Biochemical profile and bioactive potential of wild folk medicinal plants of Zygophyllaceae from Balochistan, Pakistan

Recent focus is on analysis of biological activities of extracts from plant species. Zygophyllaceae is exceedingly important angiosperm family with many taxa being used in folk medicines widely dispersed in arid and semi-arid zones of Balochistan, Pakistan. Only a small proportion of them have been scientifically analyzed and many species are nearly facing extinction. Therefore present investigation explores the biochemical and bioactive potential of fourteen folk medicinal plants usually used for treatments of different ailments. Fresh aerial parts of nine taxa and two fruit samples were collected from plants growing in arid and semi-arid zones of Balochistan and analyzed for enzymatic, non-enzymatic and other biochemical activities. Higher phytochemical activities were detected in the aerial parts. Superoxide dismutase was detected maximum in Fagonia indica, (184.7±5.17 units/g), ascorbate peroxidase in Tribulus longipetalus subsp. longipetalus (947.5±12.5 Units/g), catalase and peroxidase was higher in Peganum harmala (555.0±5.0 and 2597.8±0.4 units/g respectively). Maximum esterase and alpha amylase activity was found in Zygophyllum fabago (14.3±0.44 and 140±18.8 mg/g respectively). Flavonoid content was high in T. longipetalus subsp. longipetalus (666.1±49 μg/ml). The highest total phenolic content and tannin was revealed in F. olivieri (72125±425 and 37050±1900 μM/g. respectively). Highest value of ascorbic acid was depicted in F. bruguieri var. rechingeri (448±1.5 μg/g). Total soluble Proteins and reducing sugars were detected higher in P. harmala (372.3±54 and 5.9±0.1 mg/g respectively). Maximum total antioxidant capacity (TAC) was depicted in Z. simplex (16.9±0.01 μM/g). Pigment analysis exhibited the high value of lycopene and total carotenoids in T. terrestris (7.44±0.2 and 35.5±0.0 mg/g respectively). Chlorophyll a, b and total chlorophyll content was found maximum in T. longipetalus subsp. pterophorus (549.1±9.9, 154.3±10 and 703.4±20.2 ug/g respectively). All taxa exhibited anti-inflammatory activity as well as anti-diabetic inhibitory potential. Seed extracts of Zygophyllum eurypterum (96%) exhibited highest inhibitory potential, along with twelve other taxa of Zygophyllaceae indicated (96-76%) activity when compared with the standard drug diclofenac sodium (79%). Seeds of T. longipetalus subsp. longipetalus (85%) exhibited the highest anti-diabetic activity; other eleven taxa also exhibited inhibitory activity of α-amylase ranging from (85-69%) compared with Metformin (67%) standard drug. Phytochemical screening revealed that selected taxa proved to be the potential source of natural antioxidants and could further be explored for in-vivo studies and utilized in pharmaceutical industries as potent therapeutic agents validating their ethno-pharmacological uses.

was found in Zygophyllum fabago (14.3±0.44 and 140±18.8 mg/g respectively). Flavonoid 23 content was high in T. longipetalus subsp. longipetalus (666.1±49 µg/ml). The highest total 24 phenolic content and tannin was revealed in F. olivieri (72125±425 and 37050±1900 µM/g.  Superoxide dismutase (SOD) assay: 129 Method of [24] used to determine SOD activity by homogenizing the fresh aerial parts and fruits 130 of selected taxa in phosphate buffer (50 mM, pH 7.8), EDTA (0.1 mM) and DTT (1 mM) 131 following the procedure of [24]. Further analyzed by assessing its property to stop the 132 photochemical reduction of nitro-blue tetrazolium as explicated by [25]. One unit of SOD 133 activity was demarcated as the amount of enzyme causing 50% inhibition of photochemical 134 reduction of nitro-blue tetrazolium.
Peroxidase (POD) assay: 136 The assessment of POD activity carried out using method [26] was followed with few changes. 137 Homogenized mixture of the aerial parts and fruits prepared in1 ml phosphate buffer (50 mM, 138 pH 7.8), EDTA (0.1 mM) and DTT (1 mM). The assay solution contained 535µl distilled water, 139 phosphate buffer 50 mM (pH 7.0), Guaiacol (20 mM), H 2 O 2 (40 mM) and 15µl enzyme extract. 140 The addition of enzyme extract initiated the reaction. At 470 nm absorbance rises was noted at 141 interval of 20 sec. Absorbance change of 0.01 min −1 was demarcated as one unit POD activity.

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Enzyme activity was expressed on the basis of fresh sample weight.  Ascorbate peroxidase (APX) assay: 151 The assessment of APX activity carried out by following the [24]  sulphate (II). Absorption measured at 560nm after 5 min by using spectrophotometer.

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Pigment analysis: 178 The concentration of Lycopene, chlorophyll (a and b), Total chlorophyll and carotenoids were 179 examined by method of [32]. Samples (0.2 g) were grind in acetone (80%) and centrifuged at 10,000 g for 5 minutes. Absorbance measured at 645, 663 and 480 nm by using a 181 spectrophotometer.

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Place this mixture at room temperature for 1 hour and take reading at 765 nm. Linear regression     Protein estimation of plant samples was based on quantitative protein analysis described by [36]. 209 Aerial parts and fruits samples were homogenized in potassium phosphate (50 mM, pH 7.0).  In Vitro Anti-Diabetic Activity (enzyme α-amylase inhibition method) 214 The in Vitro anti-diabetic activity was determined by assaying the inhibitory activity of the 215 enzyme α-amylase which involves in the breakdown of starch to produce glucose [37]. In this 216 method, 1 ml of methanolic extracts of all species were tested separately and thus added to 1 ml 217 of the enzyme α-amylase in a test-tube and incubated for 10 min at 37°C. Then 1 ml of 1% starch 218 solution was added into it and again incubated for 15 min at 37°C. Then 2 ml 3, 5-dinitrosalicylic 219 acid reagent was added into it, in order to terminate the reaction. The reaction mixture was then 220 incubated in boiling water bath for 5 min and then allows it to cool at room temperature. The In Vitro Anti-Inflammatory Activity (Protein Denaturation Method) 227 The protein denaturation assay was determined using a modified method as described by [38]. 228 Briefly, the reaction mixture (0.5 mL; pH 6.3) consisted of 0.45 mL of bovine serum albumin 229 (5% aqueous solution) and 0.05 mL of distilled water. The pH was adjusted to 6.3 using a small wt.). In seeds of selected taxa no significant variation was detected the maximum TAC was in F.   (Table 3). Total Phenolic Content (TPC) was estimated, in aerial parts of selected taxa no 336 significant TPC variation was found among (Table 4). In general, highest TPC was depicted in T. 337 longipetalus subsp. pterophorus (63025±1725 µM/g. dry wt.) followed by F. bruguieri var. 338 rechingeri (54600±1350 µM/g. dry wt.). Seeds of selected plant samples showed significant 339 variation. Highest TPC was detected in Z. propinquum (69225±775µM/g. s. wt.) followed by F. 340 bruguieri var. rechingeri (66850±3900 µM/g. s. wt.) shown in Table 3. No significant Tannin 341 variation was detected among aerial parts as well as in seeds of all tested taxa (Table 2).

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However, highest amount of tannin was estimated in T. longipetalus subsp. pterophorus 343 (40375±4125 µM/g dry wt.) followed by Z. fabago (39175±4825 µM/g. dry wt.). In seeds 344 highest amount of tannin was estimated in P. hermala (47525±2575 µM/g s. wt.) followed by Z. 345 propinquum (42625±175 µM/g. s. wt.) shown in Table 3. Ascorbic Acid was observed, in aerial 346 parts of all selected taxa and significant difference found among studied taxa (Table 4). Highest 347 value of AsA was found in F. olivieri (744.2±2.7 µg/g dry wt.) followed by F. bruguieri var. 348 bruguieri. AsA content in seeds showed no significant variation. In general the highest AsA 349 content was found in F. bruguieri var. laxa (740.8±2.19 µg/g s. wt.) given in Table 3. Alpha 350 amylase activity in dry aerial parts of selected plants was assessed (Table 2) no significant 351 variation was found among the taxa of Zygophyllaceae. However, highest α-amylase activity 352 was found in Z. simplex (164.9 ±3.39 mg/g. dry wt.) followed by Z. fabago (153 ± 6.6 mg/g. dry 353 wt.). In seeds of selected taxa significant variation was found among various taxa is shown in 354 (Fig 19 f). The highest value was observed in F. bruguieri var. laxa (159.5±11.8 mg/g. s. wt.) 355 followed by F. ovalifolia subsp. pakistanica (133.9±0.37 mg/g. s. wt.) given in Table 3.  s. wt.) second highest was in F. bruguieri var. rechingeri (15.5±2.5 µM/g. s. wt.) given in Table   365 3. Reducing Sugar was measured in aerial parts of all selected plants (Table 2) there was a 366 significant difference found among all taxa. Highest value of reducing sugar was recorded in Z. 367 fabago (7.47±0.2 mg/g. s. wt.) followed by Z. propinquum and with minimum difference (7.19 368 ±0.55 mg/g. s. wt.). In seeds highest value of reducing sugar was found in T. terrestris (7.9±0.1 369 mg/g. s. wt.) given in Table 3.   Lycopene content in dry aerial parts of various taxa was measured ( Table 4). The higher value 376 was found in T. longipetalus subsp. pterophorus (9.25± 1.8 mg/g dry wt.) followed by T.  shown in (Table 4). Maximum Content of total chlorophyll was depicted in T. longipetalus 400 subsp. pterophorus (800±62.9 ug/g dry wt.) with significant difference. It was followed by T. 401 terrestris (719±1.8 ug/g dry wt.). In seeds measured among different taxa of Zygophyllaceae is 402 given in (Table 5)   Pigment analysis in fresh samples shown in S . Fig 4 a, b, c, d,  This study was carried out to assess the anti-inflammatory and anti-diabetic potential of naturally

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Anti-diabetic activity was analyzed by using amylase inhibition assay. Seeds and aerial parts of 483 selected species revealed significant differences in anti-diabetic activity when compared with 484 standard drug Metformin (Fig.6)