Effect of concurrent extreme temperatures and water deficit on the phytochemistry, antimicrobial and antioxidant activities of Portulacaria afra using four extraction solvents
Authors: Adeleye Oluwafunbi Christianah , Risenga Ida Masana 
Published: December 2023

Abstract
Background
The phyto-medicinal and ethnopharmacological significance of Portulacaria afra has been documented by a few studies, with high presence of coumarins known for anti-cancer properties. This indigenous South African plant, recognized for its succulent nature and medicinal properties, remains relatively under-researched. Furthermore, abiotic factors, seasonal changes, plant part and harvest period affect the phytochemical composition and secondary metabolites accumulation of medicinal plants. As of the present, there remains an absence of published data pertaining to the impact of environmental stress factors on crucial biological activities of P. afra. Therefore, this study aimed to investigate, for the first time, the phytochemical composition, antimicrobial and antioxidant activities of P. afra’s plant parts, using three antioxidant assays, with four extraction solvents of different polarities under concurrent extreme temperatures (hot & cold) and water deficit condition.
Results
The present study examined the simultaneous impact of varying temperatures [(mid-range high (30/40ºC); and mid-range low (10/15ºC), extreme high (35/45ºC); and extreme low (0/5ºC)] together with water deficit, on the antimicrobial, antioxidant properties, in addition to the extraction of phytochemical compounds from the methanol, ethyl acetate, n-hexane, and aqueous (60°C) extracts of P. afra The determination of combined abiotic stress-induced acclimation's impact on the qualitative and quantitative composition of certain phytochemicals was carried out. The assessment of antioxidant potential within leaf, stem, and root extracts was conducted employing the metal chelating activity assay, hydrogen peroxide scavenging (H₂O₂), and the 2,2-diphenylpicrylhydrazyl (DPPH) free radical assay for the respective plant parts.
Qualitative phytochemical investigation of extracts of all the selected plant parts, compared to the control samples, showed an increasing trend with the stronger presence of coumarins, volatile oil, tannins, glycosides, flavonoids, phenols, quinones, terpenoids, saponins, steroids, phytosteroids, as well as carbohydrates while test for amino acids was negative. The qualitative phytochemical analysis clearly revealed that the concurrent cold temperatures [ (10/15ºC and 0/5ºC)] and water deficit condition, especially after a 144-hour treatment period, had the strongest phytochemical presence across all plant parts. In contrast to the control group, methanol, and aqueous extracts from all plant sections in both concurrent extreme temperatures with water deficit settings showed the most presence of phytochemicals.
In comparison to the control group, notable increases in both total phenolic (TPCs) and total flavonoid contents (TFCs) were evident across various plant parts, with varying concentrations depending on the extraction solvent, temperature, and treatment period. In the comparative analysis of plant components, the result showed that the extracts derived from various plant parts exhibited maximal accumulation of TPCs and TFCs during the period of concurrent extreme high temperatures (35/45ºC) with water deficit conditions. This was observed in the ethyl acetate leaf and methanolic leaf extracts, after a 96-hour treatment period (9962.29±35.63 and 8598.59±14.28 mg/g, respectively).
In this study, the antimicrobial efficacy of P. afra’s plant parts were investigated by implementing the agar-well diffusion technique. The assessment focused on evaluating the inhibitory effects against three distinct microorganisms, namely Staphylococcus aureus, Escherichia coli, and Streptomyces griseus. Experiments revealed that the plant extracts under the extreme hot temperatures (35/45ºC) with water deficit condition demonstrated more potent inhibitory effect against gram-negative E. coli compared to the gram-positive microorganisms. The highest level of inhibitory effect of 21mm was recorded against gram-negative E. coli from the methanolic root extracts after a 48hr-treatment period. However, the inhibitory activities mainly observed in the mid-hot temperatures (30/40ºC) were intermediate, ranging from 11-13mm against gram-positive Streptomyces griseus and Staphylococcus aureus. Observation from this investigation showed that plant extracts under the hot temperatures [mid-range high (30/40ºC); and extreme high (35/45ºC)] with water deficit condition showed better activity against all the microorganisms.
The antioxidant activity data showed that the most significant DPPH and metal chelating antioxidant activity was found under the mid-range hot temperatures (30/40ºC) in the ethyl acetate root extracts after a 96-hour treatment period and the methanolic leaf extracts after a 144-hour treatment period (0.26±0.065 and 0.40±0.078 mg/ml respectively), in comparison with IC50 values (3.00±0.092 and 6.66±0.629 mg/ml) for control samples of ethyl acetate root and methanolic leaf extracts respectively. However, n-hexane stem extract under concurrent extreme hot temperatures (35/45ºC) and water deficit condition was the most potent for hydrogen peroxide scavenging activity (0.14±0.048 mg/ml) after a 144-hour treatment period, when compared with the control sample with IC50 values (2.94±0.017 mg/ml).
Conclusion
This investigation indicates how environmental factors in combination can influence the antimicrobial, antioxidant properties and the phytochemical profile of P. afra parts. It is evident that abiotic factors in combination impact the biosynthesis of secondary metabolites, potentially resulting in enhanced yields of phytomedicine and the production of phytocompounds such as volatile oils, coumarins, tannins, glycosides and flavonoids which are known to be lead phytocompounds for treating ailments such as respiratory diseases, cancer, ulcers, central nervous system, and cardiac ailments. Ultimately, this report agrees with a complicated and elaborate response system that varies for each plant parts, species of plant, abiotic factor, treatment/harvest period and extraction solvent. The exploration and investigation of combined abiotic stress applications for the purposeful synthesis of phytocompounds, aimed at targeted phytopharmaceutical production and the examination of associated biological properties, warrant further comprehensive research.