HPLC-DAD Based Polyphenolic Profiling and Evaluation of Pharmacological Attributes of Putranjiva roxburghii Wall.

The current study was intended to explore the phytochemical profiling and therapeutic activities of Putranjiva roxburghii Wall. Crude extracts of different plant parts were subjected to the determination of antioxidant, antimicrobial, antidiabetic, cytotoxic, and protein kinase inhibitory potential by using solvents of varying polarity ranges. Maximum phenolic content was notified in distilled water extracts of the stem (DW-S) and leaf (DW-L) while the highest flavonoid content was obtained in ethyl acetate leaf (EA-L) extract. HPLC-DAD analysis confirmed the presence of various polyphenols, quantified in the range of 0.02 ± 0.36 to 2.05 ± 0.18 μg/mg extract. Maximum DPPH scavenging activity was expressed by methanolic extract of the stem (MeOH-S). The highest antioxidant capacity and reducing power was shown by MeOH-S and leaf methanolic extract (MeOH-L), respectively. Proficient antibacterial activity was shown by EA-L extract against Bacillus subtilis and Escherichia coli. Remarkable α-amylase and α-glucosidase inhibition potential was expressed by ethyl acetate fruit (EA-F) and n-Hexane leaf (nH-L) extracts, respectively. In case of brine shrimp lethality assay, 41.67% of the extracts (LC50 < 50 µg/mL) were considered as extremely cytotoxic. The test extracts also showed mild antifungal and protein kinase inhibition activities. The present study explores the therapeutic potential of P. roxburghii and calls for subsequent studies to isolate new bioactive leads through bioactivity-guided isolation.


Introduction
Plants are nature's gifts that have been employed for the management of various healththreatening diseases since the early ages [1][2][3]. It has been revealed that approximately 80% of the population across the globe depends upon plant-based therapeutics for healthcare needs [3]. Phytoconstituents are the compounds generated by plants as a defense system against pathogens and predators. They possess certain characteristics that are helpful for the treatment of diseases including antimicrobial, antioxidant, and stimulation as well as inhibition enzymes [4]. Plants contain a wide range of constituents such as alkaloids, tannins, polyphenolics, terpenoids, etc. which are attributable to therapeutic potential [5]. Phytochemicals either isolated compounds or crude extracts, providing opportunity for x = Total weight of the dried extract, y = Total dried weight of the powdered plant material used in extraction, i.e., 500 g each part.

Phytochemical Analysis
Stock solutions of all test extracts were formulated as 4 mg/mL DMSO for phytochemical analysis.

Determination of Total Phenolic Content (TPC)
Initially, 20 µL test extract was shifted to the different wells of 96-well plate with subsequent inclusion of 90 µL Folin-Ciocalteu (FC reagent), 90 µL of sodium carbonate and absorbance was checked after incubation. Gallic acid was taken as positive control and calibration curve was plotted (y = 0.0738x + 0.086) while DMSO was taken as negative control. Results were depicted as the mean of µg gallic acid equivalent (GAE) /mg extract ± SD [21].

Determination of Total Flavonoid Content (TFC)
Initially, 20 µL test extract was shifted to different wells of the 96-well plate with subsequent inclusion of 10 µL (1M) potassium acetate solution, 10 µL (10%) aluminum chloride, and 160 µL distilled water and absorbance was checked after incubation. Quercetin was taken as a positive control and the calibration curve was plotted (y = 0.0535x − 0.0033) while DMSO was employed as negative control. Results were recorded as mean of µg quercetin equivalent (QE)/mg extract ± SD [21].

Determination of Total Antioxidant Capacity (TAC)
Initially, each test sample (100 µL) was shifted to Eppendorf tubes with subsequent addition of 900 µL TAC reagent and incubation at 95 • C for 90 min [24]. Ascorbic acid and DMSO were taken as positive control and blank, respectively. Absorbance of the reaction mixture was noted at 630 nm and a calibration curve was plotted (y = 0.0408x − 0.025) while results were illustrated as the mean of µg ascorbic acid equivalent (AAE)/mg extract ± SD.

Determination of Total Reducing Power (TRP)
TRP of test extracts was investigated by adopting a previously reported protocol [24]. At first, each test extract (100 µL) was shifted to Eppendorf tubes (Merck, Kenilworth, USA) with the subsequent addition of 200 µL phosphate buffer (0.2 mol/L, pH 6.6) and 250 µL of 1% potassium ferricyanide. Amalgam was incubated and 10% trichloroacetic acid (200 µL) was then added followed by centrifugation. Then, 150 µL aliquots from the supernatant was transferred into the wells of microtiter plate containing FeCl 3 (50 µL, 0.1%) and absorbance was recorded. Positive and negative controls were ascorbic acid and DMSO. A calibration curve was plotted (y = 0.0754x + 0.1034) while the results were demonstrated as a mean of µg AAE/mg extract ± S.D.

Antimicrobial Assays Antibacterial Assay
The antibacterial activity of test samples was investigated by disc diffusion protocol [25]. Refreshed bacterial cultures [Pseudomonas aeruginosa (ATCC15442), Staphylococcus aureus (ATCC-6538), Klebsiella pneumoniae (ATCC-1705), Escherichia coli (ATCC-25922), and Bacillus subtilis (ATCC-6633)] (Microbiologics, Saint Cloud, MN, USA) were employed to prepare lawns on nutrient agar plates. Briefly, 5 µL aliquots of every sample were transferred from stock solution (20 mg/mL DMSO) to discs. Subsequently, cefixime and roxithromycin were chosen as positive control while DMSO as a negative control. The discs were implanted on agar plates followed by incubation and zones of inhibition (ZOI) were measured (mm) around each disc. Minimum inhibitory concentration (MIC) was investigated for all the test samples showing ≥12 mm zone of inhibition. For MIC determi-nation, the bacterial inoculum was prepared with a pre-defined density (5 × 10 4 CFU/mL). Serial dilutions were prepared for each test sample in a 96-well plate by using nutrient broth. Subsequently, 195 µL of inoculum was included and the plate was then incubated. The absorbance of assay plate was recorded after 30 min (zero time reading) and 24 h of incubation and difference was determined. Percent inhibition of bacterial growth was calculated as: T s is the turbidity of the sample well and T c is the turbidity of the control well. The lowest concentration at which test samples showed ≥90% inhibition of bacterial growth was referred as MIC.

α-Amylase Inhibition Assay
This assay was conducted in accordance with a preceding protocol [24]. First of all, phosphate buffer (15 µL, pH 6.8), α-amylase enzyme (25 µL, 0.0175 U/mL), test sample (10 µL, 4 mg/mL DMSO) and starch solution (40 µL, 2 mg/mL potassium phosphate buffer) were transferred to the corresponding wells of 96 well plate. The plates were kept at 50 • C for 30 min after which HCl (20 µL, 1 M) and iodine reagent (90 µL) were added, and absorbance was checked at 540 nm. An identical protocol was adopted for the formation of negative and positive controls by substituting the test sample with a similar volume of DMSO and acarbose, respectively. Moreover, a blank was prepared by adding an equal quantity of buffer rather than test extract and α-amylase enzyme solution. %enzyme inhibition was calculated as: where, Abs = absorbance of test sample, Abb = absorbance of blank, and Abn = absorbance of negative control.

α-Glucosidase Inhibition Assay
This assay was executed by adopting a protocol demonstrated by Nair et al. [27]. Initially, a substrate solution (25 µL, 20 mM), phosphate buffer (69 µL, 50 mM, pH 6.8), test sample (5 µL, 4 mg/mL DMSO), and enzyme solution (1 µL) were transferred to the corresponding wells of 96 well plate. First reading was taken at 405 nm immediately and the amalgam was placed at 37 • C for 30 min. The reaction was quenched by sodium bicarbonate solution (100 µL, 0.5 mM) and the absorbance was again checked. Negative and positive controls were prepared by following a similar protocol just by substituting the test extract with the same amount of DMSO and acarbose, respectively. Following formula was used for the calculation of %enzyme inhibition: %enzyme inhibition = Ac − As/Ac × 100 Ac = Absorbance of control, As = Absorbance of sample This assay was executed by using brine shrimp (Artemia salina acquired from Ocean star Int., Coral Springs, FL, USA) larvae in a 96 well plate by following a preceding protocol [22]. Artemia salina eggs were allowed to hatch in a dual-chamber punctured tank containing simulated seawater. The chamber filled with eggs was enfolded with aluminum foil while another chamber was kept under a light source. The tank was kept at 30-32 • C for 24-48 h after which nauplii started moving towards the lightened chamber. Subsequently, 10 mature nauplii were taken in each well of a 96-well plate with subsequent inclusion of 150 µL seawater and corresponding volume of test samples to attain final concentrations of 200, 100, and 50 µg/mL. Seawater was added to make the final volume of each well up to 300 µL. Doxorubicin was chosen as positive control while 1% DMSO as negative control. Ultimately, 96 well plate was incubated after which dead nauplii were computed by using an inverted microscope. Following formula was used to calculate %mortality and LC 50 was calculated by graph pad prism 5 software (GraphPad, San Diego, CA, USA). % mortality = no. of dead shrimps/total no. of shrimps × 100

Protein Kinase Inhibition Assay
Mycelia fragments of Streptomyces species were immersed into sterile tryptone soy broth and kept for 24 h. Petri plates containing ISP4 medium were swabbed with refreshed culture (100 µL). Subsequently, discs loaded with a test sample (5 µL, 20 mg/mL DMSO) were implanted on seeded plates. Surfactin and DMSO-loaded discs were employed as positive and negative controls, respectively. The plates were incubated and the diameter of the bald or clear zone (mm) of inhibition was measured as an indicator of protein kinase inhibition potential [26].

Data Analysis
All the assays were conducted in a triplicate manner and the data have been demonstrated as mean ± standard deviation of triplicate analysis. LC 50 and IC 50 values were determined through Graph pad prism 5 software (GraphPad, San Diego, CA, USA) while Post Hoc Tukey HSD (Honest significant difference) test was used to analyze the results using the statistical package SPSS (IBM SPSS, Armonk, NY, USA) and p < 0.05 (95% confidence interval) was considered as significant. Correlation coefficient (R 2 ) of phytochemical activities was calculated by employing the correlation and regression function of Microsoft Excel program (Microsoft, Redmond, WA, USA).

Percent Extract Yield
Percent recovery of test extracts is summarized in Figure 1. The results demonstrated that DW and MeOH extracts of leaf possess maximum extraction efficiency, i.e., 6.3 ± 0.4% and 5.3 ± 0.3%, respectively [28,29]. During the current study, polar solvents showed maximum extraction efficiency. Water-soluble phytoconstituents mainly present in plants include flavonoids, tannins, terpenoids, quinones, etc. [30,31]. The presence of some of these metabolites might be attributable to the maximum extraction efficiency of DW and MeOH leaf extracts. The results were in conformity with a previous study in which nonpolar extracts of Ajuga bracteosa Wall. showed less extraction efficiency as compared to polar ones [25].
6.3 ± 0.4% and 5.3 ± 0.3%, respectively [28,29]. During the current study, polar solvents showed maximum extraction efficiency. Water-soluble phytoconstituents mainly present in plants include flavonoids, tannins, terpenoids, quinones, etc. [30,31]. The presence of some of these metabolites might be attributable to the maximum extraction efficiency of DW and MeOH leaf extracts. The results were in conformity with a previous study in which non-polar extracts of Ajuga bracteosa Wall. showed less extraction efficiency as compared to polar ones [25].

Total Phenolic Content (TPC)
A remarkable TPC was noted in test extracts. Maximum TPC was notified in DW extracts of stem and leaf, i.e., 55.23 ± 0.10 μg GAE/mg extract while least TPC was found in nH extracts of leaf and fruit, i.e., 2.06 ± 0.03 μg GAE/mg extract, respectively ( Figure  1). TPC notified in our research was somewhat higher than a recently published study which revealed that P. roxburghii leaf (hydroethanol, 30:70) extract contains 46.58 ± 2.52 mg/g GAE polyphenolic content [32]. Alterations in agro-climatic conditions accompanied with temperature and rainfall impart a significant impact on the amount of phytoconstituents within similar species of plants growing in different regions [33]. Hence, differences in solvent composition and plant collection sites might be responsible for the variations of estimated TPC in both studies. Current study investigated the phenolic content in fruit and stem parts of P. roxburghii as well and employed a wide range of solvents [32]. Phenolics are secondary metabolites of plants which help to counteract different pathogenic microbes and impart colors to the plants. They are widely distributed in all plant parts and hence, act as an essential component of the human diet [34]. Phenolics are highly effective to counteract oxidative stress-related ailments such as neurodegenerative diseases, cancer, diabetes, ageing, and cardiovascular diseases [35]. Phenolic compounds perform antioxidant activity by the virtue of hydrogen donating action and chelation of metal ions involved in the generation of free radicals [36,37]. The

Total Phenolic Content (TPC)
A remarkable TPC was noted in test extracts. Maximum TPC was notified in DW extracts of stem and leaf, i.e., 55.23 ± 0.10 µg GAE/mg extract while least TPC was found in nH extracts of leaf and fruit, i.e., 2.06 ± 0.03 µg GAE/mg extract, respectively ( Figure 1). TPC notified in our research was somewhat higher than a recently published study which revealed that P. roxburghii leaf (hydroethanol, 30:70) extract contains 46.58 ± 2.52 mg/g GAE polyphenolic content [32]. Alterations in agro-climatic conditions accompanied with temperature and rainfall impart a significant impact on the amount of phytoconstituents within similar species of plants growing in different regions [33]. Hence, differences in solvent composition and plant collection sites might be responsible for the variations of estimated TPC in both studies. Current study investigated the phenolic content in fruit and stem parts of P. roxburghii as well and employed a wide range of solvents [32]. Phenolics are secondary metabolites of plants which help to counteract different pathogenic microbes and impart colors to the plants. They are widely distributed in all plant parts and hence, act as an essential component of the human diet [34]. Phenolics are highly effective to counteract oxidative stress-related ailments such as neurodegenerative diseases, cancer, diabetes, ageing, and cardiovascular diseases [35]. Phenolic compounds perform antioxidant activity by the virtue of hydrogen donating action and chelation of metal ions involved in the generation of free radicals [36,37]. The presence of hydrophobic benzenoid rings enables the phenolic constituents to interact with proteins and to inhibit enzymes involved in free radical production [36,38]. These compounds can also act as neuroprotective, gastroprotective, antipyreticm, antiatherosclerotic, cardioprotective, antiulcer [39], anti-inflammatory [35], antibacterial [40], antifungal [41], antiviral [42], and antitumor agents [35,43], antidiabetic, anxiolytic [39]. Hence, the presence of a remarkable quantity of phenolic content in P. roxburghii might be accountable to its promising therapeutic potential.  Figure 1). Minimum TFC was notified in nH extracts of stem, leaf, and fruit, i.e., 1.04 ± 0.02 µg QE/mg extract. The results were in close resemblance to a previous study which revealed that the hydroethanolic extract of P. roxburghii leaf part contains 29.0 ± 2 mg/g QE flavonoid content [32]. It was found that EA leaf extract contains a greater quantity of flavonoids than MeOH extract. Flavonoids are an important class of polyphenolics containing benzo-γ-pyrone moiety and possess a wide range of therapeutic activities [44]. Flavonoids contain a flavan core, consisting of 15 carbon atoms organized in three rings. Depending upon the oxidation state of the central core, flavonoids are categorized as: anthocyanins, flavanones, isoflavones, flavones, flavanols, and flavonols. The structural differences in each subclass are due to the extent and pattern of prenylation, methoxylation, glycosylation, and hydroxylation [34]. Flavonoids can act as antimicrobial, anti-inflammatory, immunomodulatory, enzyme inhibitor, antiviral, antiparasitic, cardioprotective, cytotoxic, anti-tumor, antiaging agents [45,46]. A wide range of flavonoids have been isolated from roxburghii species such as catechin and quercetin in Rosa roxburghii Tratt. [47], isorhamnetin, rhamnazin, roxburoside from Anoectochilus roxburghii Wall. [48], and putraflavone from P. roxburghii [49]. During the present study, the flavonoid presence in polar extracts of P. roxburghii referred it to be a potential reservoir of antioxidants.

Antioxidant Assays DPPH Assay
Metabolic processes within the body and environmental factors produce free radicals. Free radicals mainly include reactive oxygen species (ROS) which cause various ailments including ageing, carcinogenesis, mutagenesis, and cardiovascular abnormalities. Antioxidants are the agents, which counteract the effects of free radicals and limit oxidative stress [74]. DPPH assay is a standard method to investigate the free radical scavenging ability of test samples [75]. The percent free radical scavenging activity (%FRSA) of test extracts was analyzed and results are given in Figure 3. Maximum %FRSA was shown by MeOH-S and DW-L extracts (86 ± 0.56%) with IC 50 values of 68 ± 0.43, 149 ± 0.21 µg/mL, respectively. Whereas, nH extracts of stem, leaf, and fruit were unable to express any free radical scavenging activity. Moreover, ascorbic acid showed 78% FRSA with IC 50 of 14.56 µg/mL. We calculated the correlation of %FRSA with TPC (R 2 = 0.8084) and TFC (R 2 = 0.0211) which demonstrates that phenolic compounds other than flavonoids are mainly attributable to free radical scavenging activity. Previously reported %FRSA of methanolic stem extracts of P. roxburghii was somewhat lower than calculated during our commenced research. Moreover, the current study revealed that DW-L extract of P. roxburghii also possesses a promising free radical scavenging potential. Highest extraction efficiency of DW-L extract provides another perspective to employ this extract as an antioxidant. Overall, our results were in accordance with a previous study which revealed that polar extracts possess better scavenging potential than non-polar extracts [25]. and reducing power of crude extracts which is in support of our current research results [24,76].
at Figure 3. Demonstration of (a) %DPPH radical scavenging activity (at 40 μg/mL concentration of test extracts) (b) TAC and TRP (μg AAE/mg) of P. roxburghii crude extracts. Assays were conducted in a triplicate manner and the data have been demonstrated as mean ± standard deviation. Significantly different means (p < 0.05) are represented by different superscripts ( a-j ).

Antimicrobial Assays
Antibacterial Assay Antibacterial property of test extracts (100 μg per disc) was investigated against gram-negative (K. pneumoniae, P. aeruginosa, E. coli) and gram-positive (B. subtilis, S. aureus) bacteria as shown in Table 2. Test extracts exhibiting zone of inhibition ≥ 12 mm were further subjected for MIC evaluation by employing the broth microdilution method. EA-L extract was highly active against B. subtilis (MIC 3.7 μg/mL), K. pneumoniae (MIC 33.3 μg/mL), and E. coli (MIC 3.7 μg/mL) with 24 ± 0.5, 20 ± 0.50, 23 ± 0.76 mm ZOIs, respectively. The ZOIs generated by the test extracts were equivalent to those produced by standards, i.e., roxithromycin and cefixime. Moreover, DMSO taken as negative control was unable to show inhibition. Overall, polar extracts showed better antibacterial potential than non-polar extracts. In a previous study, antibacterial potential of P. roxburghii MeOH leaf part was investigated, and results were in somewhat resemblance to our

Total Antioxidant Capacity (TAC)
TAC of test extracts was calculated as shown in Figure 3. Maximum TAC was shown by MeOH-S and MeOH-L extracts, i.e., 48.65 ± 0.52, 46.07 ± 0.07 µg AAE/mg extract, respectively. Minimum TAC (10.91 ± 0.05 µg AAE/mg extract) was expressed by nH-L extract. We noticed a remarkable relationship between TAC and TPC (R 2 = 0.744) which demonstrates that phytoconstituents producing antioxidant activity mainly belong to polyphenols. Moreover, the correlation between TAC and TFC (R 2 = 0.1419) shows that flavonoids are not the main contributor to the antioxidant activity of test extracts. During a previous study, the estimated TAC of P. roxburghii stem part was significantly lower than that of the current study. This might be due to the fact that varying agro-climatic conditions can impact the type and quantity of phytoconstituents [41]. Moreover, the antioxidant capacity of different parts of P. roxburghii except stems, has been investigated for the first time [9]. Plant-derived antioxidants are considered better than synthetic antioxidants as they possess better compatibility with the human body [41], hence provide a justification for the exploitation of herbal products for therapeutic appraisal.
Total Reducing Power (TRP) TRP was investigated for crude extracts and results are depicted in Figure 3. Maximum TRP was expressed by MeOH-L and DW-L extracts, i.e., 118.59 ± 0.08, 101.28 ± 0,09 µg AAE/mg extract while nH-F extract showed the least TRP, i.e., 20.48 ± 0.52 µg AAE/mg extract, respectively. A positive relation was notified between TPC and TRP (R 2 = 0.7251) in contrast to the correlation between TFC and TRP (R 2 = 0.1495) which demonstrates that phenolic constituents other than flavonoids are mainly accountable for reducing potential of plants. Reducing power of P. roxburghii stem part was previously investigated; however, the current study revealed that leaf part of this plant possesses better antioxidant potential [12]. Reducing power is mainly attributable to the presence of reductones which cleaves the free radical chain by hydrogen atom donation and participates in antioxidant action. Multiple studies have notified a close relationship between antioxidant activity and reducing power of crude extracts which is in support of our current research results [24,76].

Antimicrobial Assays Antibacterial Assay
Antibacterial property of test extracts (100 µg per disc) was investigated against gram-negative (K. pneumoniae, P. aeruginosa, E. coli) and gram-positive (B. subtilis, S. aureus) bacteria as shown in Table 2. Test extracts exhibiting zone of inhibition ≥ 12 mm were further subjected for MIC evaluation by employing the broth microdilution method. EA-L extract was highly active against B. subtilis (MIC 3.7 µg/mL), K. pneumoniae (MIC 33.3 µg/mL), and E. coli (MIC 3.7 µg/mL) with 24 ± 0.5, 20 ± 0.50, 23 ± 0.76 mm ZOIs, respectively. The ZOIs generated by the test extracts were equivalent to those produced by standards, i.e., roxithromycin and cefixime. Moreover, DMSO taken as negative control was unable to show inhibition. Overall, polar extracts showed better antibacterial potential than non-polar extracts. In a previous study, antibacterial potential of P. roxburghii MeOH leaf part was investigated, and results were in somewhat resemblance to our study. However, EA leaf extract was investigated for the first time, and it was noted that this extract possessed stronger antibacterial activity than previously reported MeOH leaf extract. Phenolics impart toxic effects to microbes either through interaction with sulfhydryl groups or proteins resulting in enzyme inhibition. Moreover, polyphenols generate heavy complexes with proteins which interact with bacterial adherence and disrupt the receptors on cell surface [26,77]. During the current study, a proficient amount of TPC and TFC was notified which might contribute to the antibacterial activity.

Antifungal Assay
During present study, MeOH-F extract (100 µg/disc) showed slight activity against A. flavus and A. fumigatus with 7 ± 0.89 and 8 ± 0.98 mm ZOIs, respectively and MIC value of >100 µg/mL. MeOH-S extract was moderately active against A. flavus with 12 ± 0.98 ZOI and >100 µg/mL MIC while other extracts were unable to show antifungal activity. Clotrimazole (10 µg/disc) showed 20 ± 0.57 to 31 ± 1.1 mm ZOIs. A previous study reported the promising antifungal activity of seed and pericarp of P. roxburghii. However, during the present study; stem, leaf, and fruit parts of P. roxburghii showed slight to moderate antifungal active. Moreover, fungal strains used in current study were different from those used in previous study. Flavonoids and tannins can form complexes with extracellular proteins present in the cell wall of fungi and rupture the fungal membrane [78,79]. P. roxburghii contains tannins (ellagic acid, gallic acid, gallocatechin, ellagi-and gallo-tannins) and flavonoids (bioflavones) which might be a contributor to antifungal activity [49].

α-Amylase Inhibition Assay
The suppression of carbohydrate cleaving enzymes, i.e., α-amylase and α-glucosidase is a promising approach to reduce the blood glucose concentration in case of diabetes [27]. Inhibition of both enzymes impedes carbohydrate digestion and enhance the time required for carbohydrate digestion, limits rate of glucose absorption and ultimately prevents postprandial rise in plasma glucose level [80]. During the present study, α-amylase inhibition potential of crude extracts was investigated and results are depicted in Figure 4. Maximum α-amylase inhibition activity was expressed by EA-F and EA-S extracts, i.e., 67.37 ± 0.05%, 61.37 ± 0.06% with IC 50 values, i.e., 14.28 ± 0.9, 33.26 ± 0.5µg/mL, respectively. Acarbose was employed as standard which showed 80.45 ± 0.76% α-amylase inhibition with IC 50 value of 34.85 ± 0.21 µg/mL. Results were in accordance with previous studies which reported that less polar extracts exhibited improved enzyme inhibition activity than highly polar extracts. These studies also notified a direct relationship between the concentration of phenolics and flavonoids with α-amylase inhibition activity [81,82]. However, the current study did not notice any significant relation of TPC (R 2 = 0.343), and TFC (R 2 = 0.0008) with α-amylase inhibition potential.

α-Glucosidase Inhibition Assay
Antidiabetic activity of P. roxburgii was further confirmed by estimation of α-glucosidase inhibition potential of crude extracts and results are depicted in Figure 4. Maximum αglucosidase inhibition (80 ± 0.78%) was shown by nH-L, nH-S, and DW-S extracts with IC 50 values, i.e., 90.21 ± 0.02, 93 ± 0.078, 94.11 ± 0.99 µg/mL, respectively. Just like α-amylase inhibition, there was no positive relationship of TPC (R 2 = 0.1971) and TFC (R 2 = 0.1231) with α-glucosidase inhibition. Acarbose and miglitol are commercially available α-glucosidase inhibitors which hinder the carbohydrate absorption and reduce post-prandial escalation in glucose levels [83]. However, these agents possess severe gastrointestinal adverse effects such as diarrhea and flatulence [84]. α-amylase inhibitors have been reported to be present in plants which helps them to combat predators [85].
to be present in plants which helps them to combat predators [85].
A detailed study was executed for the first time to investigate α-amylase and α-glucosidase inhibition potential of different parts of P. roxburghii. Previous study demonstrated that plants containing tannins such as Camelia sinensis Var., Artocarpus heterophyllus Lam., Persea Americana Mil., and Syzygium polyanthum Walp. are potent inhibitors of αglucosidase [86]. A wide range of tannins such as ellagic acid, gallic acid, gallocatechin, ellagi-and gallo-tannins are present in P. roxburghii might be involved behind the α-glucosidase inhibition activity [49]. However, further studies are needed to prove this hypothesis. Figure 4. Demonstration of (a) α-amylase inhibition and (b) α-glucosidase inhibition potential of P. roxburghii crude extracts. Assays were conducted in a triplicate manner and the data have been demonstrated as mean ± standard deviation. Significantly different means (p < 0.05) are represented by different superscripts ( a-h ).

Preliminary Toxicity Potential
Brine Shrimp Lethality Assay Brine shrimp lethality assay is a preliminary method to investigate the cytotoxic effect of test samples [87]. Brine shrimp lethality potential was noticed to be in direct relation to the concentration of test extracts ( Table 3). Out of the twelve extracts subjected for estimation of cytotoxic activity, 41.67% of the extracts (LC50 < 50 μg/mL) were considered as extremely cytotoxic while 33.33% of crude extracts (LC50 ≥ 50 but ≤ 200 μg/mL) were classified as moderately cytotoxic. Other 25% of the extracts (>200 μg/mL) were categorized as weakly cytotoxic. Overall, DW-F extract was noticed as most cytotoxic extract showing LC50 9.36 ± 0.91 μg/mL while LC50 of doxorubicin (positive control) was 5.93 μg/mL. A detailed study was executed for the first time to investigate α-amylase and αglucosidase inhibition potential of different parts of P. roxburghii. Previous study demonstrated that plants containing tannins such as Camelia sinensis Var., Artocarpus heterophyllus Lam., Persea Americana Mil., and Syzygium polyanthum Walp. are potent inhibitors of α-glucosidase [86]. A wide range of tannins such as ellagic acid, gallic acid, gallocatechin, ellagi-and gallo-tannins are present in P. roxburghii might be involved behind the α-glucosidase inhibition activity [49]. However, further studies are needed to prove this hypothesis.

Preliminary Toxicity Potential Brine Shrimp Lethality Assay
Brine shrimp lethality assay is a preliminary method to investigate the cytotoxic effect of test samples [87]. Brine shrimp lethality potential was noticed to be in direct relation to the concentration of test extracts ( Table 3). Out of the twelve extracts subjected for estimation of cytotoxic activity, 41.67% of the extracts (LC 50 < 50 µg/mL) were considered as extremely cytotoxic while 33.33% of crude extracts (LC 50 ≥ 50 but ≤ 200 µg/mL) were classified as moderately cytotoxic. Other 25% of the extracts (>200 µg/mL) were categorized as weakly cytotoxic. Overall, DW-F extract was noticed as most cytotoxic extract showing LC 50 9.36 ± 0.91 µg/mL while LC 50 of doxorubicin (positive control) was 5.93 µg/mL. Previously, brine shrimp lethality potential of methanolic seed extract was investigated and mild cytotoxicity potential was reported [88]. Undertaken study was carried out on various other parts of P. roxburghii and promising brine shrimp lethality potential of nH and DW extracts of fruit was revealed for the first time. During the present study, 100% of the test extracts exhibited LC 50 values of less than 1000 µg/mL, referring to the existence of cytotoxic phytometabolites in the subject plant.

Protein Kinase Inhibition Assay
Protein kinase regulates multiple functions of the cell cycle including apoptosis, metabolism, growth, and differentiation. In the case of human cancers, irregularities of growth factor signaling pathways were notified. Hence, protein kinase inhibition can serve as a target for drug development by overcoming the irregularities in signaling pathways [89]. Protein kinase inhibition activity of the test extracts (100 µg/disc) was investigated as well (Table 3). DW and nH fruit extracts showed 8 ± 0.4 and 7 ± 0.97 mm bald phenotype zones, respectively while no clear zone was found. The remaining extracts were unable to express any protein kinase inhibition activity. The absence of ZOI in case of DMSO confirmed the non-toxic effect of negative control. Protein kinase is involved in the regulation of various processes of cell cycle, i.e., control of cycle growth, metabolism, differentiation, and apoptosis. Abnormalities of growth factor signaling pathways have been notified in various forms of human cancer. Hence, protein kinase inhibitors capable to repair the dysregulations in these signaling pathways can act as promising targets for anticancer drug development [89]. Previously, this plant has shown significant anticancer potential against different cancer cell lines. A combined herbal therapy comprised of Punica granatum and P. roxburghii showed strong cytotoxic activity against HepG2 hepatocellular carcinoma cell line in correlation with its antioxidant effect, hence proving the significant anticancer potential of the subject plant in the current study [90]. Not only the organic solvents extracts but the green silver nanoparticles synthesized using P. roxburghii leaves extracts depicted stupendous inhibitory activity against PANC-1, HCT-116 and MDA-MB-231 cell lines showing its potential to reduce the proliferation of cancerous cells [91]. During the present study, P. roxburghii DW extract showed mild protein kinase inhibition activity. The activity is supported by the results of a previous finding in which water extracts of Sargassum oligocystum depicted significant cytotoxic and antiproliferative activity against K562 and Daudi cell lines [92]. The linkage of protein kinase inhibitory activity and anticancer effects persuaded to draw the relation between previous findings and the results of the current studies. Although, a detailed study has been conducted for the first time to investigate the protein kinase inhibition potential of multiple parts of P. roxburghii.

Conclusions
Undertaken study demonstrates the polyphenolic analysis and pharmacological potential of crude extracts. Overall, leaf part showed maximum biological activities as compared to the stem and fruit parts of P. roxburghii. In case of leaf part DW and MeOH extracts were highly active whereas EA and nH extracts also showed some biological activities. The present study suggests that P. roxburghii crude extracts are a potential reservoir of phytoconstituents instigating the considerable antioxidant, antibacterial, antidiabetic, and cytotoxic compounds. These bioactive phytoconstituents could act as unique frameworks in a quest for innovative drugs. we recommend that subsequent studies should be carried out for the isolation of bioactive compounds from P. roxburghii (especially DW-L extract), which can be considered as potential candidates for the treatment of different ailments.