{"id":61849,"date":"2024-12-30T11:14:41","date_gmt":"2024-12-30T11:14:41","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=61849"},"modified":"2025-01-06T18:37:28","modified_gmt":"2025-01-06T18:37:28","slug":"effect-of-concurrent-extreme-temperatures-and-water-deficit-on-the-phytochemistry-antimicrobial-and-antioxidant-activities-of-portulacaria-afra-jacq-using-four-extraction-solvents","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no4\/effect-of-concurrent-extreme-temperatures-and-water-deficit-on-the-phytochemistry-antimicrobial-and-antioxidant-activities-of-portulacaria-afra-jacq-using-four-extraction-solvents\/","title":{"rendered":"Effect of Concurrent Extreme Temperatures and Water Deficit on the Phytochemistry, Antimicrobial and Antioxidant Activities of Portulacaria Afra Jacq Using Four Extraction Solvents"},"content":{"rendered":"\n

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

Abiotic factors\nencompass a spectrum of challenging environmental elements that significantly\ninfluence ecological systems. These factors include, but are not limited to,\ntemperature variations, drought occurrences, salinity levels, heavy metal\nconcentrations, water stress, cold temperature exposure, anoxic conditions,\nhigh-intensity light exposure, and imbalances in nutrient availability1<\/sup>. <\/p>\n\n\n\n

The impact of abiotic\nfactors, notably temperature fluctuations and water deficits, presents a\nsignificant impediment to the reproductive processes of medicinal plants. This\ninterference consequently disrupts biosynthetic pathways, which in turn affects\nsecondary metabolites accumulation and subsequent alterations in\nphytopharmacological activities2<\/sup>.<\/p>\n\n\n\n

Elevated temperatures\ninduce heat stress, while reduced temperatures result in cold stress. The\nsubstantial impact of high temperatures extends to the composition of\nphytocompounds3<\/sup>. Elevated temperatures typically correlate with heightened\nbiosynthesis of secondary metabolites in plants, thereby enhancing the\nproduction of secondary metabolites across various plant species4<\/sup>. Conversely, high\ntemperatures inducing stress were observed to result in reduced levels of\nsecondary metabolites in plants5<\/sup>.<\/p>\n\n\n\n

Water deficit results in\na decrease of water absorption and potentials within plants, consequently\nnegatively impacting various physiological processes and altering the\nbiosynthesis of secondary metabolites6<\/sup>. Earlier research has\ndemonstrated the profound impact of water deficit or drought stress on the\ncomposition of secondary metabolites in herbal plants, leading to increased\naccumulation. An instance is the increased proportion of flavonoids and\nphenolics observed in plants as a response to the\nstress-induced by water deficit7<\/sup>. <\/p>\n\n\n\n

In adverse environmental\nconditions, plants exhibit resilience against diverse abiotic stresses through\nthe activation of adaptive mechanisms8<\/sup>. These mechanisms\nencompass the synthesis of chemicals facilitating the assembly of proteins and\ncellular structures, osmotic adjustments, maintenance of general homeostasis,\nand modulation of the antioxidant system. Additionally, this response\npotentially leads to heightened production of secondary metabolites such as\nphenolics1<\/sup>.<\/p>\n\n\n\n

According to recent\nclimate change models, it is more likely than previously thought that plants\nwill experience novel or rigorous concurrent abiotic stresses in the years to\ncome9<\/sup>. <\/p>\n\n\n\n

Earlier research\nfindings have reported that plant responses to stresses caused by combined\nantagonistic abiotic factors, are phenomenal compared to when exposed to single\nones9<\/sup>. The long-term effects of these abiotic factors\nmay significantly affect the quality and biochemical productivity of\nmedicinal plants such as P. afra<\/em> by disrupting the phytopharmacological\nactivities, metabolic, developmental, and physiological processes9<\/sup>. These have in turn\ncontributed to the need to study abiotic stress combinations within medicinal\nplant species.<\/p>\n\n\n\n

Portulacaria\nafra<\/em> is a succulent evergreen plant indigenous\nto South Africa,\nKenya, and Mozambique. The\ntaxonomic classification of Portulacaria afra<\/em> falls within the family Portulacaceae<\/em>\nor alternatively recently categorized under Didiereaceae<\/em>. <\/em>According to\nethnobotanical knowledge, and a few studies, P. afra<\/em> leaves and leaf juice is renowned for its curative\nproperties against skin diseases and oral infections10<\/sup> andphytochemical\nstudies on P. afra<\/em> tissue extracts\nrevealed a strong presence of various phytochemicals11<\/sup>. Also, it was\npreviously demonstrated that P. afra<\/em>\nextracts exhibited high levels of therapeutic properties in all plant parts,\nwith a strong presence of coumarins known for anti-cancer properties in the\nmethanolic leaf and stem extract12<\/sup>.Basson13<\/sup>, also reported how P.\nafra<\/em> leaves showed significant increase in phytochemicals and biological\nactivities after exposure to elevated CO2<\/sub>. The lack of comprehensive\nscientific documentation regarding the impact of combined environmental factors\non P. afra<\/em> is a notable limitation. This prompted the need to\ninvestigate on the impact of concurrent extreme temperatures (hot and cold)\nwith water deficit on the phytochemistry, antimicrobial  and antioxidant activities in P. afra<\/em>. <\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1: Portulacaria afra<\/em> leaves, stems, and roots<\/strong><\/p>\n

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

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

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

In accordance with\nstandard laboratory protocols, analytical-grade chemicals and solvents were\nused throughout the experimental procedures. The specific substances, namely\ngallic acid, Folin-Ciocalteau reagent, methanol, n<\/em>-hexane, ethyl\nacetate, Mueller-Hinton agar, Baird Parker agar, dimethyl sulfoxide (DMSO),\n2,2-diphenylpicrylhydrazyl (DPPH), and iron (II) chloride, were procured from Sigma-Aldrich, USA,\nbearing the corresponding CAS numbers1162-658. <\/p>\n\n\n\n

In this study, the\nwalk-in convirons (Conviron, Winnipeg, Canada; model No-PGW40)  with specifications meticulously detailed\nbearing serial N0-170028 and manufactured in China (280033R01) were used. It\noperates at 230\/400 Volts, 50 Hz frequency, and a three-phase system. The total\ninput amperage is 29.2, with the compressor drawing 65.5 Amps, while designed\nto function under high pressure at 400 PSI and low pressure at 200 PSI.\nControl, lighting, heater, fan, and drier systems require 2.0, 9.4, 4.3, 2.6\nHP, and 10.9 Amps respectively. Additionally, specific details regarding\nreceptacle, glycol pump, motor, and auxiliary amperages were included. The\nsystem\u2019s minimum circuit ampacity (MCA) is 31.2 Amps, with a maximum\novercurrent protection (MOP) set at 32 Amps, and a robust short circuit\nwithstand capacity of 5000 Amps RMS.<\/p>\n\n\n\n

Plant Material collection<\/strong><\/p>\n\n\n\n

In July 2021, cuttings of Portulacaria afra<\/em> were cultivated at the University of the Witwatersrand in Johannesburg, South Africa (26.1899\u00b0 S, 28.0319\u00b0 E) for propagation. The plant specimen was verified by a botanist and a voucher specimen (IRM 001) was placed in the institute’s herbarium centre.<\/p>\n\n\n\n

Green\ncuttings with non-lignified stems were carefully chosen from a pathogen-free\nparent plant of P. afra<\/em>. To hold the stem cuttings for roots, 225 pots\nmeasuring 2 to 2.5 litres each were filled with professional cutting mix soil\nfrom Culterrean Potting. <\/p>\n\n\n\n

The\nplants underwent a three-month acclimatization period and root system\ndevelopment in the Oppenheimer Life Sciences greenhouse facilities within the\npremises of University of the Witwatersrand in Johannesburg, South Africa (26.1899\u00b0 S, 28.0319\u00b0 E). The\nplant growth was observed and sustained by administering 500 ml of water per\npotted plant every 48 hours due to their low water requirement.  <\/p>\n\n\n\n

 Table 1: Experimental Design<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n

 <\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

Water deficit<\/p>\n<\/td>\n

\n

500ml of water every
2nd day.<\/p>\n<\/td>\n

\n

No water<\/p>\n<\/td>\n

\n

No water<\/p>\n<\/td>\n

\n

No water<\/p>\n<\/td>\n

\n

No water<\/p>\n<\/td>\n<\/tr>\n

\n

Temperatures
(\u02daC night\/day)<\/p>\n<\/td>\n

\n

25\/27<\/p>\n<\/td>\n

\n

0\/5<\/p>\n<\/td>\n

\n

10\/15<\/p>\n<\/td>\n

\n

30\/40<\/p>\n<\/td>\n

\n

35\/45<\/p>\n<\/td>\n<\/tr>\n

\n

Episodic harvesting frequency (hrs) <\/p>\n<\/td>\n

\n

Once off <\/p>\n<\/td>\n

\n

48, 96, 144<\/p>\n<\/td>\n

\n

48, 96, 144<\/p>\n<\/td>\n

\n

48, 96, 144<\/p>\n<\/td>\n

\n

48, 96, 144<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Sample size per harvest: n= 15\/harvest<\/p>\n\n\n

The conviron simulations settings used for the treatments were in accordance with the South African Department of Environmental Affairs predictions14<\/sup>, IPCC15<\/sup>, and Mbokodo16 <\/sup>records on the variations in the current and anticipated climatic conditions. The treatments were selected in terms of temperature ranges and the span of heat and cold waves.  Control temperature ranges were based on records from the South African Weather Service. The different temperature settings used were (25\/27\u00baC), (0\/5\u00baC), (10\/15\u00baC), (30\/40\u00baC), and (35\/45\u00baC). Each plant was placed in the controlled climate simulation chamber (Conviron chambers, PGW40) with the high and low temperatures set concurrently with water deficit while other parameters such as CO2<\/sub> (400 ppm), humidity (60%), light (160 nits\/level 1) pH, salinity, were kept at ambient.The lightning was programmed to operate for a duration of 12 hours daily, spanning from 6 am to 6 pm17<\/sup>.<\/p>\n\n\n\n

Five plants were\nharvested episodically 3 times every 48hrs for up to 6 days: (48hr, 96hr, 144hr)\n(Table 1). Samples were subjected to oven drying at 40\u00b0C for a period of 4 days\npost-harvest.This procedure was repeated three times for each of\nthe treatments. Where, n is [15 plants per harvest period-(48hrs, 96hrs,\n144hrs) =45 plants per treatment]; n=45 for control (25\u00b0), n=45 for (0\/5\u00b0C),\nn=45 (10\/15\u00b0C), n=45 (30\/40\u00b0C), n=45 (35\/45\u00b0C) with a total sample size of 225.<\/p>\n\n\n\n

Sample Preparation and Extraction<\/strong><\/p>\n\n\n\n

The leaves, stems, and\nroots were systematically gathered and subjected to a sequential process:\ninitial harvesting, followed by thorough washing using deionized water, and subsequent\ndrying in a hot air dryer maintained at 40\u00b0C for a duration of four days.\nPost-drying, the plant materials were finely ground into a crude powder using\nan electric grinder. This powdered form was then enclosed in foil and secured\nin an airtight container. Subsequently, it was stored in a dark cupboard at\nroom temperature (32\u00baC) until required for subsequent testing procedures18<\/sup>.<\/p>\n\n\n\n

Preparation of Extracts<\/strong><\/p>\n\n\n\n

The plant extract of the\nleaves, stems and roots were produced through sequential extraction using: 80%\nmethanol, n<\/em>-hexane, ethyl acetate, and distilled water, boiled at 60\u00b0C.\nEach extraction involved placing 3g of powdered plant material into individual\nglass bottles with cover, with the addition of 30 ml of the respective solvents19<\/sup>. The methanol, n-<\/em>hexane, ethyl acetate extracts were\nplaced on a shaker, the water extract underwent preparation\nusing a sonicator at a specific temperature (60\u00b0C) for a duration of 45 min.\nThe shaking of all extracts lasted 48 hrs, except for the water extracts. The\nwater extracts underwent centrifugation aimed at reducing their viscosity,\nfollowed by filtration through filter paper into vials. The residual\nsupernatant was removed, and subsequently, the vials were enveloped in foil and\nrefrigerated at 4\u00baC until the commencement of the tests.<\/p>\n\n\n\n

Preliminary Phytochemical Screening Tests<\/strong><\/p>\n\n\n\n

The chemical constituents\nwithin the leaves, stems, and roots extracts underwent evaluation using\nstandard colour test methods. A qualitative analysis was conducted in order to\ndetermine the presence or absence of quinones, coumarins, phenols, carbohydrates,\nterpenoids, flavonoids, glycosides, steroids, saponins, amino acids,\nphytosteroids, and volatile oils, through visual observation for\nalterations in colour or precipitation with the adoption of the procedure\ndescribed by Roghini and Vijayalakshmi20<\/sup>.  The observation for each test was  then documented. <\/p>\n\n\n\n

Evaluation of Total Phenolics and Total\nFlavonoids Contents<\/strong><\/p>\n\n\n\n

Folin-Ciocalteu reagent\nassay was used for the approximation of the total phenolics and flavonoids\ncontent in Portulacaria afra <\/em>extracts\nusing the procedure outlined by Teffo21<\/sup>. Standard solutions of\ngallic acid at varying concentrations were used to construct a calibration\ncurve. The concentrations of total phenolic content within the extracts were\nextrapolated from this curve, using the equation: y=0.9287x+0.0658, r2<\/sup>=0.9952.\nDenoting the findings as gallic acid equivalents per gram (mg GAE\/g) of the\nextract\u2019s dry weight.<\/p>\n\n\n\n

 Similarly, the total flavonoid contents of the\nplant parts were assessed using a quercetin-based standard curve, using the\nequation: y=0.2388x-0.0019, r2 <\/sup>=0.9997 with the results expressed as\nquercetin per gram (mg RE\/g) of dry weight. <\/p>\n\n\n\n

Antibacterial Activity Assay<\/strong><\/p>\n\n\n\n

The antibacterial\ninhibitory activity of Portulacaria afra<\/em>\nextracts was assessed by following the agar-well diffusion procedure described\nby Teffo22<\/sup> with a few modifications. The\nbacterial strains were obtained from Thermo-Fisher\u2019s laboratory specialists in\nJohannesburg (Pty) Ltd, South Africa, and the Mueller-Hinton (MH) agar was\npurchased from Sigma Aldrich Ltd. Briefly, Petri dishes were first sterilized and\nautoclaved prior to the addition of Mueller-Hinton agar and Baird Parker agar.\nOnce the agar solidified, sterile cotton swabs were used to uniformly inoculate\nthe surfaces of the agar with specific test microorganisms. Baird Parker agar\nplates were inoculated with Gram+ Staphylococcus\naureus<\/em> (ATCC 25923), whereas Mueller-Hinton agar plates were\ninoculated with Gram- Escherichia coli<\/em> (ATCC\n25922), and Gram+ Streptomyces griseus <\/em>and were incubated at 37\u00baC for\n24hours.<\/p>\n\n\n\n

Six wells were created\nin the inoculated media with successful growth of pathogens, using a 6 mm\nsterile borer. Subsequently, 100 \u03bcl of plant extracts were introduced into five\nof these wells, while the sixth well served as the negative control (dimethyl\nsulfoxide (DMSO). The plates were then covered and refrigerated for 30 minutes\nto allow for diffusion before being transferred to an incubator set at 37\u00b0C for\n24 hours.\n<\/strong>Following the incubation period, the diameters of the zones of\ninhibition were measured in millimetres using a ruler. The presence of an\ninhibitory zone indicated positive antibacterial activity. The experiment was\nconducted in triplicate, and the data were analysed using Microsoft Excel to\nexpress the results as mean \u00b1 standard deviation. P < 0.05 was used to\ndetermine the statistical significance of the findings.<\/p>\n\n\n\n

In vitro<\/em><\/strong> Antioxidant Activity Assessment<\/strong><\/p>\n\n\n\n

The\nassessment of antioxidant potential was conducted using the metal chelating\nactivity assay, hydrogen peroxide scavenging (H\u2082O\u2082), and the\n2,2-diphenylpicrylhydrazyl (DPPH) free radical assay for the respective plant\nparts as described below:<\/strong><\/p>\n\n\n\n

DPPH Scavenging Activity Assay<\/strong><\/p>\n\n\n\n

A 2, 2 diphenyl\npicryhydrazyl (DPPH) free radical assay described by Basson23<\/sup> was used with some\nmodifications. In the determination of the scavenging potential of DPPH, 50 mg of\nDPPH was dissolved in 100 ml of 80% methanol to create a stock solution. From\nthis stock solution, a working solution was derived at a ratio of 1:5 with 80%\nmethanol. Each extract had 5 concentrations, ranging from 10 to 50 \u03bcL, and was\nmixed with 700 \u03bcL of the working solution which was then adjusted to a final\nvolume of 1 ml with 80% methanol to create a reaction mixture. A control\nsolution was prepared using the working solution and 80% methanol, while a\nblank was generated using solely 80% methanol. These mixtures were then incubated\nin darkness for 45 minutes, after which the absorbance was measured at a\nwavelength of 517 nm using a spectrophotometer. The inhibition (%) was\nexpressed with the following equation:<\/p>\n\n\n\n

DPPH Scavenging Effect (%) = [(Absorbance of\nsample \u2013 Absorbance of blank)\/Absorbance of control] x 100<\/strong><\/p>\n\n\n\n

Metal Chelating Activity<\/strong><\/p>\n\n\n\n

The metal chelating\nantioxidant assay was carried out by following the procedure of Adusei24<\/sup> with few modifications.2 ml of\nextracts was combined with 0.25 ml of a 250 mM iron (II) chloride solution with\nan addition of 0.25 ml of ferrozine. The resulting mixture was vigorously\nshaken and left at room temperature (32\u00baC) for a duration of 10 minutes.\nFollowing this, the absorbance was quantified at a wavelength of 562 nm.\nMethanol was used as the blank, while a control group (without extracts) was\nused. The percentage of metal chelation was determined with the formula below:<\/p>\n\n\n\n

Metal chelation (%) = {(A0 \u2013 <\/em>A1)\/ A0} \u00d7 <\/em>100<\/p>\n\n\n\n

A0 represents the\nabsorbance of the control while A1 stands for the absorbance of the test\nsamples.<\/p>\n\n\n\n

Hydrogen Peroxide Scavenging (H\u2082O\u2082) Assay<\/strong><\/p>\n\n\n\n

The hydrogen peroxide\nscavenging activity was determined by adapting the methods of Bouabid25<\/sup>.<\/sup> 1 ml of extracts was\ncombined with 3.4 ml of phosphate buffer (50 mM, pH 7.4). Following this, 600\n\u03bcL of 40 mM hydrogen peroxide (30%) was introduced into the solution. The\nresulting mixture was left at room temperature (32\u00baC) for a duration of 40\nminutes. Subsequently, the absorbance of the solution was determined using a\nspectrophotometer at a wavelength of 230 nm. The hydrogen peroxide scavenging\nactivity was quantified using the provided formula:<\/p>\n\n\n\n

Scavenged H\u2082O\u2082 (%) = [(Absorbance of control \u2013 Absorbance of sample)\/Absorbance of control] x 1<\/p>\n\n\n\n

Statistical Analysis<\/strong><\/p>\n\n\n\n

The concentration values\nwere plotted against the % inhibition values in Microsoft Excel to derive a\ntrend line equation used for calculating the IC50<\/sub>. A one-way\nanalysis of variance (ANOVA) was used to determine the significant differences.\nAll experiments were conducted thrice, and data were presented as mean \u00b1\nstandard error (SE) for n = 3. <\/p>\n\n\n\n

P\n<\n0.05 was used to determine the statistical significance of the findings and\nTukey HSD post hoc tests in R Studio was used to determine where the\nsignificance lies. The significant differences (P\n< 0.05) were indicated by different letters (a, b, c, d) which were compared\nwith the control within the same group.<\/p>\n\n\n\n

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

Qualitative Screening of Phytochemicals<\/strong><\/p>\n\n\n\n

The aqueous (60\u00b0<\/sup>C), methanol, n-hexane,<\/em> and ethyl acetate extracts of P. afra <\/em>subjected to concurrent extreme hot and cold temperatures and water deficit were screened for their phytochemical constituents. For the qualitative phytochemical screening, the leaves, stems, and roots extracts were evaluated for the presence of saponins, flavonoids, glycosides, quinones, phenols, terpenoids, steroids, phytosteroids, volatile oil, carbohydrate, amino acids and coumarins. In general, phytochemicals in the plant parts varied according to the different solvents mentioned above. Increasing trends were noticed in the phytochemical presence under concurrent cold & hot temperatures with water deficit condition as shown in the heat map (Tables 2 and 3). Table 2 shows the fluctuations in the presence of phytochemical contents of the leaves, stems, and roots of P. afra<\/em>, when plant parts were subjected to concurrent cold & hot temperatures (10\/15\u00b0C and 30\/40\u00b0C) with water deficit conditions. Table 3 summarizes the results of the preliminary phytochemical screening under concurrent  cold & hot temperatures (0\/5\u00b0C and 35\/45\u00b0C) with water deficit conditions.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 2: Heat map for the phytochemical tests of Portulacaria afra extracts exposed to temperatures (10\/15\u00b0C and 30\/40\u00b0C) and water deficit<\/strong><\/p>\n<\/p>\n

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

Temp \u00baC, temperature; C, control samples; 48, 48hr treatment; 96, 96hr treatment; 144, 144hr treatment; W, hot water extract (60\u00baC) extract; M, methanolic extract; H, n<\/em>-hexane extract; E, ethyl acetate extracts. <\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 3: Heat map for the phytochemical tests of Portulacaria afra<\/em> extracts exposed to temperatures (0\/5\u00b0C and 35\/45\u00b0C) and water deficit<\/strong><\/p>\n<\/p>\n

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

Temp \u00baC, temperature; C, control samples; 48, 48hr treatment; 96, 96hr treatment; 144, 144hr treatment; W, hot water extract (60\u00baC) extract; M, methanolic extract; H, n<\/em>-hexane extract; E, ethyl acetate extracts. <\/strong><\/p>\n\n\n\n

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

Quantitative\nresults for the total phenolics (TPC) and total flavonoid content (TFC) in\nFigure 2 \u2013 9, show the variations in the phytoconstituents across all the plant\nextracts, under concurrent cold and hot temperatures [(10\/15\u00baC), (30\/40\u00baC)] and\n[(0\/5\u00baC), (35\/45\u00baC)] with water deficit condition. The highest TPC and TFC in\nthe plant extracts in comparison with the control samples were found in cold\ntemperatures (10\/15\u00baC) with water deficit settings (Figures 2 and 3\nrespectively). The above were recorded in the aqueous\nstem extracts after a 144-hour treatment period and aqueous leaf extracts after\na 48-hour treatment period (9921.55\u00b18.91mg GAE\/g and 7529.09\u00b13.12 mg QE\/g;\nFigures 2 and 3respectively) with control samples values for TPC\n(1820.67\u00b125.08 mg GAE\/g) and TFC values (592.70\u00b149.99 mg QE\/g) in the aqueous\nstem and leaf extracts respectively. ANOVA results showed a\nsignificant difference (P < 0.05) in P. afra, <\/em>Tukey test in R Studio\nrevealed where the significance lies among plant parts.<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 2: Total Phenolic content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 10\/15\u00baC and water deficit. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 3: Total Flavonoid content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 10\/15\u00baC and water deficit. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 4: Total Phenolic content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 30\/40\u00baC and water deficit. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 5: Total Flavonoid content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 30\/40\u00baC and water deficit. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 6: Total Phenolic content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 0\/5\u00baC and water deficit. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 7: Total Flavonoid content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 0\/5\u00baC and water deficit.<\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 8: Total Phenolic content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 35\/45\u00baC and water deficit. <\/strong><\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 9: Total Flavonoid content in Portulacaria afra<\/em> leaves (A), stems (B) and roots (C) extracts using four solvents under 35\/45\u00baC and water deficit.<\/strong><\/p>\n

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

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

Observed variation in the inhibitory effect of Portulacaria\nafra<\/em> extracts against the tested microorganisms are presented in Table 4 \u2013\n7. The highest level of inhibitory effect of 21mm\nagainst gram-negative Escherichia coli<\/em>\nin comparison with the control samples was observed in the methanolic root\nextracts after a 48hr-treatment period, upon exposure to combined extreme hot\ntemperatures (35\/45\u00baC) and water deficit, (Table 7). This was also followed by\na considerable effect of 14mm against Escherichia\ncoli<\/em> in the methanolic stem extracts (Table 7). Furthermore, the inhibitory\nactivities observed in the ethyl acetate extracts were intermediate, ranging\nfrom 9-12mm, with an increasing trend when compared with the control samples\n(Table 7). No inhibitory activity was observed against Escherichia coli<\/em> across all the plant\nextracts exposed to hot temperatures (30\/40\u00baC), however, intermediate\ninhibitory activities ranged from 11-13mm against gram-positive Streptomyces griseus<\/em> and Staphylococcus aureus <\/em>(Table 6). Under the cold temperatures\nsettings (10\/15\u00baC)  with water deficit\ncondition (Table  4), a slight inhibitory\neffect of 11mm was observed only in the n-<\/em>hexane\nand ethyl acetate root extracts against gram-positive Streptomyces griseus<\/em> and\nStaphylococcus aureus<\/em> after a 48hr and 96-hour treatment period\nrespectively, with a decline in activity when compared with the control\nsamples.  However, there were no\ninhibitory activities observed across all the plant extracts exposed to cold\ntemperatures (0\/5\u00baC) with water deficit condition (Table 5). From these\nfindings, the examined plant extracts exposed to temperatures [(30\/40\u00baC) and\n(35\/45\u00baC)] with water deficit condition showed better antimicrobial activity\nagainst all the tested microorganisms. ANOVA results showed a significant\ndifference (P < 0.05) in P. afra <\/em>extracts. The findings indicate that\nthe antimicrobial activity of P. afra<\/em> extracts is subject to the plant\npart, temperature and water deficit.
<\/p>\n\n\n\n

Table 4: Antibacterial activity of Portulacaria afr<\/em>a extracts against Escherichia coli<\/em>, Staphylococcus aureus, and Streptomyces griseus<\/em> under 10\/15\u00b0C with water deficit <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Zone of inhibition (millimetres) of extracts <\/strong><\/p>\n

Treatment period (hours)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

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

\n

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

\n

Water (60\u00baC)<\/strong><\/p>\n<\/td>\n

\n

Ethyl Acetate<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

16\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

14\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces griseus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Stems<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

\u00a0Streptomyces
griseus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

15\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

17\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

24\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

11\u00b1
0.3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces griseus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18\u00b1
0.6<\/p>\n<\/td>\n

\n

11\u00b1
0.1<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

C, Control sample extract; 48, 48hr-treatment extract; 96, 96hr-treatment extract; 144, 144hr-treatment extracts. Values are presented as mean \u00b1 Standard Deviation, n=3<\/p>\n\n\n

Table 5: Antibacterial activity of Portulacaria afr<\/em>a extracts against Escherichia coli<\/em><\/strong>, Staphylococcus aureus, and<\/strong> Streptomyces griseus<\/strong><\/em> under 0\/5\u00b0C with water deficit<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Zone of inhibition (millimetres) of extracts<\/strong><\/p>\n

Treatment period (hours)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Microorganism<\/strong>s<\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

Water (60\u00baC)<\/strong><\/p>\n<\/td>\n

\n

Ethyl Acetate<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

16\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

14\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces griseus<\/em><\/strong>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<\/em>\u00a0<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Stems<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces
griseus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

15\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

17\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

24\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces griseus<\/em><\/strong>\u00a0 \u00a0\u00a0<\/em>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<\/em>\u00a0<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

C, Control sample extract; 48, 48hr-treatment extract; 96, 96hr-treatment extract; 144, 144hr-treatment extracts. Values are presented as mean \u00b1 Standard Deviation, n=3.<\/p>\n\n\n

Table 6: Antibacterial activity of Portulacaria afr<\/em>a extracts against Escherichia coli<\/em><\/strong>, Staphylococcus aureus, and<\/strong> Streptomyces griseus<\/strong><\/em> under 30\/40\u00b0C with water deficit<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Zone of inhibition (millimetres) of extracts<\/strong><\/p>\n

Treatment period (hours)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

\u00a0<\/strong>Microorganisms\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

Water
(60\u00baC)<\/strong><\/p>\n<\/td>\n

\n

Ethyl Acetate<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia
coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

16
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus
aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

14
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces
griseus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Stems<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia
coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus
aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

11
\u00b10.1<\/p>\n<\/td>\n

\n

13
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

12\u00b1
0.1<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces
griseus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

11
\u00b10.1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia
coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

15
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus
aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

17
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

24
\u00b10.6<\/p>\n<\/td>\n

\n

13
\u00b10.1<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces
griseus<\/em><\/strong>\u00a0<\/em><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

C, Control sample extract; 48, 48hr-treatment extract; 96, 96hr-treatment extract; 144, 144hr-treatment extracts. Values are presented as mean \u00b1 Standard Deviation, n=3.<\/p>\n\n\n

Table 7:Antibacterial activity of Portulacaria afr<\/em>a extracts against Escherichia coli<\/em><\/strong>, Staphylococcus aureus, and<\/strong> Streptomyces griseus<\/strong><\/em> under 35\/45\u00b0C with water deficit<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

\u00a0 \u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Zone of inhibition (millimetres) of extracts <\/strong><\/p>\n

Treatment period (hours)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

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

\n

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

\n

Water (60\u00baC)<\/strong><\/p>\n<\/td>\n

\n

Ethyl Acetate<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

10
\u00b10.1<\/p>\n<\/td>\n

\n

16
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

10\u00b1
0.2<\/p>\n<\/td>\n

\n

11\u00b1
0.3<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

14\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces griseus<\/em><\/strong>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

18
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Stems<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

14\u00b1
0.1<\/p>\n<\/td>\n

\n

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

\n

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

\n

13
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

12\u00b1
0.1<\/p>\n<\/td>\n

\n

12\u00b1
0.3<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces griseus<\/em><\/strong>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0<\/em>\u00a0<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

13
\u00b10.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

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

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Escherichia coli<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

21\u00b1
0.1<\/p>\n<\/td>\n

\n

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

\n

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

\n

15\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

9\u00b1
0.1<\/p>\n<\/td>\n

\n

11\u00b1
0.2<\/p>\n<\/td>\n

\n

10\u00b1
0.1<\/p>\n<\/td>\n<\/tr>\n

\n

Staphylococcus aureus<\/em><\/strong><\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

17\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

24\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

Streptomyces griseus<\/em><\/strong>\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/em><\/p>\n<\/td>\n

\n

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

\n<\/td>\n\n

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

\n

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

\n

18\u00b1
0.6<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

0<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

C, Control sample extract; 48, 48hr-treatment extract; 96, 96hr-treatment extract; 144, 144hr-treatment extracts. Values are presented as mean \u00b1 Standard Deviation, n=3<\/p>\n\n\n

Antioxidant Activity<\/strong><\/p>\n\n\n\n

The in\nvitro<\/em> antioxidant potential of all the extracts was determined by a 2, 2 diphenylpicryhydrazyl (DPPH) free\nradical assay, hydrogen peroxide scavenging (H\u2082O\u2082), and metal chelating\nactivity assay, where IC50 <\/sub>value is the amount of extract required\nto decrease the absorbance of antioxidant radicals by 50%. Lower IC50 <\/sub>values\nindicate stronger antioxidant scavenging ability of the extracts. Figure 10 \u2013 21 graphically show the various\nchanges observed in the antioxidant activities of Portulacaria afra<\/em> extracts upon exposure to hot and cold\ntemperatures and water deficit, when compared with the control samples. Under\nthe cold temperatures settings [ (10\/15\u00baC) and (0\/5\u00baC)] and water deficit\n(Figure 10 \u201315), a significant increasing trend of antioxidant capacity was\nobserved across all plant parts when compared to the control samples.\nSimilarly, under hot temperatures [ (30\/40\u00baC) and (35\/45\u00baC)] with water deficit\nconditions (Figure 16 \u2013 <\/a>21), an increase in\nantioxidant activity was observed in some of the extracts in comparison with\nthe control samples. The highest DPPH and\nmetal-chelating antioxidant activity was observed in the ethyl acetate root\nextracts after a 96-hour treatment period and the methanolic leaf extracts\nafter a 144-hour treatment period (0.26\u00b10.065 mg\/ml; Figure 16) and (0.40\u00b10.078\nmg\/ml respectively; Figure 17), under concurrent hot temperatures settings\n(30\/40\u00baC) with water deficit. However, n<\/em>-hexane\nstem extracts under concurrent hot temperatures settings (35\/45\u00baC) with water\ndeficit conditions showed the strongest hydrogen peroxide scavenging activity\n(0.14\u00b10.048 mg\/ml; Figure 21) after a144-hour treatment period.<\/p>\n\n\n\n

Data available here reveal excellent\nantioxidant activity of all plant parts. The\nmethanolic leaf extracts showed the strongest antioxidant activity against\nferric ion radical, ethyl acetate root extracts also showed a very strong\nscavenging ability against DPPH radical, while n-<\/em>hexane stems extract\nscavenged hydrogen peroxide radical much better, indicating the strongest\nantioxidant activity of Portulacaria afra<\/em> across the three different\nassays. All IC50<\/sub> values were less than 1 mg\/ml with extracts\nshowing significant differences (P < 0.05).
<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 10: Graphical representation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity in Portulacaria afra <\/em>plant parts under 10\/15\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 11: Graphical representation of Metal chelating antioxidant activity in Portulacaria afra<\/em> plant parts under 10\/15\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 12: Graphical representation of Hydrogen peroxide antioxidant activity in Portulacaria afra<\/em> plant parts under 10\/15\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 13: Graphical representation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity in Portulacaria afra <\/em>plant parts under 0\/5\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 14: Graphical representation of Metal chelating antioxidant activity in Portulacaria afra<\/em> plant parts under 0\/5\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 15: Graphical representation of Hydrogen peroxide antioxidant activity in Portulacaria afra<\/em> plant parts under 0\/5\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 16: Graphical representation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity in Portulacaria afra <\/em>plant parts under 30\/40\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 17: Graphical representation of Metal chelating antioxidant activity in Portulacaria afra<\/em> plant parts under 30\/40\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 18: Graphical representation of Hydrogen peroxide antioxidant activity in Portulacaria afra<\/em> plant parts under 30\/40\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 19: Graphical representation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity in Portulacaria afra plant parts under 35\/45\u00b0C and water deficit.<\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 20: Graphical representation of Metal chelating antioxidant activity in Portulacaria afra<\/em> plant parts under 35\/45\u00b0C and water deficit. <\/strong>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 21: Graphical representation of Hydrogen peroxide antioxidant activity in Portulacaria afra<\/em> plant parts under 35\/45\u00b0C and water deficit.<\/strong>\n

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

Components such as root, stem, leaf, flower, fruits, and\nseed are categorised according to the synthesis, accumulation, and distribution\npatterns of phytochemicals26<\/sup>. <\/sup>In specific organs, tissues, and cells, distinct regulatory\npathways and transport routes facilitate the synthesis of diverse secondary\nmetabolites across various parts of medicinal plants. This phenomenon arises\ndue to the complexity and diversity inherent in these secondary metabolites26<\/sup>. <\/p>\n\n\n\n

Extraction solvents are important in the\nextraction of potential antioxidant compounds, and are determined by variations\nin chemical characteristics, polarities, and solubility of these compounds27<\/sup>. Hence, the solvents options for extraction of phytochemicals in\nthis study were chosen based on the polarity of the solute, for better accuracy\nin dissolving the solute27<\/sup>. The presence of phytochemicals in the leaves, stems, and\nroots extracts of P. afra <\/em>exposed to various treatments, when compared with the control, showed that the methanol\nextracts, across all the parts, in both hot & cold conditions showed the\nmost presence of phytochemicals, followed by the aqueous extracts. This is in accordance with earlier and current research\nas they are mostly recommended for phytochemical extractions28<\/sup>, due to their high polarity index of 5.1 and 10.2 respectively27<\/sup>, which also validates the use of hot water extraction in\ntraditional medicine.<\/p>\n\n\n\n

Previous findings have documented that methanol and water\ntarget sugars, amino acids, and glycosides during phytochemical extraction. On\nthe other hand, alkaloids, aglycones and glycosides are commonly extracted with\nethyl acetate while n-<\/em>hexane extracts\nwaxes, fats, fixed oils and volatile29<\/sup>. <\/p>\n\n\n\n

Tannins are complex polyphenolic biomolecules with abundant\nhydroxyls and carboxyl groups, they form strong complexes with macromolecules.\nThey are known for anti-inflammatory and curative properties, used in treating\npiles, burns, and venereal diseases, and serve as insecticides and regulators\nof plant growth30<\/sup>. <\/sup>The strong presence of tannins observed across all solvents\nin the leaf extracts could be responsible for anti-inflammatory and curative\nproperties of P. afra<\/em>, thus\nvalidating its suitability and potency for the treatment of piles, burns and\nsome venereal diseases.<\/p>\n\n\n\n

In the pharmaceutical industry,\nvolatile oils are commonly utilized flavouring agents and are known\nparticularly for their antibacterial and carminative attributesTop of Form31<\/sup>. Interestingly,\nin this study, n<\/em>-hexane, and ethyl\nacetate extracts across all plant parts exposed to hot (35\/45\u00baC) & cold\n(0\/5\u00baC) (Table 3) temperatures with water deficit showed the highest presence\nof volatile oil concentration, when compared to the other solvents. The\nconcentrations ranged from low to high, the observed outcome is not completely\nin accordance with earlier research, indicating that warmer seasons or hotter\ntemperatures present better concentrations of volatile oil and essential oils32<\/sup>. In contrast to the above reports,\nstudies have shown how low temperature enhances the production of several\ngroups of volatile terpenoids in essential oil glands on the leaf surface33<\/sup>. Similar observation was reported\nfor the increase in monoterpenes in sage leaves (Salvia officinalis L<\/em>.) and peppermint essential oil (Mentha piperita L.<\/em>) mostly under low temperatures33<\/sup>. The increase observed in the\nvolatile oil concentration across the different temperature settings in this\nstudy, could suggest the response of this plant species to the physical and\nchemical stress impacted simultaneously by the combined effect of both extreme\ntemperatures and water deficit than with individual stress. Thus, resulting to\nthe production of terpenoids, which are bioactive compounds that confer several\nbiological activities32<\/sup>. Conversely, volatile oils were produced in low to high\nquantity in all plant parts under the temperatures [hot (30\/40\u00baC); & cold\n(10\/15\u00baC)] (Table 2), while methanolic stem extracts\ndetected a strong presence of volatile oils in both treatments. <\/p>\n\n\n\n

Phenols are the most prevalent class of phytoconstituents\nthat perform protective roles by shielding plants from pathogens and\nantagonistic abiotic factors34<\/sup>. Methanolic extracts showed a high\npresence of phenols in all plant parts under the temperatures [hot (30\/40\u00baC);\n& cold (10\/15\u00baC)] (Table 2), with water deficit when compared with the\ncontrol samples. Meanwhile, the concentrations of terpenoids and\nsteroids, also showed an increase in comparison with the control sample\nextracts across all plant parts under both temperatures with water deficit.<\/p>\n\n\n\n

Coumarins present in all plant parts of Portulacaria afra<\/em>, which is commonly used in cancer treatment,\nrenal cell carcinoma and blood diseases has caught the attention of many\nresearchers for its photochemotherapy and therapeutic application in cancer35<\/sup>. Results as\nshown in the heat map (Table 3) suggest that the production of coumarins is\nbest under hot (35\/45\u00baC) & cold (0\/5\u00baC) temperatures with water deficit.\nThe methanolic and aqueous extracts of all plant parts, showed a significant\nincrease in comparison to the control in both hot (35\/45\u00baC) and cold (0\/5\u00baC)\ntemperatures with water deficit (Table 3). Plant carbohydrates are\nimportant biochemical indicators of temperature acclimation and cold tolerance\ndevelopment33<\/sup>. Therefore, the carbohydrate\nconcentration across all plant extracts, compared to the control samples showed\nan increasing trend only in the methanolic extracts. This observation shifted\nfrom absent to considerable amounts in both hot (35\/45\u00baC) & cold (0\/5\u00baC)\ntemperatures with water deficit (Table 3). While in the methanolic stem\nextracts exposed to temperatures [hot (30\/40\u00baC); & cold (10\/15\u00baC)]\ntemperatures with water deficit (Table 2), an increasing pattern was also\nnoted.<\/p>\n\n\n\n

Studies have shown that water stress may amplify the accumulation of\nphytochemicals36<\/sup>, contrary to the belief of the negative impact of severe\nwater deficit. This has been extensively researched to establish its effects on\nthe secondary metabolite profile, frequently leading to increasing output in\nmost cases, which indicates higher quality37<\/sup>. The qualitative phytochemical data from this study,\nhowever, is attributed to the simultaneous impact of both extreme hot and cold\ntemperatures with water deficit stress.<\/p>\n\n\n\n

A broad class of polyphenols with the chemical structure\nbenzoyl-pyrone are called flavonoids. They are prevalent everywhere in plants,\nproduced by the phenylpropanoid pathway and perform a wide range of\npharmacological actions38<\/sup>. It is believed that the organoleptic properties of fresh\nfruits, fruit juices, and wine are also significantly influenced by phenolic\nchemicals, including tannins and flavonoids. Therefore, their potential impact\non human health will primarily depend on the environmental variables that\nregulate their amounts and quality of accumulation in plants.<\/p>\n\n\n\n

It was observed that hot temperatures (35\/45\u00baC) with water\ndeficit increase the accumulation of total phenolics and total flavonoids in Portulacaria afra <\/em>(Figures 8 and 9), in comparison with\ncold temperatures (0\/5\u00baC) and water deficit. Al-Huqail1<\/sup> reported similar higher\naccumulation of total phenolics content (TPC) in plants under stress, especially in basil plants (Ocimum basilicum L.<\/em>) treated with high\ntemperature, as well as an increase in total phenolics content (TPC) and total\nflavonoids content (TFC) concentrations in response to water stress and a\nhighly significant increase in flavonoids under high climate temperature\ncondition. The increase in the contents of the chemical compounds analysed,\nagrees with data in the literatures indicating that this is a response to the\ngeneration of reactive oxygen species. Reactive oxygen species (ROS) are\nproduced by plants in response to environmental stress factors such as shade,\nexcessive salt levels, extreme temperatures, drought, or water deficit, which\nwhen present in excess can lead to oxidative stress.  Moreover, a vast variety of substances, such\nas arylpyrones and styrylpyrones, stibenes, tannins, coumarins, flavonoids,\nlignins, and lignans, are included in the category of phenolics and they are\nproduced in response to abiotic stress7<\/sup>. Consequently, they protect the plants and increase their\ntolerance against various abiotic and biotic stresses7<\/sup>. Phenolic compounds, due to their\nredox attributes, act as antioxidants, effectively neutralizing singlet oxygen,\ndisplaying notable pharmacological significance. They have demonstrated\nantioxidative, anticarcinogenic, antibacterial, and anti-inflammatory effects\nin previous findings1<\/sup>, which supports the traditional therapeutic use of P.\nafra<\/em>. Additionally, exogenous factors are responsible for the biosynthesis\nand accumulation of phenolic compounds while endogenous factors, developmental\nstage, and tissue differentiation39<\/sup> are responsible for the site of synthesis in plants. As a\nresult, variation in the climatic, biotic, and environmental factors\nexperienced during seasons can be used to explain the observed differences\nacross seasons. Hence, the content of phenolic compounds and their\ntemperature-dependent fluctuations as seen in this study, suggest that there is\na link between their distinct functions in each unique plant parts, and the\nstimuli causing their synthesis.<\/p>\n\n\n\n

Lately, there has been a significant rise in antibiotic\nresistance, creating a growing therapeutic challenge. Using medicinal plant\nmaterials is one way to lessen antibiotic resistance40<\/sup>. <\/sup>Plants create a wide range of\nchemicals to defend themselves against different diseases. Plant extracts with\ntargets locations different from those used by antibiotics are anticipated to\nbe effective against microorganisms with drug resistance41<\/sup>.<\/p>\n\n\n\n

The antimicrobial activity results of P. afra<\/em> extracts showed that the antibacterial activity of the extracts is subject to temperature with water deficit (Table  4 \u2013 6). The inhibitory activity observed in the methanolic root and stem extracts (Table 7) against Escherichia coli<\/em>, are similar to the report of previous research demonstrating maximum antibacterial activity against Escherichia coli<\/em> by the leaves of Amaranthus spinosus L.<\/em> during the summer month May-June42<\/sup>. The antibacterial activity observed could be correlated with the maximum building up of bioactive compounds in the methanolic stem and root of P. afra<\/em> as a response to the concurrent effect of hot temperatures (35\/45\u00baC) with water deficit. Conversely, the other extracts across the plant parts were not active against Staphylococcus aureus <\/em>and Streptomyces griseus. <\/em>A similar trend was observed in Harpephyllum caffrum <\/em>bark extracts where the highest inhibitory activity was detected with plant material collected during summer (December) with increasing antibacterial activity against gram-negative bacteria whereas it declined against gram-positive bacteria43<\/sup>. Meanwhile in hot temperatures (30\/40\u00baC) with water deficit (Table 6), no positive antibacterial activity was observed against Escherichia coli<\/em> across all the plant extracts. However, intermediate inhibitory activities, ranging from 11-13mm were observed against gram-positive Streptomyces griseus<\/em> and Staphylococcus aureus. <\/em>Here, the different fractions of the extracts appeared to be more effective against the gram-positive microorganisms. These results can be explained by studies that have proven that gram-negative bacteria have an outer membrane permeability barrier, which makes them more resistant to antimicrobial treatments than gram-positive bacteria44<\/sup>. This might also explain the inhibitory effect seen against S. aureus <\/em>and S. griseus <\/em>to the extracts and non-effectiveness of the tested fractions against E. coli<\/em>.<\/p>\n\n\n\n

The slight inhibitory effect of 11mm observed only in the n-<\/em>hexane and ethyl acetate root extracts\nagainst gram-positive Streptomyces\ngriseus<\/em> and Staphylococcus aureus<\/em>\nunder cold temperatures (10\/15\u00baC) with water deficit (Table 4), is partly in\nline with findings from Ncube45<\/sup>, where the best antibacterial activity was observed in\nwinter season from dichloromethane bulb extracts against S. aureus.<\/em> The observed variation in efficacy to inhibit\nmicroorganism growth may be as a function of the distinct cell wall layers\ncharacteristic of each microorganism46<\/sup>. The outcomes of this study\nmight be linked to various factors, including the chemical composition of\ndistinct solvents, temperature variations, specific plant parts used, microbial\nproliferation, and the extent of exposure of the test microorganisms46<\/sup>. <\/p>\n\n\n\n

Antioxidants inhibit and delay oxidation process, by\ntransferring electrons to free radicals to limit fluctuations and to prevent\nfurther reaction47<\/sup>. The antioxidant activity and medicinal power of plants are\nsignificantly dependent on the secondary metabolites present in them, which in\nturn results to a reduction of oxidative stress by absorbing and neutralising\nfree radicals48<\/sup>. Depending on the amount of antioxidant molecules present\nin plants, which are greatly influenced by their growth environment, plants\nhave various antioxidant properties49<\/sup>.<\/p>\n\n\n\n

Natural antioxidants attract attention in research, and food\nproduction, for their therapeutic properties and defence against oxidative\nstress-induced diseases50<\/sup>. In fact, they are most beneficial for use since they are\nmostly free of negative side effects in comparison to the synthetic ones49<\/sup>.<\/p>\n\n\n\n

Antioxidant potential of Portulacaria\nafra<\/em> leaves, stems, and roots extracts were investigated through a 2, 2\ndiphenylpicryhydrazyl (DPPH) free radical assay, hydrogen peroxide scavenging\n(H\u2082O\u2082) and metal chelating activity assay. <\/p>\n\n\n\n

The variations observed in Figure 10 \u2013 21 in this study could potentially be because of the\ndissimilarities in the concentration or potency of active compounds in each part of P. afra<\/em> under these stress conditions51<\/sup>. In addition, different antioxidant\nenzymes have temperature sensitivity, and they can only function at specific\ntemperatures52<\/sup>. Their actions also vary according to the crop varieties,\ntheir growth phases, and the growing season\u2019s tolerance or susceptibility to\ntemperature53<\/sup>. Moreover, the availability of various precursors that the\nplant needs to produce the active components can vary depending on the\ntemperature and time of the year54<\/sup>.<\/sup><\/p>\n\n\n\n

Thus, the results from this study are similar to Basson13<\/sup>, where accumulation of\nphytochemicals and biological activities in P. afra<\/em> were found to either\nremain constant or increased after exposure to elevated CO2<\/sub>.\nConsequently, these results suggest that the responses in the biological\nactivities in the plant parts under concurrent hot and cold temperatures with\nwater deficit condition serve as an adaptive and protective mechanism of P. afra <\/em>against the concurrent stressors\nexposed to in this study.<\/p>\n\n\n\n

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

In conclusion, the phytochemical content, and biological\nactivities in Portulacaria afra<\/em> were\nsignificantly influenced by extreme temperature simulations and water deficit.\nThe observed variability in plant responses provided significant insights into\nthe diverse reactions of various plant parts to distinct environmental stimuli\nand extraction solvents. These include the modulation of secondary metabolites,\nalterations in antibacterial properties, and adjustments in antioxidant\nactivities, highlighting fluctuations in response to the combined abiotic\nfactors. It is evident that abiotic factors in combination may influence the\nbiological activities of P. afra<\/em>, which shows the adaptation and\nresilience of the plant reflected in its ability to still provide therapeutic\ntreatment against targeted diseases. Consequently, optimum\ntemperatures and water deficit conditions are recommended for peak\nconcentrations of phytochemicals and biological activities in P. afra<\/em>.\nAlthough the results provide valuable insights, one\nlimitation of this study is the use of one plant species, which may affect the generalizability\nof the findings for\ncombined temperature and water deficit stress on plants. This is because plant\nresponses are species dependent. More diverse plant\nspecies would be necessary to further confirm the trends observed both in this\nstudy and previous research in order to strengthen the overall validity of the\nconclusions. Further studies are still recommended for the characterization of\nthe bioactive compounds in these extracts for in vivo<\/em> studies. <\/p>\n\n\n\n

Acknowledgements<\/strong><\/p>\n\n\n\n

Authors are Grateful to South Africa\u2019s\nNational Research Foundation (NRF) for supporting this work. Special\nappreciation to Michael Tobin, Gontse and Mokgadi for assistance with\nLaboratory related work.<\/p>\n\n\n\n

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

This work was supported by South Africa\u2019s\nNational Research Foundation (NRF) under grant number:TTK201129577193<\/p>\n\n\n\n

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

The author(s) do not have any conflict\nof interest.<\/p>\n\n\n\n

Data Availability Statement- <\/strong><\/p>\n\n\n\n

This statement does not apply to this\narticle<\/p>\n\n\n\n

Ethics Statement- <\/strong><\/p>\n\n\n\n

This research did not involve human participants, animal subjects,\nor any material that requires ethical approval.<\/p>\n\n\n\n

Informed Consent Statement<\/strong><\/p>\n\n\n\n

This study did not\ninvolve human participants, and therefore, informed consent was not required<\/p>\n\n\n\n

Clinical Trial Registration-<\/strong><\/p>\n\n\n\n

This\nresearch does not involve any clinical trials<\/strong><\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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