Determination of DPPH Radical
Scavenging Activity

CPQ Nutrition (2019) 3:4
Experimental Paper

In Vitro Evaluation of the Antioxidant and Urease Inhibition Activities of Three Triterpenes from the Flowers of Alstonia Scholaris

Nighat Sultana^1* & Muhammad Saleem^1,2

¹Pharmaceutical Research Center, PCSIR Laboratories Complex, Karachi-75280, Pakistan
²Departments of Biotechnology, University of Karachi, Karachi, Pakistan

*Correspondence to: Dr. Nighat Sultana, Pharmaceutical Research Center, PCSIR Laboratories Complex, Karachi-75280, Pakistan.

Copyright © 2019 Dr. Nighat Sultana. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: 16 March 2019
Published: 10 April 2019

Keywords: In Vitro Evaluation; Urease Inhibition; Flowers

Reinvestigation of the flowers of Alstonia scholaris of Pakistan origin have resulted in the isolation of three triterpenoids, two of the ursane type, 3β-acetate- 24-nor- urs-4, 12-diene ester triterpene (1), 3β -hydroxy- 24-nor-urs-4,12,28-triene triterpene (2) and one of the oleanane type 3,28- β -diacetoxy-5-olea-triterpene (3) together with two known triterpenes α-amyrin acetate (4) and ursolic acid (5). These compounds were analyzed for antioxidant and antiurease inhibition tests. Compounds 2 and 4 were subjected to enzymatic bioassays. Compounds 2 and 4 were found to be antioxidant with IC₅₀ 107±0.39 and >500μM respectively, while compounds 2 and 4 were also found to be urease inhibitors with IC₅₀ 175 ± 0.26 and 218 ± 0.14μM, respectively.

Figure 1: Some selected mass fragments of 1

Figure 2: Some selected mass fragments of 2

Figure 3: Some selected mass fragments of 3

The genus Alstonia comprises about twelve species. Alstonia scholaris Linn. R. Br. belongs to family Apocynaceae [1], grows throughout India, in deciduous and evergreen forests, also in plains [2,3]. Its timber is a non-durable hardwood, suitable for light indoor construction purposes, pulp and paper production. The wood has been used for school blackboards, hence the name ‘scholaris’. They are milk bearing shrubs or trees, with large, entire, generally whorled leaves, and terminal cymes of white flowers.

The plant Alstonia scholaris has been used in different system of traditional medication for the treatment of diseases and ailments of human beings. It is reported to contain various alkaloids, flavonoids and phenolic acids. The flowers of Alstonia scholaris are known to contain lupeol and β-amyrin [4]. The bark contains alkaloids: ditaine, echitenine, echitamine (ditamine) and echitamidine together with triterpenes β-amyrin and lupeol [5-9]. It has been reported as antimicrobial, antiamoebic, antidiarrhoeal, antiplasmodial, hepatoprotective, immunomodulatory, anti-cancer, antiasthmatic, free radical scavenging, antioxidant, analgesic, anti-inflammatory, anti-ulcer, anti-fertility, anaemia, chronic diarrhea, dysentery, menstrual disorders, malarial fever, colic and acute arthritis and wound healing activities [10-14]. There are also reports available for the traditional use of this plant for its cardiotonic, anti-diabetic and anti-arthritic properties. The bark yields a tonic and antiseptic medicine. A concentrated decoction of trunk bark is used as a wash in furunculosis and impetigo, and as a gargle in dental caries. The bark, leaves and milky exudates of Alstonia scholaris are used in India [1-3].

3β -Acetate-24-nor-urs-4,12-diene ester triterpene (1), C₃₁H₄₈O₂, was obtained as a white solid mass by column and preparative thin-layer chromatography of the ethanolic extract of A. scholaris flowers.

The molecular composition of 1 was determined as C₃₁H₄₈O₂ by high-resolution electron-impact mass measurements of the M⁺ m/z 452.3652 (calcd. 452.3654) which indicated eight degrees of unsaturation in the molecule.

The ¹H-NMR spectrum [11,12] (CDCl₃, 500MHz) of 1 showed five three-proton singlets (δ 2.11, 0.85, 0.95, 0.84, 1.23) and two methyls as doublets (δ 0.80, J_29,19 = 6.5Hz, 0.91, J_30,20= 6.5Hz) indicating the presence of five tertiary methyls and two secondary methyls in the molecule as expected in a pentacyclic triterpenoidal skeleton (see experimental section).

The ¹³C-NMR spectra (broad-band decoupled and DEPT [11,12] CDCl₃, 125MHz) of 1 exhibited signals for all 31 carbons and further supported the formula derived by mass spectrometric observations. DEPT spectra showed the presence of seven methyl, ten methylene, seven methine and (by difference from the broad-band decoupled spectrum) seven quaternary carbons (see experimental section).

Two-dimensional NMR techniques such as COSY 45º, HOHAHA, HMQC and HMBC [12-14] were used to obtain more structural information. The Homonuclear Hartmann Hahn spectrum (HOHAHA) [12-15] recorded with mixing delay of 100ms showed that the C-3 methine proton is coupled with four protons i.e. with the C-2 and C-1 methylenic protons (δ 1.23/1.25 and 1.61/1.81) respectively [16-18]. These observations led to define the structure of 1 as 3β -acetate-24-nor-urs-4,12diene ester triterpene.

Figure 4: Selected multiple bond interactions of 1 observed in the HMBC experiment

3β-hydroxy-24-nor-urs-4,12,28-triene triterpene (2), C₃₁H₄₈O, was obtained as a white amorphous substance by column and preparative thin-layer chromatography of the ethanolic extracts of A. scholaris. The molecular composition was determined as C₃₁H₄₈O by high-resolution electron-impact mass measurements of the M₊ (m/z 436). The UV spectrum recorded in methanol showed terminal absorption only, while the infrared spectrum displayed absorptions for hydroxyl (OH, 3415 cm_-1) and C = C (1630 cm_-1) groups.

The ¹H-NMR spectrum [11,12] (CDCl₃, 500MHz) of 2 showed four three-proton singlets at δ 0.84, 0.95, 0.85, 1.23 and two methyls as doublets (δ 0.80, d, J = 6.8 Hz, 0.91, J = 6.6 Hz) indicating the presence of four tertiary methyls and two secondary methyls in the molecule as expected in a pentacyclic triterpenoidal skeleton. The downfield region of the spectrum contained only three signals i.e. a broad double doublet at δ 3.54 (J_3α,2β = 12.4Hz, J_3α,2α = 4.5Hz), four close doublets two at δ 4.57 (d, J_23α,23β = 2.4Hz), 4.68 (d, J_23β,23α = 2.4Hz) and another two at 4.54 (d, J_31α,31β = 2.5Hz) δ 4.65 (d, J_31β,31α = 2.5Hz) and a tripplet at δ 5.11 (t, J = 3.26Hz), which could be assigned to the hydroxy-bearing C-3 methine, C-23, 31 methylene and the vinylic C-12 protons, respectively. The chemical shift and coupling constants of the C-3 methine signal indicated an equatorial (β) orientation of the OH group [19].

The ¹³C-NMR spectra (broad-band decoupled and DEPT [11,12] CD3OD, 100MHz) exhibited signals for all 31 carbons and further supported the formula derived by mass spectrometric observations. The ¹³C-NMR broad-band spectrum (CDCl₃, 100MHz) of 2 showed the resonances of 31 carbon atoms in the molecule. DEPT spectra showed the presence of six methyl, eleven methylene, seven methine and (by difference from the broad-band decoupled spectrum) seven quaternary carbons. The downfield signals at δ 79.01 and 124.34 were due to the hydroxy-bearing C-3 and vinylic C-12, respectively. The six methyl carbons resonated at δ 15.72, 17.48, 28.06, 23.53, 28.11 and 19.74 in the ¹³C-NMR spectra. The assignments to the various carbons in the molecule are presented in experimental section.

Two-dimensional NMR techniques such as COSY 45º, HOHAHA, HMQC and HMBC [11-13] were used to obtain more structural information. The one-bond ¹³C/¹H couplings were determined by the HMQC spectrum see experimental section. The Homonuclear Hartmann Hahn spectrum (HOHAHA) [15,16,20- 23] recorded with mixing delay of 100ms showed that the C-3 methine proton is coupled with four protons i.e. with the C-2 and C-1 methylenic protons (δ1.23/1.25 and 1.61/1.81) respectively. The another two downfield exocyclic methylene protons appearing in the COSY-45º spectrum (4.54 and 4.65) not only displayed geminal coupling interactions but also gave strong cross peaks with the C-32 proton (δ 1.64/4.54, 4.65) in the same 2D experiment indicating the presence of the isopropene protons. These observations led to define the structure of 2 as 3β-hydroxy-24-nor-urs-4,12,28-triene triterpene.

3,28-β-diacetoxy-5-olea- triterpene (3), C₃₃H₅₂O₄, was obtained as a white amorphous compound by column and preparative thin-layer chromatography of the ethanolic extract of A. scholaris flowers. The UV spectrum recorded in methanol showed terminal absorption only, while the infrared spectrum displayed absorptions for C=O of ester group (1735cm^-1) and C = C (1630cm^-1) groups [19,24-26]. The molecular composition was determined as C33H52O4 by high-resolution electron-impact mass measurements of the M+ (m/z 530).

The ¹H-NMR spectrum (CDCl₃, 500MHz) of 1 showed nine three-proton singlets at δ 2.13, 2.10, 1.15, 1.08, 0.97, 0.95, 0.94, 0.85 and 0.84 indicating the presence of nine tertiary methyls in the molecule as expected in a pentacyclic triterpenoidal skeleton. The downfield singlets at δ 2.10 and 2.13 were due to the C-31, C-33 acetate methyls. The downfield region of the spectrum also contained two signals i.e. a double doublet at 4.50 (dd, J_3α,2α= 6.12, J_3α,2β= 14.5Hz) and a broad double doublet at δ 5.62 (J6,7α = 4.0Hz, J_6,7β= 12.5Hz), which could be assigned to the hydroxy-bearing C-3 methine and the vinylic C-6 protons, respectively [27]. A comparison of ¹H-NMR chemical shifts of 1 with 3α-hydroxy-D-friedoolean-5-ene was also done [10].

The ¹³C-NMR spectra (broad-band decoupled and DEPT [10,12] CDCl₃, 100MHz) of 3 exhibited signals for all 33 carbons and further supported the formula derived by mass spectrometric observations. DEPT spectra showed the presence of nine methyl, ten methylene, five methine and (by difference from the broadband decoupled spectrum) nine quaternary carbons. The downfield signals at δ 80.94, 121.70 and 170.96 were due to the acetate-bearing C-3, vinylic C-6 and acetate carbonyl C-28 respectively. The eight methyl carbons resonated at δ 31.87, 31.49, 29.20, 20.00, 19.39, 19.04, 18.04 and 12.05 in the ¹³C-NMR spectra. Two-dimensional NMR techniques such as COSY- 45º, HOHAHA [17,18,28], HMQC and HMBC [13] were used to obtain structural information.

The Heteronuclear Multiple Quantum Coherence [13] (HMQC) spectrum of 3 displayed cross peaks between directly coupled carbon-proton pairs. HMQC interactions are presented in experimental section.

The Heteronuclear Multiple Bond Connectivity [13] (HMBC) spectrum was also very informative in determining the position of the double bond between C-5 and C-6, since the C-6 vinylic proton (δ 5.62) showed long-range interactions with C-4 (δ 40.45) and C-9 (δ 51.24). The C-3 proton (δ 4.50) showed coupling with C-5 (δ 140.78).

Figure 5: Selected multiple bond interactions of 2 observed in the HMBC experiment

Figure 6: Selected multiple bond interactions of 3 observed in the HMBC experiment

α-Amyrin acetate (4) M.P. 166-170 was isolated as a white amorphous solid from the pet. ether extract of A. scholaris by column and thin-layer chromatography. The high resolution electron-impact mass spectrum of 4 showed the molecular ion-peak at m/z 468.3708, which corresponded to the molecular formula C₃₂H₅₂O₂. 13C-NMR chemical shifts of C-12 and C-13 at δ 124.33 and 139.63 respectively suggested that 4 belongs to Δ12-α amyrin series of triterpenoids with ester group in A/B rings.

Ursolic acid (5) was isolated as a greenish white powder from the pet. ether extract of A. scholaris by column and thin-layer chromatography. Compounds 4 and 5 were isolated for the first time from the flowers of this plant [4] and had not been previously isolated.

These compounds were analyzed for antioxidant and antiurease inhibition test. Compounds 2 and 4 were found to be antioxidant with IC₅₀ 107±0.39 and > 500μM respectively, while compounds 2 and 4 were also found to be urease inhibitors with IC₅₀ 175 ±0.26 and 218±0.14μM, respectively Table 1. The quantity of 1 and 3 was short, hence its activity could not be checked. BHA (IC₅₀ = 44.3±0.09μM) and thiourea (IC₅₀ = 21.0±0.08) were used as a positive control [29,30].

Table 1: In vitro quantitative inhibition of antioxidant and urease by compounds 1, 2 and 4a

Experimental Section

General Experimental Procedures
The mass spectra were recorded on a Jeol HX-110 instrument. The ¹H- and ¹³C-NMR spectra were recorded in CDCl₃ at 500, 400 and 125, 75MHz, respectively, on a Bruker AM-500, 400 NMR spectrometer. The UV and IR spectra were recorded on Shimadzu UV-240 and JASCO A-320 spectrophotometers, respectively. Optical rotations were measured on a polatronic D Polarimeter. The purity of the compounds was checked on TLC (si-gel, Merck PF254, 0.25mm thickness). Melting points were determined in glass capillary tubes using Buchi 535 and Gallenkamp 30/MF-370 melting point apparatus.

Plant Material
The flowers of Alstonia scholaris, (5kg) were collected from university campus Kashmir in October 2006. A voucher specimen (AKUH # 58106) was deposited in the Herbarium of Department of Botany, University of Azad Kashmir.

Extraction and Isolation
Air-dried flowers of Alstonia scholaris (5kg dry weight) were extracted with MeOH (50L). The MeOH extract was concentrated to a gum (822gm), dissolved in distd. water and extracted thoroughly with pet. ether (25L). The pet. ether soluble portion was evaporated under reduced pressure to yield a gum (66.92gm) which was chromatographed on a si-gel column (Merck, 70-230 mesh, 2025.01gm). The elution of the column was initiated with pet. ether. The combined column sub-fractions 1-8 (5.91gm) obtained by elution with 5:95 ethyl acetate-pet. ether, which showed similar tlc behaviour upon spraying with ceric sulphate reagent were combined and again subjected to CC using silica gel (type 60, 70-230 mesh, 200.10gm) and column was eluted with pet.ether- ethyl acetate (99:1). The sub-fractions 6-30 (1.86gm), which showed similar tlc behaviour were combined and further purified on preparative tlc plates using a solvent system of pet.ether- ethyl acetate (98:2) to afford pure compound 1 (19.5mg, 9.5 x 10^-5% yield). The fractions obtained on elution of the column with n-hexane-ethyl acetate (10:90) were checked on TLC. Fractions 7-18 showed similar behaviour on TLC were combined and further purified by preparative TLC (Merck PF₂₅₄ ,0.2mm) using CHCl₃ as eluent to afford pure compound 2 (28mg, 1.4 x 10^-4 % yield). Elution of the major column which was loaded with 66.92g of pet.ether soluble material was eluted with 30% ethyl acetate -pet.ether yielded an impure mixture (7.83gm). This mixture was again subjected to CC (silica gel, 70-230 mesh, 60.20gm). The sub-fractions 6-30 (1.86gm), which showed similar tlc behaviour were combined and further purified on preparative tlc plates using a solvent system of pet.ether-ethyl acetate (90:10) to afford pure compound 3 (19.5mg, 9.5 x 10^-5% yield). The fractions obtained with 30: 70 ethyl acetate-pet.ether yielded an impure compound 4, which was further purified by preparative tlc using a solvent system of pet. ether- ethyl acetate (70:30) to obtain pure 4 (20mg, 10 x 10^-4 % yield). Fractions 99-145 (1.91gm) obtained by elution with pet. ether- ethyl acetate (35: 65, 500ml each) were collected and again subjected to CC (70-230 mesh, 60.20gm). The sub-fractions 7-18 (0.92gm, 500ml each) obtained with 80: 20 hexane- ethyl acetate showed similar behaviour on TLC (Ceric sulphate active) were combined and further purified on preparative TLC (Merck PF₂₅₄ ,0.2mm) using hexane- ethyl acetate 70:30 as eluent to obtain pure 5 (28mg, 1.4x 10^-4 % yield, with R_f = 0.1).

3β-acetate-24-nor-urs-4, 12-diene ester triterpene (1)
White solid substance, [α]²⁹_D = 40, UV: (MeOH) λ_max nm: terminal absorbtion only, IR: (CHCl₃) ν_max cm^-1, 1732 (C=O), 1630 (C=C), HREI MS: m/z 234.1618 (calcd. 234.1619), 218.2032 (calcd. 218.2034), 174.1406 (calcd.174.1408), F.D. 452, MS: m/z 452.3652 (calcd. 452.3654); 1H-NMR: (CDCl₃, 500MHz), ¹³C-NMR (CDCl₃, 125MHz) δ; data is presented in Tables-1, 2.

Table 2: ¹H-NMR (# δ ppm) data of compounds 1, 2 and 3

Table 3: ¹³C-NMR (# δ ppm) data of compounds 1, 2 and 3

♣ Multiplicity assignments based on DEPT experiment # One-bond heteronuclear correlations determined by HMQC experiment

3β-Hydroxy-24-nor-urs-4, 12, 28-triene triterpene (2)
White amorphous substance, [α]²⁹_D = 40 (CHCl₃), UV: (MeOH) λ max nm: only terminal absorbtion, IR (CHCl₃) ν max cm_-1 hydroxyl (OH, 3415cm^-1) and C = C (1630cm^-1) groups, HREI MS: m/z 192.1513 (calcd. 192.1514, C₁₃H₂₀O) and m/z 244.2191 (calcd. 244.2190, C18H28), 174.1406 (calcd. 174.1408, C₁₃H₁₈ fragment c); F. D. 436; MS: m/z 436; 1H-NMR: (CDCl₃, 500MHz), 13C-NMR (CD₃OD, 100MHz) δ; data is presented in Tables 1-2.

3,28-β-Diacetoxy-5-olea-triterpene (3)
White amorphous compound; [α]_D²⁹ = 64.5º (CHCl₃); UV: (MeOH) λ_max nm: only terminal absorbtion, IR (CHCl₃) ν max cm^-1 (C=O, 1735cm^-1), (C=C, 1630cm^-1); EIMS: m/z 530, HREI MS: m/z 208.1462 (calcd. 208.1463), 304.2403 (calcd. 304.2402), 148.1252 (calcd. 148.1251) and 244.2191 (calcd. 244.2190); 1H-NMR (500MHz, CDCl₃sub>); δ: ¹³C-NMR (100MHz, CD₃OD) data is presented in Tables 1-2.

α-Amyrin acetate (4)
White amorphous solid; M. P. = 166-170ºC, [α]_D²⁹ = + 83.00º (CHCl3); UV λ^max (MeOH) nm, only terminal absorption; IR (CHCl₃) cm^-1; 2850 (C-H), 1630 (C=C),1715 (C=O), 1136 and 1369 (C-O) cm^-1 EI-MS gave m/z at 469 (M• + H, C₃₂H₅₂O²), 454 (M• + H -CH₃), 409 (42%), 394 (28%), 357 (15%), 298 (11%), 273 (39%), 267 (38%), 249 (68%), 232 (24%), 218 (100%), 203 (73%), 189 (75%), 175 (66%), ¹H-NMR showed signals at δ ppm= 0.78 (s, 1 x CH₃), 0.86 (s, 2 x CH₃), 0.87 (s, 2 x CH₃), 0.97 (s, 1 x CH₃), 1.00 (s, 1 x CH₃), 1.06 (s, 1 x CH₃), 2.02 (s, COCH₃), 4.50 (m, 1-3β-H ), 5.11 (t, J= 3.6 Hz, C-12H), 13C-NMR showed signals at 171.0, 139.71,124.41, 81.02, 59.16, 55.35, 47.73,42.82, 39.73, 39.72, 39.62, 38.56, 36.89, 34.82, 33.36, 31.32, 28.79, 28.45, 28.18, 26.68, 23.68, 23.44, 23.29, 23.29, 21.34, 18.32, 17.55, 16.94, 16.79, 15.77, ¹H-NMR (CDCl₃, 500MHz) δ: ¹³C-NMR (CDCl₃, 125MHz) [31-33].

Ursolic acid (5)
Greenish white powder, [α]_D²⁹ = 59 (c = 0.3, pyridine); M. P. 283-285 ºC, UV λ_max (MeOH) nm; only terminal absorption, EIMS m/z: 456.4 [M]^+•, 438.4, 423.4, 300.3, 248.3, 203.2, 133.1; HRMS m/z: 456.3603 (C₃₀H₄₈O₃), IR (CHCl₃) ν_max; ¹H-NMR (CDCl₃, 400MHz); ¹³C-NMR (CDCl₃, 125MHz) [29].

Determination of DPPH Radical Scavenging Activity
The free radical scavenging activity was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH) [30]. The solution of DPPH of 0.3mM was prepared in ethanol. Five microlitres of each sample of different concentration (62.5μg - 500μg) was mixed with 95μl of DPPH solution in ethanol. The mixture was dispersed in 96 well plate and incubated at 37ºC for 30min. The absorbance at 515nm was measured by microtitre plate reader (Spectramax plus 384 Molecular Device, USA) and percent radical scavenging activity was determined in comparison with the methanol treated control. BHA is used as standard.

DPPH scavenging effect (%) = Ac - As/Ac × 100

Where, Ac = Absorbance of Control (DMSO treated)
As = Absorbance of Sample

Urease Assay and Inhibition
Reaction mixtures comprising 25μL of enzyme (Jack bean Urease) solution and 55μL of buffers containing 100mM urea were incubated with 5μL of test compounds (1mM concentration) at 30ºC for 15 min in 96-well plates. Urease activity was determined by measuring ammonia production using the indophenol method as described by Weatherburn [34]. Briefly, 45μL each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70μL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The increasing absorbance at 630nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200μL. The results (change in absorbance per min) were processed by using SoftMax Pro software (Molecular Device, USA). All the assays were performed at pH 8.2 (0.01M K₂HPO₄.3H₂O, 1mM EDTA and 0.01M LiCl₂). Percentage inhibitions were calculated from the formula 100-(ODtestwell/ODcontrol) x100. Thiourea was used as the standard inhibitor of urease.

Acknowledgement
The authors wish to thank the Ministry of Science and Technology, government of Pakistan for providing the spectroscopic facilities for the current study.

Bibliography