Three new α-glucosidase inhibitor benzo-isochromenes from Datura stramonium

Abstract Three new α-glucosidase inhibitory benzo-isochromenes were isolated from the chloroform fraction of Datura stramonium. Their structures were established with the help of modern spectroscopic techniques and were assigned the names as 1,6,8-triimethoxy-2-methyl-3,4-dihydrobenzoisochromene-5,10-diol, 3,6-dimethoxy-5-hydroxy-4-methylbenzoisochromene-9,11-dione and demethylflavasperon for compounds 1-3 respectively. The α-glucosidase inhibiting activity of compound 2 showed strong inhibition with an IC50 value of 27.5 µM, while compound 1 exhibited moderate activity with IC50 value of 60.2 µM compared to positive control (acarbose). Graphical Abstract


Introduction
Datura stramonium is a widely distributed plant in all parts of the tropical Oromia region. It possesses poisonous flower cereal yields and has many traditional uses against various diseases (Soni et al. 2012). D. stramonium is also recognized by other names like thorn apple, devil's trumpt, hells bell, angle trumpt, moonflower and others (Devi et al. 2011). D. stramonium is recognized well for both poisonous and medicinal values. It is reported for possessing psychoactive properties. It is related to Solanaceae family and demonstrates anticholinergic properties due to alkaloids like hyoscyamine, atropine and scopolamine (Padavala and Devaraj 2016). The plant is reported in old literature as early Sanskrit and Chinese literature reveals its narcotics properties. D. stramonium has also been recognized as a remedy for periods problems (Ma et al. 2006).
D. stramonium or thorn apple is reported as a drug of exploitation and has uses as unintentional ending of living beings. Indications related to D. stramonium poisoning comprised extreme thirst, fast heat beat, urinary retention, dry skin, loss of cognizance, misperception and hallucinations (Soni et al. 2012;Baynesagne et al. 2017).
In herbal medicines, the plant display very important role e.g. leaves and seeds are used as sedating, antitussive, antispasmodic etc. (Okwu and Igara 2009). Occasionally D. stramonium seeds are added to beer/wine to increase the outcome of drunkenness. It is used by women of many countries to relive labor pain; particularly Maxican women use it frequently (Alabri et al. 2014). The use of D. stramonium has also been reported in medicines for treating bruises and injuries, asthma, muscle contraction and painful menstruation (Arulvasu et al. 2010). The plant has uses in treating mucus, diarrheal problem, epilepsy, hemorrhoids, ulcers, hysterics and wounds. Furthermore the D. stramonium is used against cough and to overcome laryngitis and tracheitis (Dabur et al. 2004).
The leaves and seeds of D. stramonium can also be used in overcoming sleeplessness, downheartedness, asthma and relaxing smooth muscles of respiratory tube (Iqbal et al. 2017). The larvicidal activity of D. stramonium demonstrate its capacity to yield high amount of amino acids particularly alanine, glutamatephenylalanine, and tyrosine (Devi et al. 2011).
Different structurally complex and potent natural products have been isolated so for from D. stramonium. Among the various, fang and his collaborators isolated two new structurally potent steroids from D. stramonium (Fang et al. 2013). The plant has atropine alkaloid bearing methylated nitrogen which act as a bronchodilator mediated via inhibition of cholinergic airway smooth muscle (Padavala and Devaraj 2016). In recent times a number of pharmacological studies have been accompanied on Datura stramonium. Literature reveals that different extracts of this plant demonstrated antimicrobial activity, antifungal activity, hypoglycemic and antimutagenic activities (Bellila et al. 2011).
The D. stramonium is recognized for cytotoxic activity which revealed the presence of withanolide type steroids that assists in preventing humanoid cancer cell growth (Ma et al. 2006). The plant contains two major alkaloids namely Hyoscyamine and Hyoscine which have uses in ophthalmology and also used as preauesthetic agents in surgery. In addition, D. stramonium is considered as a chief commercial source of hyoscyamine (Mallick and Sasmal 2000). The D. stramonium seed's oil is considered as a good source of proteins and useful minerals (Oseni 2011). D. stramonium extracts have also been evaluated for their acaricidal functions on T. urticae (Rinc on et al. 2019). Considering the poisonous and medicinal values of extracts and compounds reported from D. stramonium, objective of the present research was to isolate new chemical constituents from stem, fruits and leaves of D. stramonium and subsequently examine their anti-diabetic potential.

Results and discussion
The present study begins with isolation of compounds from D. stramonium which resulted into the following three compounds given in Figure 1 (Note: see Figure 1 in a separate file).
Compound 1 was isolated as a yellow powder. The HRESIMS spectrum of the compound 1 gave an [M þ H] þ peak at m/z at 321.1326, corresponding to the molecular formula of C 17 H 20 O 6 (with eight degrees of unsaturation). Analysis of its 1 H and 13 C NMR data in association with DEPT experiments deduced that compound 1 also possessed benzo-isochromene skeleton and had close resemblance with rubrofusarine (Megawati et al. 2017). In comparison to the NMR data of Rubrofurasine, the compound showed some variations in chemical shift at Carbon 2, Carbon 3 and Carbon 4. Moreover, the NMR data of compound 3 revealed that methoxy group replaced the phenolic hydroxyl group at C-8 of Rubrofurasine, and this variation was also confirmed by HMBC experiment by showing correlation with dc 157.1 (Table S1, See supporting information for the Table S1 (109.6, 113.5, 124.9, 134.5, 136.4, 149.3, 157.1 and 157.2). The methylene carbon was further confirmed by DEPT experiment ( Figure S3). Finally, based on the analyzed data the structure of the compound 1 (Figure 1) was identified as 1,6,8-triimethoxy-2-methyl-3,4-dihydrobenzoisochromene-5,10-diol (Tianaka and Takido 1988). Compound 2, yellow powder, assigned molecular formula of C 15 H 12 O 6 based on its HRESIMS data, which displayed peak for [M þ H] 7). The structure was further confirmed by HMBC experiment showing correlation from H-13 (3.50) to C-3 (167.6), H-14 (3.43) to C-6 (167.9), H-2 (6.81) to C-1 (91.7), C-3 (164.7), C-4 (135.9) and C-8a (97.0) and H-7 (6.42) to C-5 (151.7), C-6 (167.9) and C-8a (97.0). The connectivity and the chemical shift of the protons and protonated carbons were established based on the HSQC experimental data ( Figure S9). Finally, the complete structure of compound 2 (Figure 1) was established by the combination of NMR and mass spectral data which was in close agreement with the reported data for benzo-isochromene and the compound 2 was named as 3,6-dimethoxy-5-hydroxy-4methylbenzoisochromene-9,11-dione. The NMR spectral data for the isolated compounds 1-2 are presented in Table S1 (See supporting information).
Compound 3 was isolated as a yellow coloured amorphous for which the HRESIMS analysis (m/z 259.0596 [M þ H] þ ; calcd. for C 14 H 11 O 5 þ ; 259.0601) data suggested a molecular formula as C 14 H 10 O 5 . The 1 H NMR and 13 C NMR data of compound 3 revealed that it was structurally related to Flavasperon (Sakai et al. 2008) except that hydroxyl groups replaced the methoxy groups of flavasperon. This variation was confirmed by the absence of peaks for methoxy groups in 1 H and 13 C NMR spectra and also by 2 D NMR and Mass spectra. Hence the name of compound 3 was assigned as demethylflavasperon (Lee et al. 1997;Bandara et al.2015). The NMR spectral data for the isolated compound 3 is presented in Table S2 (See supporting information).

a-Glucosidase inhibitory activity
The a-glucosidase inhibition property of the isolated natural products was carried out while using acarbose as a positive control (Table 3, Note: See the Table 3 in supporting information). Compound 2 showed strong inhibition with an IC 50 value of 27.5 ± 0.3 mM. Compound 1 exhibited moderate activity with IC50 values of 60.2 ± 0.4 mM compared to positive control (acarbose), while compound 3 was found least active. The results indicated that the number and position of the -OH groups affect the inhibition property as shown by the difference in inhibition of compound 1 and 3. Methylation of phenolic hydroxyl functions effectively decreases the inhibition activity of the compounds as shown by the weak activity of compound 3 compared to compounds 1 and 2.

Plant materials
The plant materials were collected from D.I.Khan. The plant was identified by Dr. Saddiq Khan, Faculty of Agriculture, Gomal University D.I.Khan, KP. Pakistan, and the voucher specimen number 00117 (RAW) was submitted to national herbarium, National Agricultural Research Centre (NARC), Islamabad.

Extraction and fractionation
The methanol extract of the plant was concentrated with rotary evaporator to get crude which was then suspended in 20% MeOH-H 2 O. The suspension was then fractionated with n-hexane, chloroform, ethyl acetate to get the corresponding n-hexane (15 g), Chloroform (26 g) and ethyl acetate (44 g) fractions. The chloroform extract was separated into 7 sub-fractions (A-H) by silica gel column chromatography by gradient elution of hexane/CH 2 Cl 2 (from 90:10 to 00:100) and then CH 2 Cl 2 /MeOH (from 100:00 to 00:100).

Purification of the compounds
Fraction G was further purified by silica gel column chromatography by gradient elution of CH 2 Cl 2 /MeOH (from 100:00 to 80:20) to get four sub fractions (G1-G4). Fraction G-2 was purified by semi preparative HPLC (75% MeOH-H 2 O flow rate 1.0 ml/min; C18, 10 Â 250 mm, 5 mm) to afford compound 3. Fr. G4 was further purified by HPLC (75% CH 3 CN-H 2 O flow rate 1.0 ml/min; C18, 10 Â 250 mm, 5 mm) to get two pure Compound 2. Fraction F was subjected to sephadex LH-20 eluted with CH 2 Cl 2 /MeOH (1:1) to get five sub fractions (F1-F5). Fr. F-3 was further purified by semi preparative HPLC (65% MeOH-H 2 O flow rate 1.5 ml/min; C18, 10 Â 250 mm, 5 mm) to afford pure compound 1 (15 mg).     3.5. a-Glucosidase inhibitory activity method. The a-glucosidase inhibition property was carried out according to the standard protocol with slight modifications. All the activities were performed by using 10 mM KH 2 PO 4 -, K 2 HPO 4 buffers and pH 7.0. The enzymatic solution was prepared as 2 Units/mL in 2 mL buffer solution. The reaction mixture comprised phosphate buffer, pH7.0 (150 mL), 20 mL of enzymatic solution, 20 lL of DMSO or inhibitor (test sample dissolved in DMSO (10 lmol/mL)), and 20 mL of the substrate (p-nitrophenylglycoside, 1.5 mg/mL). The inhibitors were pre-incubated with the enzyme at 37 C for 20 minutes and the substrate was then added. The reaction was examined through spectrophotometer by observing the absorbance at 400 nm for an interval of one minute. Calculation was performed as per the given equation g(%) ¼ [(B À S)/B] Â 100%, where B represents assay medium with DMSO and S represents assay medium with inhibitor. All the analysis were performed in triplicate. The reported IC50 was an average value of the two independent experiments.

Conclusion
The isolation studies of finding new chemical constituents from chloroform fraction of Datura stramonium plant material and their subsequent anti-diabetic activity provided three new anti-diabetic benzo-isochromenes (1-3). Structural formulae of the obtained compounds were established with the help of modern Mass and NMR spectral techniques. The a-glucosidase inhibiting activity results demonstrated that all the three compounds were active as anti-diabetic where compound 2 exhibited highest inhibition (IC 50 ¼ 27.5 ± 0.3), compound 1 showed moderate and compound 3 showed lowest activity, respectively. This study also suggests that D. stramonium is a promising source of bioactive natural products responsible for prevention of diabetes mellitus and its complications. Since these in-vitro results are of a preliminary nature therefore further in-vivo investigations will be carried out in due course, to exploit in detail medicinal importance of Datura stramonium.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.