leaves and underground stems. Only the roots had insignificant under 600ppm & 40\/30 \u02daC against the control (p<\/em> > 0.05). The control leaf extract had a greater zone of inhibition value against E. coli<\/em> (18.33 \u00b1 0.88 mm), and all harvested samples under 600ppm & 40\/30 \u02daC had zones of inhibition between 11 and 12 mm, also showing significance against the control (p<\/em> < 0.05; Table 5). The underground stems and roots also had an intermediate activity in both control (15.33 \u00b1 0.88 mm- underground stems; 13.33 \u00b1 0.88 mm- roots) and 600ppm & 40\/30 \u02daC samples (p<\/em> < 0.05; Table 5). <\/p>\n\n\n\n

600ppm and 45\/35 \u02daC<\/strong> <\/p>\n\n\n\n

 All of the harvested plant samples under 600ppm & 45\/35 \u02daC showed a good antibacterial activity against S. aureus <\/em>(Table 4). The leaves had stronger activity under treatment than the control, while the underground stem and root extracts had slightly larger zones of inhibition under treatment compared to the control (p<\/em> < 0.05; ANOVA). An interesting pattern was noted in the antibacterial activity of the leaves under 600ppm & 45\/35 \u02daC against E. coli <\/em>(Table 5). The activity greatly decreased between the control and 48 hours (6.00 \u00b1 3.00 mm; p<\/em> < 0.05) indicating bacterial resistance. The leaves then increased in their activity, from intermediate at 96 hours to susceptible at 144 hours (20.33 \u00b1 1.20 mm; p<\/em> < 0.05). Slight variations in the zones of inhibition of the underground stem and root extracts were reported, but both plant parts maintained a good antibacterial activity from all harvest periods under elevated CO2 <\/sub>and high temperatures (p<\/em> < 0.05). <\/p>\n\n\n\n

800ppm and 40\/30 \u02daC <\/strong> <\/p>\n\n\n\n

A similar pattern was as the previous two treatments was observed in 800ppm & 40\/30 \u02daC, where all plant parts showed a moderate growth inhibitory effect against S. aureus <\/em>(Table 4). The leaves and underground stems both displayed significance (p<\/em> < 0.05), while the harvested roots were not significant to the control (p<\/em> > 0.05). Each plant part showed a consistent zone of inhibition between 48 and 192 hours under 800ppm & 40\/30 \u02daC, showing overall significance against E. coli<\/em> (p<\/em> < 0.05; Table 5). The leaves had a range between 12 and 16 mm, the underground stems, being the most consistent, around 16-17 mm, and the roots, between 15-16 mm. <\/p>\n\n\n\n

800ppm and 45\/35 \u02daC<\/strong> <\/p>\n\n\n\n

The leaves and underground stems had small variations in the zones of inhibition. Though the leaves harvested under 800ppm & 45\/35 \u02daC had shown an increase in activity compared to the control, the underground stems displayed significantly smaller diameters than the control (p<\/em> < 0.05). The root extracts from samples harvested at 96 (18.00 \u00b1 0.58 mm) and 192 hours (17.67 \u00b1 0.33 mm; p<\/em> < 0.05) had greater zones of inhibition than the control. Overall, a good antibacterial activity was reported from the plant extracts (Table 4). The leaves, underground stems, and roots all displayed an intermediate antibacterial activity against E. coli<\/em> under 800ppm & 45\/35 \u02daC (p<\/em> < 0.05). The control leaf extract was found to be stronger in activity, as the zone of inhibition was higher than all the extracts from the elevated conditions. The underground stems and roots had greater zones of inhibition at 96 hours (16.67 \u00b1 0.67 mm) and 48 hours (16.33 \u00b1 0.33 mm) compared to their respective control (Table 5). <\/p>\n\n\n\n

Table 4: The effect of simultaneous elevated CO2<\/sub> and temperatures on the inhibitory activity of B. natalensis<\/em> against S. aureus<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment<\/strong><\/p>\n<\/td>\n

\n

Harvest period<\/strong><\/p>\n<\/td>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

Underground stems<\/strong><\/p>\n<\/td>\n

\n

Roots<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

N\/A<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

15.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

600ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

13.00b<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

12.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

14.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

13.33b<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

11.33b<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

13.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

17.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

10.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

11.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

15.33ab<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

12.67b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

12.67a<\/sup> \u00b1 1.20<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

\n<\/p><\/td>\n

\n

N\/A<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

15.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

600ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

15.67a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

12.33bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

15.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

17.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

10.67c<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

14.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

12.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

11.67bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

12.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

16.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

13.33ab<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

14.67a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n<\/td>\n\n

N\/A<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

15.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

800ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

12.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

12.00b<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

13.00a<\/sup> \u00b1 1.00<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

11.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

13.67a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

11.67a<\/sup> \u00b1 1.67<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

10.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

15.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

13.33a<\/sup> \u00b1 1.20<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

10.67a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

13.67a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

12.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

\n<\/p><\/td>\n

\n

N\/A<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

15.67b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

800ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

11.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

10.67c<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

13.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

11.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

13.33ab<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

18.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

11.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

11.67bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

12.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

12.67a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

11.33bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

17.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are expressed as mean (mg\/ml) \u00b1 SE. Mean values of different superscript letters in the same column indicates significance (p<\/em> < 0.05).<\/p>\n\n\n

Table 5: The effect of simultaneous elevated CO2<\/sub> and temperatures on the inhibitory activity of B. natalensis<\/em> against E. coli<\/em>.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Treatment<\/strong><\/p>\n<\/td>\n

\n

Harvest period<\/strong><\/p>\n<\/td>\n

\n

Leaves<\/strong><\/p>\n<\/td>\n

\n

Underground stems<\/strong><\/p>\n<\/td>\n

\n

Roots<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

18.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

13.33ac<\/sup> \u00b1 0.88<\/p>\n<\/td>\n<\/tr>\n

\n

600ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

11.67b<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

14.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

12.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

11.00b<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

13.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

10.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

11.67b<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

12.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

11.33bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

11.00b<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

15.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

18.33ab<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

15.33ab<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

13.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n<\/tr>\n

\n

600ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

6.00c<\/sup> \u00b1 3.00<\/p>\n<\/td>\n

\n

13.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

12.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

16.33ab<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

14.33ab<\/sup> \u00b1 1.20<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

20.33a<\/sup> \u00b1 1.20<\/p>\n<\/td>\n

\n

15.33ab<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

13.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

11.33bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

17.00a<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

13.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

18.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

15.33ac<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

13.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n<\/tr>\n

\n

800ppm & 40\/30 <\/strong>\u02da<\/strong>C<\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

14.67bc<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

16.33bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

16.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

16.33ab<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

16.67a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

15.33ac<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

16.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

15.67a<\/sup> \u00b1 1.20<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

12.67c<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

16.67bc<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

15.67a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

18.33a<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

15.33ac<\/sup> \u00b1 0.88<\/p>\n<\/td>\n

\n

13.33ac<\/sup> \u00b1 0.88<\/p>\n<\/td>\n<\/tr>\n

\n

800ppm & 45\/35 <\/strong>\u02da<\/strong>C <\/strong><\/p>\n<\/td>\n

\n

48 h<\/p>\n<\/td>\n

\n

14.00ab<\/sup> \u00b1 0.58<\/p>\n<\/td>\n

\n

13.33bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

16.33a<\/sup> \u00b1 0.33<\/p>\n<\/td>\n<\/tr>\n

\n

96 h<\/p>\n<\/td>\n

\n

15.00ab<\/sup> \u00b1 1.00<\/p>\n<\/td>\n

\n

16.67a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

15.33a<\/sup> \u00b1 0.67<\/p>\n<\/td>\n<\/tr>\n

\n

144 h<\/p>\n<\/td>\n

\n

12.67b<\/sup> \u00b1 0.67<\/p>\n<\/td>\n

\n

12.33b<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

15.00ac<\/sup> \u00b1 1.15<\/p>\n<\/td>\n<\/tr>\n

\n

192 h<\/p>\n<\/td>\n

\n

14.00ab<\/sup> \u00b1 1.53<\/p>\n<\/td>\n

\n

13.67bc<\/sup> \u00b1 0.33<\/p>\n<\/td>\n

\n

12.33bc<\/sup> \u00b1 0.88<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Values are expressed as mean (mg\/ml) \u00b1 SE. Mean values of different superscript letters in the same column indicates significance (p<\/em> < 0.05).<\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

South\nAfrica\u2019s geographical location makes it extremely vulnerable to climate change\ndue to its warm, arid conditions. It has been predicted that these conditions\ncould worsen in coming years.26<\/sup> Subsequently, the increase in CO2<\/sub>\ncould affect phytochemical biosynthesis, further impacting medicinal plant\nbioactivity.27<\/sup> The current study aimed to assess the implications of\nsimultaneous elevated CO2<\/sub> and high temperatures on the phytochemical\nprofile and bioactivity of B. natalensis<\/em>.\nThe qualitative screening of phytochemical compounds revealed that majority of\nthe compounds in both control and samples from the experimental conditions were\npresent in the leaves followed by the roots and the underground stems. The\noccurrence of phytocompounds having a higher presence was also the same (leaves\n> roots > underground stems). One possible explanation could be a result\nof the functional morphology of the leaves, which serve as the primary organ\nfor photosynthesis, but also temporarily stores secondary metabolites.28<\/sup>\nThe duration of exposure to the elevated conditions could have also influenced\nthe accumulation of phytochemicals.29, 30<\/sup> It is important to note\nthat qualitative screening of phytochemicals is merely indication of the\npresence of compounds, and further quantitative analyses are recommended to\nassess their quantity. <\/p>\n\n\n\n

The concentrations of total phenolics, flavonoids,\ntannins and proanthocyanidins mostly increased after 48 hours when B. natalensis<\/em> was exposed to various\ntreatments of elevated CO2<\/sub> and high temperatures. This response\ncould be an adaptive strategy to the changing environmental conditions, as\nsecondary metabolism is favoured over primary metabolism.31<\/sup> An\ninteresting discovery was that the underground stems contained the highest\noverall concentrations of the quantified phytochemical groups, followed by the\nroots. B. natalensis<\/em> is a geophyte\nsucculent, meaning it possesses a large stem or corm underground that is mainly\nused as a storage organ and comes in handy in situations of environmental\nstress.32<\/sup> This reason could explain why a higher phytochemical\ncontent was reported from the underground stems. Apart from their anchoring\nfunction, roots also play a storage role in plants.33<\/sup> The\nconcentration of total tannins in B.\nnatalensis<\/em> in both control and experimental treatments was comparatively\nhigher than the TPC, TPAC and TFC. Secondary metabolites are generally involved\nin plant defense against abiotic and biotic factors which in turn induces\noxidative stress.34<\/sup> Tannins are said to have a comparatively greater\nantioxidant activity than other phytochemical compounds because of their higher\npolymerization and molecular weight.35<\/sup> Tannins also have an affinity\nfor chelating metal ions such as iron, lead, and copper, which contributes to\ntheir pharmacological applications, especially antimicrobial activity.35-38<\/sup>\nThis possibly indicates that tannins could be the greatest contributor to the\nspecies\u2019 adaptive strategies as well as their antioxidant and antimicrobial\nproperties, considering their higher content under elevated CO2<\/sub> and\nhigh temperatures. Tannins are further classified into two groups, namely\nhydrolysable tannins and condensed tannins or proanthocyanidins.39<\/sup>\nSince the total proanthocyanidin content of B.\nnatalensis<\/em> was assessed, and results showed that their concentration was\nlower compared to the total tannins, this accumulation could allude that\nhydrolysable tannins such as ellagitannins and gallotannins are produced in\ngreater concentrations than proanthocyanidins. Furthermore, the hydrolysable\ntannins could be the driving force in the defense mechanism of B. natalensis<\/em> under concurrent elevated\nCO2<\/sub> and high temperatures. <\/p>\n\n\n\n

The plant antioxidant system, which consists of\nenzymatic and non-enzymatic antioxidants, plays a crucial role in the\nprotection against environmental stress. Phytochemicals, which make up the\nnon-enzymatic antioxidants, would normally accumulate as an adaptive response\nto environmental factors. Under various elevated CO2<\/sub> and high\ntemperature treatments, B. natalensis<\/em>\nshowed a comparatively stronger scavenging activity against hydrogen peroxide\nand iron radicals than DPPH. The underground stems greatly improved their DPPH\nscavenging activity compared to the control, whereas the leaves and roots\nmanaged to balance their potential between an excellent and a good activity.\nAll three plant parts mostly exhibited an excellent H2<\/sub>O2<\/sub>\nscavenging activity from each treatment, although, the IC50<\/sub>\nconcentrations were slightly higher than their respective controls. A similar\nresponse was noted from the iron chelating assay. Overall, the leaves were\nfairly better at quenching the radicals than the underground plant organs.\nSeveral reports have mentioned that the antioxidant activity varies when plants\nare exposed to elevated CO2<\/sub> and high temperatures. Alia (2019)40<\/sup>\nreported that the DPPH activity of Hibiscus\nsabdariffa<\/em> var. UKMR-2 significantly increased under 800 \u03bcmol\/mol compared\nto 400 \u03bcmol\/mol. One study discovered that the ferric reducing potential (FRAP)\nof basil (Ocimum basilicum<\/em> L.) and\npeppermint (Mentha piperita<\/em> L.)\nincreased under 620 ppm in comparison to the plants under 360 ppm respectively.41<\/sup>\nAnother experiment focusing on the influence of temperatures on Houttuynia cordata<\/em>, found that the\nincrease in temperature showed an increase in the total phenolic and total\nflavonoid contents at 35 \u02daC compared to 30\/25 \u02daC, 25\/20 \u02daC, 20\/15 \u02daC and 15\/10\n\u02daC respectively.42<\/sup> A longer exposure to high temperatures was\nreported to decrease the antioxidant capacity of Heracleum sosnowskyi.<\/em>43<\/sup> The results showed the lowest\nDPPH and ABTS activities when the plants were exposed to 30 \u02daC for seven days\ncompared to a shorter period of exposure at the same temperature and 35 \u02daC for\ntwo days respectively. This indicates that the variation of the antioxidant\nactivity of B. natalensis<\/em> under\nelevated CO2<\/sub> and high temperatures could also be influenced by the\nperiod of exposure. This further shows that the species could be well prepared\nfor the changing climate in the near future, as the antioxidant system seems to\nbe in optimal condition. The improvement of the antioxidant activity could also\nresult in a positive change in the medicinal properties of B. natalensis<\/em>. <\/p>\n\n\n\n

The underground stems and roots had a consistent antibacterial\nactivity against S. aureus<\/em> and E. coli<\/em> under combined elevated CO2<\/sub>\nand high temperatures, exhibiting a moderate activity (10-19 mm). The leaves\nshowed a significant improvement under the experimental treatments against S. aureus<\/em>, with each of the harvested\nmaterial having an intermediate inhibitory activity compared to the control, which\nshowed complete bacterial resistance. It would seem as though neither elevated\nCO2<\/sub> and temperatures nor the duration of exposure, greatly\ninfluences the antibacterial activity of B.\nnatalensis<\/em>, except for the inhibitory activity of the leaves against S. aureus<\/em>. However, the changes in the\nphytochemical profile and the antioxidant activity could be responsible for the\nconsistency of the species\u2019 antibacterial activity.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Climate change seems to induce\nunprecedented effects on the adaptability of medicinal plants. Future\nconditions could greatly alter the biochemical and physiological responses to\nelevated CO2<\/sub> and temperatures, which further raises the importance\nof conducting environmental stress research on one of the most vulnerable\nclasses of plants on Earth. The combined effects of elevated CO2<\/sub> and\ntemperatures on B. natalensis<\/em> for\nthis study revealed that the short-term exposure to these conditions greatly\ndetermined the overall medicinal activity at any given harvest period, from all\ntreatments. The changes in the phytochemical profile and the antioxidant\nactivity also provides an idea on how the species respond the harsh conditions,\nwhich further explains how their defence mechanisms react with the aim of\nacclimatisation and sustainability. In some of the harvest periods of the\nvarious treatments, the bioactivity showed promising improvement from the\nspecies\u2019 antioxidative and antibacterial analyses, which gives insight on the\npossibility of the species to treat other conditions which have yet to be\nassessed. From a pharmaceutical standpoint, these results could lead to the introduction\nof the cultivation of the species in controlled-growth environments, where the\nbioactive compounds can be manipulated based on the interests of quantity in\norder to develop a multitude of health-promoting products that are naturally\nsourced, reducing the amount of synthetic products. <\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

The authors would like to thank the\nUniversity of the Witwatersrand, Johannesburg\u2019s School of Animal, Plant and\nEnvironmental Sciences Laboratory of Medicinal Plants, for providing facilities\nto perform this study. <\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The author(s)\ndeclares no conflict of interest.\n<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

Gratitude is extended to the National\nResearch Foundation (NRF- Grant number: MND210614614611021) for their financial\nsupport in this study.<\/p>\n\n\n\n

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