{"id":56808,"date":"2024-03-20T11:00:13","date_gmt":"2024-03-20T11:00:13","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=56808"},"modified":"2024-04-03T05:02:27","modified_gmt":"2024-04-03T05:02:27","slug":"the-in-vitro-assessment-of-antidiabetic-activity-of-the-plant-extracts-obtained-from-portulacaria-afra-jack-grown-under-concurrent-extreme-temperatures-and-water-deficit-conditions","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no1\/the-in-vitro-assessment-of-antidiabetic-activity-of-the-plant-extracts-obtained-from-portulacaria-afra-jack-grown-under-concurrent-extreme-temperatures-and-water-deficit-conditions\/","title":{"rendered":"The In Vitro Assessment of Antidiabetic Activity of the Plant Extracts Obtained from Portulacaria afra Jack. Grown under Concurrent Extreme Temperatures and Water-deficit conditions"},"content":{"rendered":"\n

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

The significant role of herbal plants in the field of\nmedicine is due to their inherent therapeutic efficacy. Despite the advancements\nin modern medicine, numerous diseases, including diabetes mellitus and its\nassociated complications caused by the production of advanced glycation end\nproducts, are being treated with medicines of plant origin1<\/sup>. In developing nations, where most\nof the population has low resources and no access to contemporary treatments,\nplants remain important in the management of diabetes1<\/sup>. Due to the negative side effects\nof using insulin and oral hypoglycemic medications, the desire for alternate\nmethods of treating diabetes, such as plant-based medications, has increased in\nindustrialized nations2<\/sup>.<\/p>\n\n\n\n

According to WHO recommendations, the\nuse of plant-derived hypoglycemic treatments within traditional medicinal\npractices holds significant importance3<\/sup>. The antihyperglycemic effects of\nthese treatments are due to their capacity to enhance pancreatic tissue\nfunctionality through increased insulin secretion or decreased intestinal\nglucose absorption. Therefore, treatment with herbal medicines protects \u03b2-cells\nand minimizes glucose fluctuation.<\/p>\n\n\n\n

Diabetes mellitus (DM) is a lethal\nhealth condition marked by impaired insulin synthesis, which is either\nhereditary or acquired, and by diminished organ response to secreted insulin.\nIncreased blood glucose levels brought on by such a shortage can harm various\nbodily systems, including blood vessels and nerves4<\/sup>.<\/p>\n\n\n\n

The World Health Organization\n(W.H.O.) predictions show that the number of diabetic patients globally would\nsurge from 171 million in 2000 to exceeding 366 million by 20305<\/sup>. While According to data from\nStatistics South Africa6<\/sup>, non-communicable diseases accounted for 58% of the\ncountry’s increase in mortality rates between 1997 and 2018. Of these, 12% of\nadult patients had diabetes, making the condition the second leading cause of\ndeath in South Africa. These predicted increases in global\ndiabetes prevalence represent a significant burden on the health system.<\/p>\n\n\n\n

The primary objective of treating\ndiabetes mellitus is to successfully lower the increased blood sugar levels. To\nreduce post-prandial hyperglycemia, the predominant therapeutic strategies for\nmanaging diabetes centre on impeding the breakdown of dietary starch via\ncarbohydrate hydrolysing enzymes such as \u03b1-amylase and \u03b1-glucosidase, alongside\nstimulating or enhancing insulin activity within target tissues through the\nutilization of oral hypoglycemic agents7<\/sup>.<\/p>\n\n\n\n

Furthermore, the reduction of\npost-prandial hyperglycemia is achieved by delaying glucose absorption by\nblocking \u03b1-amylase or \u03b1-glucosidase in the gastrointestinal system. This\nprocedure slows down the breakdown of carbohydrates, reducing the pace of\nglucose uptake which in turn dampens the postprandially induced rise in plasma\nglucose8<\/sup>. Acarbose, miliglitol, and\nvoglibose, are some of the inhibitors currently in clinical use, these\ninhibitors’ primary negative effects are gastrointestinal, including bloating,\nstomach pain, diarrheal, and flatulence9<\/sup>.<\/p>\n\n\n\n

Secondary metabolites with high\ntherapeutic values like phenolics, flavonoids, glycosides, coumarins, saponins,\nterpenoids, alkaloids, and others are typically found in plants and contribute\nto their pharmacological qualities10<\/sup>. Also, plants contain antioxidants\nwhich are known to have therapeutic potentials for various diseases. The\nmain function of natural antioxidants is to activate endogenous antioxidants to\ncombat oxidative damage11<\/sup>.<\/p>\n\n\n\n

Different factors such as\nenvironmental conditions, seasonal variations, plant parts, treatment\/harvest\nperiod and extraction solvents affect both the growth of plants and the biosynthesis\nof phytochemicals production, chemical composition, antioxidant, and\nantidiabetic activities of plants12<\/sup>. Additionally, recent studies have shown that plant\nresponses to stresses caused by combined antagonistic abiotic factors, are\nphenomenal compared to when exposed to single ones.<\/p>\n\n\n\n

In this study we have\nselected Portulacaria afra,<\/em> a succulent medicinal plant anda\nmember of the Portulacaceae <\/em>or Didiereaceae <\/em>family.<\/em> It is commonly known as elephant bush (English), spekboom, and\nporkbush (Afrikaans). P. afra<\/em> is native to South Africa, Kenya, and\nMozambique, it thrives mostly in subtropical biomes. 13<\/sup>. P. afra<\/em> has recorded high phytochemical contents and\npharmacological evidence have shown its antioxidant and antimicrobial\nproperties Tabassum et al.<\/em>14<\/sup> <\/sup>and Adeleye & Risenga 15<\/sup>. The in-vitro<\/em> antioxidant activity observed in P. afra <\/em>whole plant extracts gives a preliminary prediction for the\nantidiabetic activity. To the best of our knowledge, there is no scientific\ndocumentation on the antidiabetic potential of Portulacaria afra <\/em>leaf, stem, and root extracts. <\/p>\n\n\n\n

Hence, the aim of our study\nis to assess the \u03b1-amylase inhibitory potential of the leaf, stem, and root\nextracts of P. afra<\/em> using four\ndistinct extraction solvents characterized by different polarities, and to\ninvestigate the effect of concurrent extreme temperatures (hot & cold) and\nwater deficit on the antidiabetic activity of the different parts. This study is essential for\ndetermining exposure duration and temperature with water deficit-dependent effects\non the plant extracts for the optimal antidiabetic activity, which could\npotentially be useful in drug production as a remedy for diabetes.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Portulacaria afra<\/em> leaf and stem.     <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
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Figure 2: Portulacaria afra<\/em> root (arrow).<\/strong><\/p>\n

<\/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

The chemicals and solvents\nutilized in this investigation were of analytical variety. The following items\nwere procured from Sigma-Aldrich, USA, under CAS Number 1162-65-8: methanol, n-<\/em>hexane,\nethyl acetate, dimethyl sulfoxide (DMSO), potato starch, 3,5-dinitro salicylic\nacid (DNSA), sodium potassium tartrate, and sodium hydroxide.<\/p>\n\n\n\n

The Conviron system specifications\nfor Model No. PGW 40, Serial No. 170028, manufactured in China (280033R01) are\nas follows: Voltage – 230\/400V, Frequency – 50Hz, Phase – 3, Total Input\nAmperage – 29.2A, Compressor Rated Load Amperage (R.L.A) – 65.5A, High Design\nPressure – 400 PSI, Low Design Pressure – 200 PSI, Control Amperage – 2.0A,\nLighting Amperage – 9.4A, Heater Amperage – 4.3A, Fan Horsepower – 2.6 HP,\nDrier Amperage – 10.9A, along with additional specifications for Receptacle\nAmperage, Glycol Pump Amperage, Motor Amperage, and Auxiliary Amperage. The\nMinimum Circuit Ampacity (MCA) required is 31.2A, with a Maximum Overcurrent\nProtection (MOP) of 32A, and a Short Circuit With-Stand Capacity of 5000 Amps\nRMS.<\/p>\n\n\n\n

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

Portulacaria afra<\/em> specimens were propagated via cuttings in July 2021 at the University of the Witwatersrand, Johannesburg, South Africa. <\/em><\/p>\n\n\n\n

Cutting and growing of plants<\/strong><\/p>\n\n\n\n

Healthy cuttings displaying characteristics such as vibrant\ngreen leaves, absence of drooping, and no signs of fading foliage were\nselectively collected from a disease-free parent plant. Cuttings measuring\n40\u201345 cm in length and 4 cm in thickness were harvested using sterile secateurs\nand subsequently transplanted into the greenhouse environment for cultivation16<\/sup>.<\/p>\n\n\n\n

A total of 225 pots, each with a capacity ranging from 2 to\n2.5 litres, were used to contain stem cuttings for root development. These pots\nwere filled with Culterrean Potting’s professional cutting mix soil, chosen for\nits superior drainage, moist environment maintenance, and absence of\npotentially harmful pathogens detrimental to the initial rooting phase of the\ncuttings.<\/p>\n\n\n\n

The cuttings were delicately\ninserted and secured within the partially filled potting mix soil before being\nsurrounded by additional potting mix soil17<\/sup>.<\/p>\n\n\n\n

The plants were further kept in the\ngreenhouse at the University of Witwatersrand, Johannesburg, South Africa, for\nthree months to allow them to adapt, develop new root systems, and produce new\nleaves along the stems18<\/sup>. In the greenhouse setting, plants monitoring was conducted\nwhile receiving a consistent watering regimen of 500 ml administered every two\ndays due to their low-frequency watering requirements16<\/sup>.<\/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<\/p>\n<\/td>\n

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

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

\n

Temperatures<\/p>\n

(\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 Harvest frequency<\/p>\n

(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<\/strong><\/p>\n\n\n

The settings for the conviron simulations used for the treatments were derived from forecasts regarding changes in the present and future climate conditions by the South African Department of Environmental Affairs 19<\/sup>, IPCC 20<\/sup>, and Mbokodo et al.<\/em> 21<\/sup> records. The temperature settings used in this study were chosen based on the length of heat and cold waves from earlier predictions, [(Control (25\/27\u00baC); mid-range high (30\/40\u00baC); and mid-range low (10\/15\u00baC); extreme high (35\/45\u00baC); and extreme low (0\/5\u00baC)].<\/p>\n\n\n\n

According to Janda et al<\/em>.22<\/sup>, each plant was placed in a\ncontrolled climate simulation chamber (Conviron chambers, PGW40) with water\ndeficit and high and low temperatures set simultaneously. Ambient conditions\nfor CO2 <\/sub>(400 ppm), humidity (60%) and light (160 nits\/level 1), pH,\nand salinity were maintained. It was arranged for the lights to be on for 12\nhours per day, from 6 am to 6 pm while the South African Weather Service’s\nrecords23<\/sup> were used to determine the control\ntemperature ranges.<\/p>\n\n\n\n

The plants were exposed to\ntreatments and not watered for up to 144 hours (6 days), and the control\nsamples were placed under 25o<\/sup>C (ambient) and watered every second\nday with 500ml of water24<\/sup>. The controls were maintained at a maximum nighttime\ntemperature of 25\u00b0C (7pm to 5am) and a maximum daytime temperature of 27\u00b0C (5am\nto 7pm). Plants (A, B, C, and D) while receiving treatment did not receive any\nwater. Samples for treatments A and B were subjected to cold temperatures of 0\nand 10\u00b0C at night and 5 and 15\u00b0C during the day. For the C and D treatments, samples\nwere exposed to hot temperatures of 30 and 40\u00b0C maximum at night and 35 and\n45\u00b0C maximum during the day.<\/p>\n\n\n\n

Five plants were harvested for all\nplant parts episodically 3 times for up to 6 days (144hrs), where plants were\nharvested every 48hrs (48, 96, 144) (Table 1). Harvested samples were oven\nairdried under 40\u00b0C for 2 to 3 days (Loveys et\nal<\/em>., 2002). This procedure was repeated three times for each of the\ntreatments. Where, n=45 control (25\u00b0), n=45 for (0\/5\u00b0C), n=45 (10\/15\u00b0C), n=45\n(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 leaf,\nstem, and root of P. afra<\/em> were\ngathered and cleansed using deionized water. Subsequently, these specimens were\nsubjected to a four-day drying process at 40\u00b0C in a hot air dryer. Following\nthis, an electric grinder was employed to finely powder the dried plant\nmaterial. The powdered samples were then sealed in foil and stored in an\nairtight container within a dark cabinet at room temperature (32\u00baC) until\nfurther analysis commenced25<\/sup>.<\/p>\n\n\n\n

Plant extract preparation<\/strong><\/p>\n\n\n\n

The crude plant extract was obtained\nby employing a series of sequential solvent extractions. A mixture of 80%\nmethanol, n<\/em>-hexane, ethyl acetate, and 100% distilled water (60\u00b0C), were\nchosen based on their respective polarities, was utilized26<\/sup>. Each extraction involved placing\n3g of powdered plant material into individual containers and adding 30 ml of\nthe designated solvent27<\/sup>. The methanol, n<\/em>-hexane, and ethyl acetate\nextractions were put on a shaker, while the aqueous extracts underwent\nsonication at a temperature of 60\u00b0C for 45 minutes. The shaking process of all\nsolvents lasted for 48 hours, except for the aqueous extracts28<\/sup>. Aqueous extracts underwent\ncentrifugation to reduce viscosity, after which all extracts were filtered\nusing filter paper into vials29<\/sup>. The resulting supernatant was discarded, and the vials\nwere sealed with foil, and then refrigerated at 4\u00baC before subsequent analyses.<\/p>\n\n\n\n

Preparation of buffer and reagent<\/strong><\/p>\n\n\n\n

The buffer and reagents were\nprepared according to the methodology outlined by Kamtekar et al<\/em>.30<\/sup> and Geethalakshmi et\nal<\/em>.3<\/sup> with slight modification.<\/p>\n\n\n\n

The phosphate buffer solution was\nprepared by initially adding 800 ml of distilled water to a suitable container.\nSubsequently, 20.214 g of Sodium Phosphate Dibasic Heptahydrate powder and\n3.394 g of Sodium Phosphate Monobasic Monohydrate were successively introduced\ninto the 800 ml distilled water. The pH of the resulting solution was adjusted\nto the desired level of 6.9 using either HCl or NaOH. Distilled water was then\nincrementally added until the total volume reached 1 L.<\/p>\n\n\n\n

For the preparation of the DNSA\nreagent, 1 g of 3,5-dinitrosalicylic acid was dissolved in 50 ml of distilled\nwater while shaking with a magnetic stirrer on a hot plate set at 90-95\u02daC.\nFollowing this, 30 g of Potassium sodium tartrate tetrahydrate was dissolved in\n20 ml of distilled water and gradually combined with the previous solution,\nresulting in the appearance of a milky yellow colour. Subsequently, 20 ml of\n2(N) sodium hydroxide solution was added, leading to the development of a\ntransparent orange colour. The final volume was adjusted to 100 ml using\ndistilled water. After complete dissolution, the solution underwent filtration\nthrough filter paper, followed by transfer into a dark glass bottle for storage\nat room temperature (32\u00baC).<\/p>\n\n\n\n

Evaluation of in vitro<\/em> Antidiabetic Activity                                               <\/strong><\/p>\n\n\n\n

In vitro<\/em> inhibitory \u03b1-amylase<\/em> assay<\/strong><\/p>\n\n\n\n

The determination of \u03b1-amylase\ninhibitory activity was conducted following the methodology outlined by Kumar et\nal<\/em>.31\n<\/sup>with\nfew alterations. The leaf, stem, and root extracts of P. afra<\/em> underwent\ndrying, whereas aqueous extracts were subjected to lyophilization at -85\u00ba C.<\/p>\n\n\n\n

The solvent-extracted residues were\ndissolved in a solution of 10% dimethyl sulfoxide (DMSO). Subsequently, all\nresulting extracts were standardized to a concentration of 100 mg\/ml.\nTriplicates of five serial dilutions were meticulously prepared for each\nextract, encompassing a concentration range of 20 to 100 mg\/ml.<\/p>\n\n\n\n

In this study, 500 \u03bcl of plant\nextract was subjected to incubation alongside 500 \u03bcl of \u03b1-amylase solution for\na duration of 10 minutes at a room temperature of 32 \u00b0C. The \u03b1-amylase\nsolution, comprising 2 units\/ml, was prepared by dissolving 0.001 g of\n\u03b1-amylase in a solution containing 100 ml of 0.02 M sodium phosphate buffer (pH\n6.9) and 6.7 mM sodium chloride. Following this initial incubation period, 500\n\u03bcl of a 1% starch solution, derived from the mixture of 1 g of potato starch\nwith 100 ml of distilled water, heated and stirred for 15 minutes, was\nintroduced into the mixture. Subsequently, the combined solution underwent a\nfurther 10-minute incubation period at room temperature (32\u00b0C).<\/p>\n\n\n\n

To cease the reaction, 1 ml of DNSA\nreagent was introduced to the mixture, followed by a 5-minute incubation in a\nhot water bath (85\u00b0C). The termination of the reaction was indicated by a\nvisible change in colour of the reaction mixture to orange red after the\nstipulated 5-minute incubation, upon which the mixture was removed from the\nwater bath and allowed to cool to room temperature. The volume was then\nadjusted to 5 ml with distilled water. Blank experiments were conducted by\nsubstituting the enzyme with buffer, while the plant extract served as the\npositive control. Subsequently, the absorbance of the resultant solution was\nmeasured at 540 nm using a spectrometer. The\nabsorbance was measured with a spectrometer at 540 nm. The inhibition (%) which\nis the sample concentration (mg\/ml) required to decrease the absorbance by 50 %\nof alpha amylase was expressed with the following equation: <\/p>\n\n\n\n

(Ac+<\/sup>) \u2013 (Ac–<\/sup>) \u2013 (As – Ab)<\/strong> \u00f7 (Ac+<\/sup>) \u2013 (Ac–<\/sup>) \u00d7 100.<\/strong><\/p>\n\n\n\n

Where, Ac+ <\/sup>is the\nabsorbance of 100% enzyme activity (only solvent with enzyme),<\/p>\n\n\n\n

Ac–<\/sup> is the absorbance of\n0% enzyme activity (only solvent without enzyme),<\/p>\n\n\n\n

 As is the absorbance of test sample with\nenzyme,<\/p>\n\n\n\n

Ab that absorbance of test sample\nwithout enzyme.<\/p>\n\n\n\n

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

The concentration values were\ngraphed against the % inhibition values employing Microsoft Excel to derive a\ntrend line equation which facilitating the calculation of IC50<\/sub>. Statistical analysis was done with\na one-way analysis of variance (ANOVA) to ascertain significant differences.\nAll experiments were conducted in triplicate, and data were presented as mean \u00b1\nstandard error (SE) when n = 3. Statistical significance was determined at P\n< 0.05, with Tukey test HSD in RStudio used to pinpoint specific significant\ndifferences.<\/p>\n\n\n\n

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

Plants are exposed to a variety of\nbiotic and abiotic elements as they grow and develop, and in response, they\nactivate their defence mechanism. These fluctuating and sometimes harmful\nexternal environmental factors interact with plants32<\/sup>. Being non-motile organisms, plants\nhave complex alternate defence mechanisms that use a wide range of chemical\ncompounds as coping mechanisms for stressful situations. Secondary metabolites\nhave a significant impact on how plants adjust to their environment32<\/sup>. The capacity of plants to\nsynthesize these metabolites is nearly infinite.<\/p>\n\n\n\n

Environmental factors, seasonal\nvariations, plant part, and harvest\/treatment period,\naffect the chemical composition, antioxidant, and antidiabetic activities of\nmedicinal plants33<\/sup>.<\/p>\n\n\n\n

Maintaining close to normal levels\nof glycemic control in both the fasting and post-prandial phases is the key\ntherapeutic objective for diabetic patients. Numerous natural resources have\nbeen studied in relation to inhibiting the generation of glucose from\ncarbohydrates in the gut or absorbing glucose from the intestine7<\/sup>. The \u03b1-amylase (\u03b1-1, 4-glucan-4-\nglucanohydrolases) is one of the main secretory products of the pancreas and\nsalivary glands and is present in bacteria, plants, and higher animals. It aids\nin the digestion of starch and glycogen. Inhibiting their activity in the human\ndigestive system is thought to be useful in managing diabetes by reducing the\nabsorption of glucose produced when these enzymes break down starch7<\/sup>.<\/p>\n\n\n\n

In this study, four extraction solvents were used to prepare P. afra<\/em> leaf, stem, and root extracts under concurrent extreme hot and cold temperatures, mid-range high (30\/40\u00baC); and mid-range low (10\/15\u00baC), extreme high (35\/45\u00baC); and extreme low (0\/5\u00baC) with water deficit conditions. The extracts were evaluated for in vitro<\/em> antidiabetic activity using the in vitro<\/em> inhibitory \u03b1-amylase assay, where IC50 <\/sub>value is the amount of extract required to inhibit \u03b1-amylase activity by 50%. Lower IC50 <\/sub>values indicate stronger antidiabetic ability of the extracts, which was used to detect the potential antidiabetic effect of P. afra<\/em> extracts. P. afra<\/em> parts showed significant differences, with extracts showing significant differences (P < 0.05). The antidiabetic activity of each part of P. afra<\/em> extract at different treatment periods is shown in Figures 3-6.  A notable increasing trend of antidiabetic capacity was seen across all parts when compared to the control samples under simultaneous cold temperatures [mid-range low (10\/15\u00baC); and extreme low (0\/5\u00baC)] with water-deficit conditions. As shown in Figure 3, the aqueous leaf extracts under mid-range cold temperatures (10\/15\u00baC) showed the strongest antidiabetic activity with IC50 <\/sub>value of (2.33\u00b10.832mg\/ml) amongst the plant\u2019s parts after a 48-hour treatment period while the IC50   <\/sub>value for the control sample was (208.818\u00b10.064mg\/ml). Figure 4 shows the antidiabetic activity under concurrent extreme cold temperatures (0\/5\u00baC) and water deficit, where ethyl acetate stem extracts showed the highest inhibitory action with IC50 <\/sub>value of (2.85\u00b10.111mg\/ml) against \u03b1-amylase after a 96-hour treatment period, while the IC50 <\/sub>value for the control sample was (324.16\u00b10.111mg\/ml). The variation observed in the activity among the plant organs at the different treatment periods suggests a possible corresponding shift in the accumulation of some compounds responsible for the activity12<\/sup>. Since most of these phytochemicals are produced in response to external stimuli such as light intensity, moisture\/water deficit stress, and temperature amongst others34,35<\/sup>.<\/p>\n\n\n\n

Similarly, Figures 5 and 6 show observed variations in the\n\u03b1-amylase inhibitory activity of the plant extracts under\nconcurrent hot temperatures [mid-range high (30\/40\u00baC); and extreme high\n(35\/45\u00baC)] with water deficit conditions. A shift in antidiabetic activity was observed\nin the extracts in comparison with the control samples. The antidiabetic\nactivity of the aqueous stem extract under the mid-range hot temperatures\n(30\/40\u00baC) showed the strongest inhibitory activity with IC50<\/sub> <\/sub>(1.70\u00b10.666mg\/ml) after a 48-hour\ntreatment period while a similar trend was observed also in the aqueous stem\nextracts under the extreme hot temperatures (35\/45\u00baC) with the highest\ninhibitory activity with IC50<\/sub> (1.99\u00b10.626mg\/ml)\nafter a 96-hour treatment period, with IC50<\/sub> <\/sub>(10.61\u00b10.127mg\/ml) for the control\nsample.<\/p>\n\n\n\n

Considering all these, the overall strongest inhibitory\nactivity against \u03b1-amylase enzyme activity under both cold and hot temperatures\nwith water deficit condition was found in the aqueous stem extracts under the\nmid-range hot temperatures (30\/40\u00baC); with IC50<\/sub> value (1.70\u00b10.666mg\/ml) after a\n48-hour treatment period. Similar result was observed in Usman et al<\/em>. 33 <\/sup>where the oil from (hot-dry) season harvested\nplants showed stronger antidiabetic activity than the essential oil from the\nharvested plants during the (wet-cold) season. Research by Chelladurai and\nChinnachamy10<\/sup> also reported the antidiabetic\nactivity of aqueous stem extract of Salacia\noblonga<\/em> to be effective against \u03b1-amylase.<\/p>\n\n\n\n

The marked variation in antidiabetic activity in\nthis study may also be attributed to the polar and non-polar compounds present in the plant parts based on\nthe extraction solvent. From a wide array of\nexperiments, it has been established that methanol and water targets sugars,\namino acids, and glycosides during phytochemical screening. Conversely,\nalkaloids, aglycones and glycosides are extracted with ethyl acetate while n-<\/em>hexane extracts waxes, fats, fixed\noils and volatile36,37<\/sup>. <\/p>\n\n\n\n

The high presence of\nvarious phytochemicals like tannins, terpenoids, phenolics and flavonoids previously\ndetected in P. afra<\/em> parts from earlier findings 14, 15<\/sup>, have potential\ninhibitory effects on \u03b1-amylase enzyme due to their ability to bind with\nproteins, which in turn contributes to the lowering of postprandial\nhyperglycemia38<\/sup>.<\/p>\n\n\n\n

Additionally, an extensive\nrange of compounds, including arylpyrones and styrylpyrones, stibenes, tannins,\ncoumarins, flavonoids, lignins, and lignans, fall within the phenolics\ncategory, being synthesized in reaction to abiotic stress39<\/sup>.<\/p>\n\n\n\n

 This correlates to this study\u2019s observations\nand might explain the observed inhibitory effects across the different extracts\nof P. afra<\/em>.<\/p>\n\n\n\n

(More information advised\nto be written for Results)<\/p>\n\n\n\n

The in vitro<\/em> antidiabetic potential of all the extracts was determined by <\/em><\/strong>in vitro<\/em> inhibitory \u03b1-amylase assay, where IC50 <\/sub>value is the amount of extract required to inhibit \u03b1-amylase activity by 50%. Lower IC50 <\/sub>values indicate stronger antidiabetic ability of the extracts, which was used to detect the potential antidiabetic effect of P. afra<\/em> extracts. Figures 3-6 experimentally reveal the various changes observed in the antidiabetic activities of P. afra<\/em> plant parts\u2019 extracts with different solvents and treatment periods, under hot and cold temperatures [mid-range high (30\/40\u00baC); and mid-range low (10\/15\u00baC), extreme high (35\/45\u00baC); and extreme low (0\/5\u00baC)] concurrently with water deficit conditions. P. afra<\/em> plant parts showed significant differences, with extracts showing statistically significant differences (p < 0.05), and were indicated by different letters (a, b, c, d) within the same column (Tukey\u2019s post-hoc test).
<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 3: Graphical representation of antidiabetic activity in P. afra<\/em> plant parts under 10\/15\u00b0C with water deficit conditions. <\/strong>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\nFigure 4: Graphical representation of antidiabetic activity in P. afra<\/em> plant parts under 0\/5\u00b0C with water deficit conditions. <\/strong>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\nFigure 5: Graphical representation of antidiabetic activity in P. afra<\/em> plant parts under 30\/40\u00b0C with water deficit conditions. <\/strong>\n

\u00a0<\/p>\n

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

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\nFigure 6: Graphical representation of antidiabetic activity in P. afra<\/em> plant parts under 35\/45\u00b0C with water deficit conditions. <\/strong>\n

\u00a0<\/p>\n

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

Conclusions<\/strong><\/p>\n\n\n\n

The present study explores the\npotential antidiabetic activity of P. afra<\/em> leaf, stem, and root with four\nextraction solvents and with focus on the inhibitory effects on\n\u03b1-amylase under extreme temperatures with water deficit condition. The results\nof this study also highlighted the effect of concurrent extreme temperatures\nwith water deficit condition, treatment period, plant part and extraction\nsolvent on the antidiabetic potential of P.\nafra.<\/em><\/p>\n\n\n\n

The antidiabetic potential of P. afra <\/em>leaf, stem and root extracts\nwith the control samples were characterized by their inhibitory effect on\n\u03b1-amylase activity. P. afra<\/em> leaf, stem and root have significant\ninhibitory effects against \u03b1-amylase enzyme. A marked variation in the\nantidiabetic activity were observed among the plant parts based on the extraction\nsolvent and treatment period for all temperature ranges with water deficit\ncondition. Of the extracts tested from P.\nafra<\/em>, aqueous stem extracts under mid-range hot temperatures after a\n48-hour treatment period showed the strongest inhibition on the enzyme\n(\u03b1-amylase) across all treatments, which may be attributed to the polar\ncompounds present based on the solvent extraction and the temperature exposure duration.\nThis study is the first to report the antidiabetic potential of the leaf, stem,\nand root of P. afra<\/em> with the effect\nof concurrent extreme temperatures and water deficit.<\/p>\n\n\n\n

Conclusively, the results of the\nstudy indicate that P. afra<\/em> leaf,\nstem and root possess great antidiabetic potential from the in vitro<\/em> assay performed. Therefore, the\nantidiabetic property can be credited to the rich phytochemicals and antioxidant\nactivity possessed by the plant, according to earlier research. For future\nproduction of this species, this work has shown how environmental factors may\ninfluence the antidiabetic potential of specific plant sections. Subsequent research\nis necessary to isolate antidiabetic compounds and conduct in vivo<\/em> studies.<\/p>\n\n\n\n

Acknowledgements<\/strong><\/p>\n\n\n\n

Authors are grateful to God, and\nSouth Africa\u2019s National Research Foundation (NRF) for supporting this work.<\/p>\n\n\n\n

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

The authors declare that there are\nno conflicts of interest.<\/p>\n\n\n\n

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

This work was supported by South Africa\u2019s National Research Foundation (NRF) under grant number: TTK201129577193<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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