HPTLC Fingerprint Profile and Identification of Antidiabetic and Antioxidant Leads from Bauhinia rufescens L

Diabetes is one of the world’s major health problems, and many reports have supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. The present study aims to evaluate the antidiabetic and antioxidant activity of Bauhinia rufescens , a plant used in Sudanese folkloric medicine for the treatment of diabetes. It was also aimed to identify isolates and characterize the bioactive antidiabetic and antioxidant compounds using bioactivity-guided fractionation followed by high-performance thin-layer chromatography (HPTLC) autobiography, liquid chromatography-mass spectrometric analysis, and nuclear magnetic resonance (NMR). Two potential compounds were successfully isolated and identiﬁed which may provide new leads for more potent analogues in drug discovery.


Introduction
In recent years, the interest in folk medicine from different cultures has increased significantly in industrialized countries due to the fact that many prescription drugs worldwide have originated from the tropical flora [1].
Diabetes mellitus is a group of disorders with multiple etiologies resulting from a defect in insulin secretion, insulin action, or both. Insulin deficiency in turn leads to chronic hyperglycemia (very high blood glucose levels) with disturbances in carbohydrate, fat, and protein metabolism. [2] Treatment of diabetes mellitus is considered as the main global problem, and successful treatment has yet to be discovered. Traditional medicines might provide useful sources for the development of new drugs for treatment of diabetes mellitus and its complications [3]. Medicinal plants, mostly with antioxidant activity, are the major source of drugs for the treatment of oxidative stress-induced diabetic complications since they have no or only few side effects [4]. Several plant extracts with hypoglycemic properties and protective activities act either through their positive impact on glucose homeostasis in diabetic patients or by lowering blood pressure and improving the renal and cardiovascular functions [5,6]. e most common herbal active ingredients used in treating diabetes are flavonoids, tannins, phenolic, and alkaloids that improve the performance of pancreatic tissues by increasing the insulin secretion or decreasing the intestinal absorption of glucose [7]. e existence of these compounds implies the importance of evaluating the antidiabetic properties of these plants [8].
Plants belonging to the genus Bauhinia have been reported as having potent antioxidant, antidiabetic, anti-inflammatory [9,10,11], and other biological properties. [12,13]. e leaves and stem bark of these plants are used in different phytopreparations to lower blood glucose levels, and they are also frequently used in folk medicine to treat infectious diseases, and several experimental studies have confirmed their antimicrobial potential [14,15,16,17,18]. e plant Bauhinia rufescens Lam common name Kharoub (family: Fabaceae) has been used traditionally in Sudan, Nigeria, and Senegal for various pharmacotherapeutic purposes. However, till date, there are no reports on scientific validation of its traditional claim neither its bioactivity. Hence, it was thought worthwhile to determine its antioxidant and antidiabetic potential and isolate and characterize the bioactive compounds. C-NMR spectra were carried out on the Bruker AM 500 spectrometer (Germany) operating at 700 MHz ( 1 H-NMR) and 125 MHz ( 13 C-NMR) in a spectroscopic grade DMSOd 6 .

Chromatographic Conditions.
Separation was performed on a UPLC system equipped with a binary solvent delivery system, an auto-sampler, a column manager, and a tunable MS detector (Waters, Manchester, UK). e system was operated under the Empower software (Waters, USA). Data acquisition has been done in positive modes. Chromatography was performed using acetonitrile (A) and 0.5% v/v formic acid in water (B) as the mobile phases on the monolithic capillary silica-based C 18 column (ACQUITY UPLC (R) BEH C 18 1.7 μm, 2.1 × 100 mm), with the precolumn split ratio 1 : 5 and a flow rate 10 μL/min at ambient temperature.
Different solvent systems were investigated in order to obtain suitable elution in TLC plates. Toluene : ethyl acetate : formic acid (5 : 4 : 1, v/v/v) was finally selected as the specific solvent.

Plant Material.
e plant was collected from Northern Kordofan and authenticated at the herbarium of the Medicinal and Aromatic Plants Research Institute (MAPRI), and a voucher specimen was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum.

Preparation of Plant Extract.
Air-dried leaves powder (1 kg) was exhaustively extracted with 500 ml petroleum ether using a soxhlet apparatus. e extract was then concentrated under reduced pressure using a rotary evaporator. e remaining marc was dried and extracted using 500 ml chloroform and then by 500 ml methanol. Both chloroform and methanol extracts were concentrated under reduced pressure using a rotary evaporator.

Fractionation of the Active Extract.
e methanol extract (active extract) was fractionated with ethyl acetate, chloroform, and n-butanol (250 ml for each) sequentially after suspending it in water.  [19]. In a 96-well plate, the test extracts were dissolved in DMSO and allowed to react with 300 μM ethanolic solution of 2, 2-di (4-tertoctylphenyl)-1-picryl-hydrazyl stable free radical (DPPH) for half an hour at 37°C. After incubation, the absorbance was measured at 517 nm against a blank of DPPH in DMSO using a multiplate reader spectrophotometer. Propyl gallate was used as a standard drug at a concentration of 0.5 mM. All tests were run in triplicate, and percentage radical scavenging activity (%RSA) by samples was determined in comparison with a DMSO-treated control group using the following formula:

Antioxidant
where RSA is the radical scavenging activity percent, A c is the absorbance of the control, and A t is the absorbance of the tested extract at 517 nm. e obtained data were statistically analyzed and presented as mean ± standard deviation of the mean.

Bioautography Method.
e extracts were run on the TLC silica gel G 60 F 254 plate using the specific solvent system. Plates were then air-dried, and visualization was conducted by spraying the plate with DPPH (0.001 g in 95% methanol/ ethanol).

In Vitro Assay Using TLC Bioautographic Method (α-Amylase Inhibitors).
e in vitro assay of antidiabetic activity of plant extracts was determined according to Washim Khan et al., a standard method [20]. e ethyl acetate fraction (active fraction) was spotted on the TLC plate and developed using toluene : ethyl acetate : formic acid (5 : 4 : 1 v/v/v) as the mobile phase. After solvent migration, the TLC plate was dried and dipped into a previously prepared enzyme solution (alpha-amylase (200 unit) which was dissolved in a buffer solution (pH 7.15) and kept at 4oC). Incubation in a humid desiccator was firstly performed for 30 min at room temperature for the primary reaction between the enzyme and enzyme inhibitors, followed by another 15 min for enzyme substrate reaction and then airdried and finally dipped in an iodine solution for the visualization. 2 Advances in Pharmacological and Pharmaceutical Sciences

In Vivo Assay of Plant Extracts.
e in vivo assay of antidiabetic activity of plant extracts was also determined according to Washim Khan et al. [20]. Diabetes was induced in the rats by the administration of streptozotocin (in citrate buffer PH 5.5) intraperitoneally at a dose of 60 mg/kg. ree days later, the blood glucose level was measured. Animals with a blood glucose level around 300 mg/100 ml of blood were used. To test the effect of extracts in normal and streptozotocin diabetic rats, the animals were fasted for 12 hours and then were administered the extracts in doses of 1 g/kg intraperitoneally. Two hours later, the animals were anaesthetized with ether and blood was collected via a cardiac puncture. Blood glucose level was measured using the Reflotron instrument and the provided glucose kits (Roche Diagnostics).

Qualitative Analysis of the Active Fraction
2.9.1. LC-MS Analysis. LC-MS analysis was performed with the same methods followed by Washim Khan et al. [21]. Sample solutions (5 mg/mL) were filtered through a 0.2 µM PTFE membrane filter and injected into the UPLC-MS chromatograph. Separation was achieved by stepwise gradients from 5% B to 100% B for 20 min. e flow rate of the nebulizer gas was set to 500 L/h, for cone gas it was set to 50 L/h, and the source temperature was fixed to 100 oC. e capillary voltages and cone voltage were set to 3.0 and 40 KV, respectively. For collision, argon was employed at a pressure of 5.3 y 10 −5 torr. e accurate mass and composition for the precursor ions and for the fragment ions were calculated using the Mass Lynx V 4.1 software incorporated in the instrument. Data obtained from UPLC-MS were processed by Mass Lynx V4.1 (Waters, USA) and further used for metabolomics analysis of different samples. Separated metabolites present in different samples were tentatively identified based on their m/z ration and on the literature.

High-Performance
in-Layer Chromatography (HPTLC) Analysis. HPTLC analysis was performed using Washim Khan et al. methods [20], where 5 μl of the active fraction solution was separately applied to a 6 mm wide band on the HPTLC plates using a Camag Linomat-V automated TLC applicator with the nitrogen flow providing a delivery speed of 150 nL/s from the syringe. Plates were developed in a Camag Twin through a glass tank presaturated with the selected solvent system for 40 min. e plate was developed horizontally in a Camag horizontal developing chamber (10 × 10 cm) at room temperature. e plate was scanned at different wavelengths such as 254 nm, 354 nm, and 454 nm. After heating the plate at 100 oC for 5 min, derivatization of the chromatogram was done with 5% anisaldehyde sulphuric acid in methanol. e scanning was carried out at 565 nm with a Camag TLC scanner III using the Wincats1.2.3 software.

Isolation and Structural Elucidation of Active
Compounds. Isolation of some active antidiabetic and antioxidant principles was achieved by preparative thin-layer chromatography (PTLC), and structure determination of the isolated compound was achieved by NMR. e chemical shift values are expressed in δ (ppm) units using (TMS) as an internal standard, and the coupling constants (J) are expressed in Hertz (Hz). Standard pulse sequences were used for generating COSY, HMQC, and HMBC spectra (2D experiments).

Results and Discussion
Although insulin therapy and oral hypoglycemic agents are the first line of treatment for diabetes mellitus, they have some side effects and fail to significantly alter the course of diabetic complications [22]; therefore, medicinal plants had been claimed to provide another source of diabetes treatment. Many reports have supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. Bioactivity-guided fractionation led to isolation and identification of different compounds with antidiabetic and antioxidant activity, and most of them are phenolic compounds, especially flavonoids. e antioxidant property of flavonoids was the first mechanism of action studied, in particular with regard to their protective effect against cardiovascular diseases [23]. Flavonoids have been shown to be highly effective scavengers of most types of oxidizing molecules, including singlet oxygen and various free radicals that are probably involved in several diseases [24]. Mechanism of antioxidant action can include suppressing reactive oxygen species formation, either by chelating trace elements involved in free radical production, scavenging reactive species, and upregulating or protecting the antioxidant natural defenses [25]. Antioxidant activity of flavonoids has beneficial effect in treatment of diabetes, and especially many reports have supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. In the present study, the antioxidant and antidiabetic activities of our selected plant were performed for the first time.

DPPH Radical Scavenging Assay of Plant Extracts.
e radical scavenging ability measured by the DPPH assay showed that extracts exhibited extremely large variation in their capacity to inhibit DPPH. As summarized in Table 1, the highest effect of free radical neutralization was obtained by the methanolic extract. It showed strong antioxidant activity (89%), almost similar in effect to the standard propyl gallate (92 %).

Antidiabetic Assay of Plant Extracts.
e three different extracts of Bauhinia rufescens, petroleum ether, chloroform, and methanol extracts, were administered in a dose of 1 g/kg intraperitoneally into normal fasted rats and at the same above dose to streptozotocin-treated rats. e methanol extract clearly decreased the blood glucose level in normal fasted rats and streptozotocin-treated rats by 27 ± 3.1% and 16.9 ± 1.7%, respectively, after two hours following administration, while the chloroform and petroleum ether extracts did not show significant effect on the glucose level (Table 2).

Fractionation of Active Methanol Extract and Activity
Evaluation.
e obtained results revealed that the methanol extract has the best antioxidant and antidiabetic activities compared to the other extracts. Accordingly, the methanol extract was fractionated by a separatory funnel using different solvents. e fractions were then subjected to different tests to quantify their activities, isolate, and identify the active compounds. e antioxidant activity of the four fractions was assayed using the DPPH radical scavenging assay. Table 3 summarizes the obtained results which revealed that the ethyl acetate fraction has potent antioxidant activity (90%).
TLC bioautographic method was then performed to detect the antioxidant compounds and the alpha -amylase enzyme inhibitors which reflect the antidiabetic activity. e active ethyl acetate fraction was spotted on the TLC plate and developed using the selected solvent. By spraying the plate with DPPH (0.001 g in 95% methanol/ethanol), a bright yellow or cream spot was shown against a purple background, which indicates the presence of compounds with antioxidant activity (Figure 1). Furthermore, to investigate the antidiabetic activity, the TLC plate was spotted with the active fraction, eluted, and dipped in an iodine solution. α-Amylase inhibitors were visible on the TLC plate by the appearance of a yellowishgreyish spot on a dark brown background (Figure 2). (1) and (2). From antidiabetic and antioxidant test results (Figures 1 and 2), it is clear that compounds 1 and 2 possess antidiabetic activity beside their activity against oxidative stress. For the isolation of these active compounds, the ethyl acetate fraction was subjected to preparative thinlayer chromatography (PTLC) using toluene : ethyl acetate : formic acid (5 : 4 : 1, v/v/v) as the mobile phase. e structures of the isolated compounds were determined by 1 H-and 13 C-NMR spectra. 1 H-NMR spectrum of compound 1 (Table 4) indicated the presence of two singlet aromatic protons, and each proton integrated for one proton at δ 6.44 and δ 6.77 and were assigned to H-6 and H-8,   (2) (1)    Advances in Pharmacological and Pharmaceutical Sciences respectively. Other aromatic proton areas were assigned for ring-B protons, and two doublet signals at δ 7.77 and δ 6.90 were assigned to H-6′and H-5′, respectively. 1H-NMR spectrum also indicated the presence of a group of signals ranging between δ 5.2 and -5.27 attributed to H-1″, H-2″, and H-3″ in rhamnose sugar. Furthermore, a multiplet protons signal appeared at δ 3.11-3.97 which assigned to the remaining rhamnose sugar, H-4″and H-5''. e glucose sugar protons, H-1‴, H-2‴, H-3‴, H-4″, H-5‴, and H-6‴, appear at δ ranging between 3.11 and 4.69. 13 C-NMR spectrum (Table 4) indicated the presence of ten quaternary carbons including one carbonyl group at δ 178.21 assigned to C-4. e downfield shift of C-4 indicated the presence of a hydroxyl group at C-5 [26]. Four oxygenated aromatic carbons at δ 166.0, 162.88, 150.00, and 144.00 were assigned to C-7, C-5, C-4′, and C-3′, respectively. e other five remaining quaternary carbons at δ 159.06, 158.08, 134.70, 120.73, and 105.99 were assigned to C-9, C-2, C-3, C-1′, and C-10, respectively. 13 C-NMR spectrum experiment also indicated the presence of a group of signals ranging between δ 70.01 and 71.79 which were assigned for sugar protons. 1 H-NMR and 13 C-NMR spectra of compound 1 were in full agreement with that reported for rutin (structure shown in Figure 3) [27]. 1 H-NMR spectrum of compound 2 (Table 5) showed that the spectrum is different from the previous identified  Advances in Pharmacological and Pharmaceutical Sciences compound, in presence of two signals at δ 8.01 and δ 6.90 attributing to H-2′ and H-3′, respectively. 13 C-NMR spectrum (Table 5) showed the presence of one oxygenated aromatic carbon at δ160.41 in ring-B carbons. Two equivalent signals at δ130.96 and 131.00 were attributed to C-2′ and C-3′, respectively. e presence of two equivalent signals indicated clearly the presence of only one hydroxyl group in ring-B. erefore, compound 2 was  Advances in Pharmacological and Pharmaceutical Sciences

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Advances in Pharmacological and Pharmaceutical Sciences 13  C-NMR spectra were in full agreement with that reported for kaempferol-3-O-rutinoside (structure shown in Figure 4) [27].

Standardization and High-Performance in-Layer Chromatography (HPTLC) Fingerprint Analysis.
e active ethyl acetate fraction was developed in a high-performance thin-layer chromatographic (HPTLC) plate using toluene : ethyl acetate : formic acid (5 : 4 : 1, v/v/v) as the mobile phase ( Figure 5), followed by TLC scanning at 254 and 366 nm. HPTLC chromatogram (2D and 3D chromatogram) revealed the presence of different compounds with R f values ranging between 0.11 and 0.90 (Figures 6 and 7). e result showed the presence of flavonoid rutin which appear at R f 0.11, when compared with the standard, which can be used as a biomarker for standardization and quality control. In general, the finger printing can be useful in checking the adulterant. erefore, it can be useful for the evaluation of different marketed herbal drugs formulated in dosage forms.

Identification of Phytoconstituents from Ethyl Acetate Fraction by LC-MS.
e UPLC/MS analysis results in identification of more than thirteen predicted compounds, and most of them are phenolic compounds, especially flavonoids (Table 6 and Figures 8 and 9).

Conclusion
In our present study, two promising antidiabetic leads, namely, rutin and kaempferol-3-O-rutinoside, had been successfully isolated and characterized from Bauhinia rufescens for the first time. ese results can be considered as the starting point for future studies. Toxicological studies may further be planned to develop it in the form of a new drug for management of diabetes.

Data Availability
e data used to support the finding of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.