HPLC-DAD finger printing, antioxidant, cholinesterase, and α-glucosidase inhibitory potentials of a novel plant Olax nana

Background The medicinal importance of a novel plant Olax nana Wall. ex Benth. (family: Olacaceae) was revealed for the first time via HPLC-DAD finger printing, qualitative phytochemical analysis, antioxidant, cholinesterase, and α-glucosidase inhibitory assays. Methods The crude methanolic extract of O. nana (ON-Cr) was subjected to qualitative phytochemical analysis and HPLC-DAD finger printing. The antioxidant potential of ON-Cr was assessed via 1,1-diphenyl,2-picrylhydrazyl (DPPH), 2,2-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) and hydrogen peroxide (H2O2) free radical scavenging assays. Furthermore, acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities were performed using Ellman’s assay, while α- glucosidase inhibitory assay was carried out using a standard protocol. Results The qualitative phytochemical analysis of ON-Cr revealed the presence of secondary metabolites like alkaloids, flavonoids, tannins, sterols, saponins and terpenoids. The HPLC-DAD finger printing revealed the presence of 40 potential compounds in ON-Cr. Considerable anti-radical activities was revealed by ON-Cr in the DPPH, ABTS and H2O2 free radical scavenging assays with IC50 values of 71.46, 72.55 and 92.33 μg/mL, respectively. Furthermore, ON-Cr showed potent AChE and BChE inhibitory potentials as indicated by their IC50 values of 33.2 and 55.36 μg/mL, respectively. In the α-glucosidase inhibition assay, ON-Cr exhibited moderate inhibitory propensity with an IC50 value of 639.89 μg/mL. Conclusions This study investigated Olax nana for the first time for detailed qualitative phytochemical tests, HPLC-DAD finger printing analysis, antioxidant, anticholinesterase and α-glucosidase inhibition assays. The antioxidant and cholinesterase inhibitory results were considerable and can provide scientific basis for further studies on the neuroprotective and anti-Alzheimer’s potentials of this plant. ON-Cr may further be subjected to fractionation and polarity guided fractionation to narrow down the search for isolation of bioactive compounds.


Background
Humans have used medicinal plants as a remedy against various diseases since time immemorial [1]. The presence of various bioactive components in different parts of plants make them an important resource for the treatment of various diseases. Furthermore, the side effects associated with some of the synthetic medications and antibiotic resistance demands intermittent research for alternative solutions [2,3]. In addition, to their direct therapeutic use, medicinal plants have been explored to fabricate various nanoscaled materials for potential biomedical applications [4][5][6][7][8][9]. Natural products based alternative therapies are still practiced in many countries and globally approximately 80% of population trust on herbal medicine as a primary source of therapeutic remedies [10]. In the last decade, a revival has been observed in the use of medicinal plants and herbal medicines. It is forecasted that the global market for herbal medicine will expand over time due to the preference of consumers for natural medicines [11]. Moreover, by the year 2017, the worldwide herbal supplements and remedies market is forecasted to reach $107 billion [12].
Free radicals are implicated in the progression of several disorders like, cancer, ischemic heart diseases, neurodegenerative diseases, diabetics, reperfusion injuries, arthritis and atherosclerosis [31]. Free radicals from various sources like toxins, environmental pollutants and deep fried foods cause abnormal genes expression and proteins synthesis which initiate degenerative reactions in the body [32]. In the living systems, generation of free radicals occur via oxidation process, which are nullified to non-radical forms in the human body by natural antioxidants. Hence, apart from our own immune system which acts against these free radicals, natural antioxidants are of prime importance to counter them [33]. Synthetic antioxidants like tertiary butyl hydro quinone, propyl gallate, butylated hydroxytoluene and butylated hydroxyanisole are associated with adverse effects to human health. Hence, natural antioxidants form plants are found to be best alternative for synthetic antioxidants [33]. Many published studies validate the potential antioxidant potential of medicinal plants crude extracts and of isolated pure compounds [34,35]. Flavonoids and phenolics are considered as potent antioxidants due to the presence of hydroxyl groups and conjugated ring structures, which via hydrogenation or complexation scavenge free radicals [36].
Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by behavioral turbulence, cognitive dysfunction and imperfection in the routine activities. Its prevalence is high among individuals of age above sixty years [37]. Currently only five drugs have been approved for clinical use, among which four are cholinesterase inhibitors [38]. The cholinesterase's including acetyl cholinesterase (AChE) and butyrycholinesterase (BChE) catalytically metabolize acetylcholine (ACh) in the synaptic cleft [39]. Acetylcholine is an important neurotransmitter involved in the transmission of impulses across the synapse and is vital in the acquisition and storage of memory. The level of ACh has been found to be depleted in AD [40]. Among the options is the use of inhibitors of AChE and BChE which will restore the activity of ACh at the synapse. Among the clinically approved cholinesterase inhibitors, two are from natural products including galantamine and rivastigmine. Consequently, natural products are under consideration for the development of more useful cholinesterase inhibitors [41].
Diabetes is a major health problem which is associated with high blood glucose level in the body. The α-glucosidase enzyme plays a key role in the control of glucose level inside the body as they are related to postprandial glucose excursion in a person suffering from diabetes [42,43]. α-Glucosidase breakdown carbohydrates into glucose and therefore increases the glucose level inside the body. Henceforth, α-glucosidase inhibition (AGI's) is considered as a popular strategy for controlling post prandial glucose level. Many plants have been researched for natural AGI's like Punica granatum, Pine bark and Andrographis paniculata [44].
To the best of our information there is no single report on the phytochemical investigation and pharmacogonostic features (anticholinesterase, antioxidant and α-glucosidase inhibitory potential) of O. nana. Therefore, the present research was undertaken to investigate and scientifically validate the use of O. nana aqueous methanolic extract against ailments such as diabetes and neurogenerative diseases.

Plant collection and extraction
O. nana whole plant was collected from Swat district of Khyber Pakhtunkhwa, Pakistan in June 2016 (Fig. 1). The plant was authenticated by botanical taxonomist Syed Afzal Shah (PhD Candidate) and Dr. Mushtaq (plant taxonomist) at the Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan. A sample was deposited at MOSEAL Laboratory, Department of Biotechnology, Quaid-i-Azam University with a voucher number MOSEAL-344. The plant was gently rinsed with running distilled water followed by shade drying for 10 days. Subsequently, the dried leaves were grinded via cutter mill to obtain a fine powdered material that weigh~1 kg. In a 1 L of 70% aqueous methanol, 300 g of the powdered plant was soaked for 5 days in a shaking incubator (37°C), with frequent sonication (60 Hz for 5 mints every day) using a sonicator (Elmasonic E 60 H -Cousins UK). The extraction step was repeated thrice, with the addition of extract to the original one and filtered via muslin cloth, ultimately followed by final filtration through Whatman® qualitative filter paper, Grade 1. Furthermore, in a rotary evaporator (EYELA N-1300S-W 115 V, Tokyo Rikakikai Co., LTD), the crude methanolic extract (ON-Cr) was concentrated, ultimately resulted in a~20 g mass of dark brownish semisolid [45].

Phytochemical analysis Preliminary phytochemical tests
For the identification of various secondary metabolites including alkaloids, flavonoids, glycosides, saponins, sterols, tannins, terpenoids and anthraquinones, qualitative phytochemical tests were performed following standard protocols as reported previously [46].

Determination of alkaloids (Dragendorff's method)
The crude methanolic extract (0.2 g) was taken in a conical flask and sulphuric acid (2%) was added. The conical flask was placed on a hot water bath for 2 min and then cooled. The sample was then filtered and treated with Dragendorff's reagent in a test tube. The orange red color precipitate in the test tube showed the presence of alkaloids.

Test for flavonoids
In a conical flask, dried powdered material was boiled with 10 mL of distilled water in a hot water bath for about 5 min and then filtered. After cooling the mixture, few drops of sodium hydroxide solution (20%) was added to 1 mL of the cooled filtrate, which changed the color to yellow. Further addition of 2% HCl changed the yellow color of mixture to colorless.

Test for tannins
To 2 g of sample, 20 mL of distilled water was added and boiled for about 5 min in a hot water bath. The boiled solution was filtered immediately and then cooled at room temperature. A 1 mL of filtrate was taken and added to 5 mL of distilled water. Then 10% of ferric chloride (2-3 drops) was added. The appearance of bluish-black precipitate or brownish-green color showed the presence of tannins.

Test for anthraquinones
A 2 g powdered plant sample was taken and macerated with ether. The solution was shaken and checked, the appearance of red, violet or pink color in the aqueous layer indicated the presence of anthraquinone.

Test for Saponins
The powdered plant material (0.2 g) along with 10 mL of distilled water was taken in a conical flask and boiled in a water bath for about 10 min. The hot mixture was then filtered and allowed to cool. A 1 mL of filtrate along with 2 mL of distilled water was taken in a test tube and vigorously shaken for 2 min. The formation of froth indicates the presence of saponins in the filtrate. Olive oil was then added drop wise to the froth and after shaking the mixture, a formation of emulsion was observed.

Test for glycosides
To a test tube containing a mixture of concentrated sulphuric acid (1 mL), aqueous plant extract (5 mL) and glacial acetic acid (2 mL), a drop of ferric chloride was added. The concentrated sulphuric acid remained beneath the mixture. The appearance of brown ring indicated the presence of cardiac glycoside.

Test for Terpenoids (Salkowski's test)
For the identification of Terpenoids, powdered plant material was dissolved in 5 mL of aqueous solution and boiled in a water bath and then allowed to cool. The solution is filtered, and 2 mL of chloroform was added to the filtrate in a test tube; followed by addition of 3 mL of concentrated sulphuric acid. The appearance of reddish brown interface indicated the presence of terpenoid in the sample.

High performance liquid chromatography-Diaode Array detection (HPLC-DAD) analysis Samples preparation
The extract sample for HPLC was prepared by mixing 20 mL of hydromethanolic mixture (1:1 v/v) with 1 g of ON-Cr. The resultant mixture was heated in a water bath at 70°C for 1 h with subsequent cooling. Thereafter, the sample was centrifuged for 10 min at 4000 rpm. Finally, 2 mL of test sample was filtered through a Whatman filter paper into HPLC vials.

HPLC-DAD procedure
An Agilent 1260 infinity high-performance liquid chromatography (HPLC) system provided with ultraviolet array detector (UVAD), quaternary pump, auto-sampler and degasser was used. Separation was done using an Agilent Zorbax Eclipse XDB-C18 column. A gradient system consisting of solvent B (methanol: acetic acid; deionized water, 100: 20: 180, v/v) and solvent C (methanol: acetic acid: deionized water, 900: 20: 80, v/v) was used. The gradient program was started with 100% B at 0 min, 85% B at 5 min, 50% B at 20 min, 30% B at 25 min, and 100% C for 30-40 min [47]. Using this sequence, elution occurred after 25 min. For analysis of compounds, the ultraviolet array detector (UVAD) was set at 280 nm and the spectra were recorded from 190 to 500 nm. Two standard compounds, rutin and pyrogallol were detected in the HPLC analysis.

Antioxidant assays
1,1-diphenyl, 2-picrylhydrazyl (DPPH) free radical scavenging assay ON-Cr was tested for DPPH free radical scavenging assay as previously reported [35,48]. Briefly, 0.004% DPPH reagent solution was prepared and added to increasing concentration of ON-Cr extract (125, 250, 500 and 1000 μg/mL) with subsequent incubation for 30 min. The absorption of reaction mixture was measured spectrophotometrically (UV-3000 O.R.I. Germany) at 517 nm. Ascorbic acid was used as positive control. The percent scavenging activity of samples was calculated using the following equation: The experiment was performed in triplicate. The IC 50 values were calculated using GraphPad Prism® (version 4.0, Sandiego, CA, USA).
2, 2-Azinobis [3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) free radical scavenging assay For further evaluation of Olax nana antioxidant potentials, ABTS free radical scavenging assay was performed [41,49]. The principle of this assay is based on the test sample ability to scavenge ABTS free radical cations, ultimately leads to reduced absorbance at 734 nm. After mixing, K 2 S 2 O 8 (2.45 mM) and ABTS (7 mM), the solution was kept in dark for 12-16 h to form ABTS free radical cations. A 300 μL of ON-Cr test sample (1000 μg/mL-31.25 μg/mL) was gently mixed with ABTS solution (3.0 mL) in a cuvette. The absorbance of the mixture was measured after every 1 min, for a period of 6 min continuously on a UV-VIS spectrophotometer (UV-3000 O.R.I. Germany). As a positive control, ascorbic acid was used. The assay was performed in triplicate and calculation of percent inhibition was done using the following equation: The percent inhibition and EC 50 value (extract concentration required for 50% reduction of ABTS radicals) were then calculated for expression of antioxidant potential.
Hydrogen peroxide (H 2 O 2 ) free radicals scavenging assay As per our previously reported method, the H 2 O 2 scavenging activity of ON-Cr was elucidated [50]. A solution of H 2 O 2 (2 mM) was prepared in phosphate buffer (50 mM, pH 7.4). In a 0.3 mL of phosphate buffer solution (50 mM), 0.1 mL of test sample was added, which was followed by addition of 0.6 mL of H 2 O 2 and then vortexed. The absorbance of solution was measured at 230 nm after a period of 10 min (UV-3000 O.R.I. Germany), in comparison to blank. The following equation was used for the calculation of H 2 O 2 free radical scavenging activity: Hydrogen peroxide scavenging activity ¼ 1−sample absorbance absorbance of control Â 100

Anticholinesterase assays
The Ellman's assay was performed to assess the AChE and BChE enzymes inhibition potentials of ON-Cr [51]. The principle of anticholinesterase assay is based on hydrolysis of acetylthiocholine iodide and butyrylthiocholine iodide by respective enzymes, leading to the formation of 5-thio-2-nitrobenzoate anion, which complexes with DTNB to form a yellow color compound, after evaluation by UV-VIS spectrophotometer (UV-3000 O.R.I. Germany).
Preparation of solutions Phosphate buffer solution (0.1 M and pH: 8.0) was prepared as previously reported [48]. For adjusting the pH, potassium hydroxide was used. The final concentrations of 0.03 U/mL and 0.01 U/mL were obtained for AChE (518 U/mg solid) and BChE (7-16 U/mg) after respective dilution in the freshly prepared buffer pH 8.0. Using distilled water, DTNB (0.0002273 M), acetylthiocholine iodide and butyrylthiocholine iodide (0.0005 M) solutions were prepared and stored in a refrigerator (8°C). The respective dilutions were also prepared for galantamine (positive control) in methanol.
Spectroscopic analysis In a cuvette containing 205 μL of ON-Cr extract, 5 μL of enzyme solution was added, followed by addition of 5 μL DTNB reagent. In a water bath, the resulting solution was incubated for 15 min at 30°C, with addition of substrate solution (5 μL) subsequently. Furthermore, using a UV-VIS spectrophotometer (UV-3000 O.R.I. Germany) the absorbance was calculated at 412 nm. As a standard inhibitor of cholinesterase, 10 μg/mL of galantamine was used as positive control, while the components other than ON-Cr extract were considered as negative control. At 30°C for 4 min of reaction time, the absorbance values at specific intervals were noted. The assays were conducted in triplicate, while the change in absorption rate with time gave calculations for the % enzyme activity and % enzyme inhibition by galantamine and ON-Cr sample.

αglucosidase inhibitory assay
The protocol of McCue et al., was adopted for the assessment of ON-Cr α-glucosidase inhibitory potential [52]. An enzyme solution was prepared by mixing 120 μL of phosphate buffer (0.1 M, pH 6.9) with α-glucosidase enzyme (20 μL of 0.5 unit/mL). The solution of substrate having pnitrophenyl-α-D-glucopyranoside was prepared in phosphate buffer (0.1 M, pH 6.9). The ON-Cr test sample was prepared in a concentration range of 31.25-1000 μg/mL followed by mixing with enzyme solution and incubation for 15 min at 37°C. Furthermore, to the mixture of enzyme and test sample, substrate solution (20 μL) was added and incubated under the same conditions. The reaction was completed by the addition of sodium carbonate solution (80 μl of 0.2 M). Using a UV-VIS spectrophotometer (UV-3000 O.R.I. Germany), the absorbance of samples was recorded at 405 nm. As a positive control, acarbose was used while the sample devoid of α-glucosidase was used as a blank. The experiment was performed in triplicate, while the following equation was used for the calculation of percent inhibition: %inhibition ¼ control absorbance−sample absorbance control absorbance Â 100 The concentration of ON-Cr extract which inhibited substrate hydrolysis via AChE and BChE enzymes by 50% (IC 50 value) was calculated from the dose response curve [53]. The kinetics of enzyme activity was measured in the presence of increasing concentrations of extract.

Statistical analysis
All the assays were performed three times, while values were represented as ± S.E.M. One-way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for the analysis of antioxidant activity and enzyme inhibition assays. At 95% confidence interval, a P value of <0.05 was considered as statistically significant. Lineweaver-Burk plots (1/v versus 1/[s]) where v is reaction velocity and [s] is substrate concentration were plotted from assays using a range of plant extract concentrations. The V max and K m values were determined using Michaelis-Menten kinetics.

Phytochemical analysis
The preliminary phytochemical analysis of ON-Cr revealed the presence of various secondary metabolites like alkaloids, flavonoids, tannins, sterols, saponins and terpenoids. Table 1 highlights the observational results of all the phytochemical tests. In the HPLC-DAD analysis of ON-Cr, several components were separated in different ratios but gallic acid derivative (25.57%), hydroxybenzoic acid derivative (17.81) and rutin (5.35%) were compounds present in higher concentrations as summarized in Fig. 2 and Table 2. The highest concentrated signals were observed at retention times of 6.9, 5.8, 5.2, 6.3 min which were 25.57, 17.81, 14.41 and 8.19% respectively.

Antioxidant assays
In the DPPH free radical scavenging assay, ON-Cr at the concentrations of 31.25, 62.50, 500 and 1000 μg/mL demonstrated % inhibition not significantly different when compared to the positive control (ascorbic acid) (P > 0.05) at the same concentration. As shown in Fig. 3, the IC 50 value of ON-Cr was 71.46 μg/mL, while that of ascorbic acid was 31.31 μg/mL. Interestingly, in ABTS free radicals scavenging assay, ON-Cr only at the concentration of 62.05 μg/mL demonstrated significantly different % inhibition in comparison to that of ascorbic acid (P < 0.001 at 95% confidence interval). While at the concentrations of 31.25, 125, 250, 500 and 1000 μg/mL, the % inhibition values were not significantly different in comparison to ascorbic acid at similar concentrations (P > 0.05 at 95% confidence interval). As shown in Fig. 4, the IC 50 value of ON-Cr was 72.55 μg/mL, while that of ascorbic acid (positive control) was 32.37 μg/mL. In the H 2 O 2 anti-radicals assay, ON-Cr at the concentrations of 31.25, 62.50 μg/mL demonstrated % inhibition significantly different in comparison to that of ascorbic acid (P < 0.001 at 95% confidence interval). While at the concentrations of 125, 250, 500 and 1000 μg/mL, the % inhibition values were not significantly different when compared to ascorbic acid at similar concentrations (P > 0.05 at 95% confidence

Anticholinesterase assays
In the acetylcholinesterase inhibitory activity, the IC 50 value for ON-Cr was 33.2 μg/mL, while that of  (Fig. 6). Furthermore, in butyrylcholinesterase inhibitory activity, ON-Cr demonstrated potential IC 50 value of 55.36 μg/mL, while that of galantamine was 24.99 μg/mL (Fig. 7). The strong inhibitory propensity of ON-Cr was further corroborated from the Linewear-Burk plots (Fig. 8)

αglucosidase inhibitory assay
The percent inhibition of α-glucosidase via ON-Cr was found to be dose-dependent. The IC 50 value of ON-Cr was 639.89 μg/mL, while that of acarbose used as positive control was 61.19 μg/mL. The percent inhibition of α-glucosidase of ON-Cr in a concentration range of 31.25-1000 μg/mL is shown in Table 3. The Linewear-Burk plots for the inhibition of α-glucosidase by ON-Cr and galantamine is shown in Fig. 9. The dissociation constant (K m ) and V max values of ON-Cr for α-glucosidase were 31.11 μg and 51.01 μg/min, respectively, which were comparable to that of positive control, acarbose (30.19 μg and 76.17 μg/min), thus showing potent inhibitory proclivity against α-glucosidase.

Discussion
In the present study, detailed phytochemical investigation and pharmacogonostic activity of Olax nana aqueous methanolic extract has been reported for the first time.
Presence of various secondary metabolites like alkaloids, flavonoids, tannins, sterols, saponins and terpenoids have been found in the preliminary phytochemical analysis. Interestingly, in the HPLC-DAD results the highest concentrated signals were of gallic acid derivatives, hydroxybenzoic acid derivatives and rutin. Previously, Olax scandens has been reported for the presence of various essential phytochemicals like oleanolic acid, octacosanol, aleanolic acid and β-sitosterol etc. [54].   Furthermore, chemical investigation of Olax mannii Oliv also reported to contain useful flavonoid glycoside and derivatives [30]. Free radicals are generated during metabolic processes in the body and are implicated in a variety of disorders including neurodegenerative disorders, coronary heart disease, cancer, diabetes and immune-suppression [55,56]. The hydrogen peroxide and singlet oxygen are included in the list of non-free radicals, while nitric oxide, lipid peroxyl, superoxide and hydroxyl are common free radicals [57]. Naturally, the lethal effect of free radicals is nullified in our body via various defense mechanisms, including the protective antioxidant system and chain breaking antioxidants generation [58]. Furthermore, extensive tissue injury occurs when free radical generation rate surpasses the limit of natural scavenging mechanisms. Hence, many diseases including neurogenerative disorders can be cured with the help of drugs having free radical scavenging ability. Plants  Acetylcholinesterase inhibitory activity (AChE) of aqueous methanolic extract of Olax nana using Ellmen's assay. One-way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical significant different between the standard drug (galantamine) and test sample at the same concentration, at 95% confidence interval. ns: values not significantly different in comparison to % inhibition of the standard drug (P >0.05) at the same concentration Fig. 8 Butyrylcholinesterase inhibitory activity (BChE) of aqueous methanolic extract of Olax nana using Ellmen's assay. One-way ANOVA followed by Bonferroni's multiple comparison post hoc test was used for statistical significant different between the standard drug (galantamine) and test sample at the same concentration, at 95% confidence interval. ns: values not significantly different in comparison to % inhibition of the standard drug (P >0.05) at the same concentration are a valuable resource of antioxidants which have been reported to protect from free radicals induced damage [59]. Significant anti-radical activities was revealed by ON-Cr in the DPPH, ABTS and H 2 O 2 free radical scavenging assays with IC50 values of 71.46, 72.55 and 92.33 μg/mL, respectively.
In the treatment of neurological disorders, potential inhibitors of acetylcholinesterase (AChE) and butyrylesterase (BChE) enzymes are of prime importance. These enzymes are involved in the catabolism of acetylcholine (ACh), hence have applications in the treatment of Alzheimer's disease (AD) [60], along with many other neurological disorders [61]. AD is connected with memory impairment, change of behavior and cognitive dysfunction [62]. A decline in the amount of ACh via malfunctioning of various biochemical pathways ultimately leads to AD [63]. AChE along with BChE result in the termination of signal transmission carried out in the synapse via ACh. Hence, by inhibiting these key metabolizing enzymes, we can treat AD and many other neurological disorders [64]. Unfortunately, the available drugs are associated with hepatotoxicity along with many adverse side effects and show effectiveness only in mild type of AD [65]. Hence, it's of prime importance to explore novel remedies for AD, which is effective and have less side effects propensities. Plants are widely explored for potential therapeutic compounds effective in the treatment of neurological disorders [66]. Many research groups including ours have also explored the potential of medicinal plants as valuable source for the treatment of neurodegenerative disorders especially AD [35,67]. In the current study, very potent acetylcholinesterase (AChE) and butyrylesterase (BChE) inhibition potential was demonstrated by ON-Cr. Interestingly, in both acetylcholinesterase and butyrylcholinesterase inhibitory activities of ON-Cr, all the concentrations (31.25, 62.50, 125, 250, 500 and 1000 μg/mL) demonstrated % inhibition values not significantly different in comparison to galantamine (standard drug) at the same concentration (P > 0.05 at 95% confidence interval). Previously, Olax Subscorpioidea is also reported to possess neuroprotective, anxiolytic and anti-depressant properties [18,21]. Significant results noticed in the antioxidant and anticholinesterase assays of ON-Cr may be attributed to the predominant presence of Gallic acid derivatives among the identified compounds (Table 2), which is a common poly phenol and strong antioxidant having promising neuroprotective potentials [68,69]. The plant extracts also contain ascorbic acid which is a strong antioxidant. Quercetin and its derivatives are extensively studied as neuroprotective and antioxidant agents [70][71][72]. ON-Cr contains several quercetin derivatives which may be responsible for the antioxidant and cholinesterase inhibitory potentials of extracts samples. Pyrogallol, another polyphenolic compound was identified via comparison with the standards. This compound is a potent auto-oxidant and stimulates the activation of indigenous antioxidant system [73]. Rutin has a well established antioxidant and neuroprotective potentials [74,75]. The current antioxidant and anti-cholinesterase potentials of ON-Cr can be attributed to the presence of these compounds. Yet further studies  regarding the isolation and testing of the plant samples will validate its potential use as a neuroprotective agent in folk medicine.
In the early stage of diabetes mellitus type 2, postprandial hyperglycemia occurs due to impaired secretion of insulin after meal. It is believed that free radicals may cause hyperglycemia and may result in complications, like retinopathy, nephropathy, and memory impairment [76]. The dietary carbohydrates in our meal are digested by a group of enzymes known as glucosidases. Acarbose is one of the key inhibitor of glucosidase, which ultimately downregulate the process of carbohydrate digestion and delays its absorption into the blood. Hence, potential inhibitors of glucosidases can ultimately prevent type 2 diabetes mellitus development via lowering of glucose after meal [77]. Plants have been reported as a great source of these type of inhibitors [78,79]. Our results showed interesting inhibition of αglucosidase via ON-Cr. The dissociation constant (Km) and Vmax values as reported by ON-Cr and acarbose were comparable to each other (Fig. 9). To the best of our knowledge, this is the first report of α-glucosidase inhibition via any Olax specie.

Conclusions
This present research is an exclusive report on the phytochemical composition, antioxidant, cholinesterase and α-glucosidase inhibitory potentials of Olax nana methanolic extract. The results of enzyme inhibitions and anti-radical activities were highly significant. These results indicate that Olax nana, is highly enriched with potential antioxidant compounds which can be exploited as potential therapy for the treatment of many disorders. In light of the potential biomedical application as demonstrated by the above results, further studies on the bioassay-guided isolation of potential active compounds and their therapeutic evaluation is recommended.