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Short Note

a-Glucosidase enzyme inhibitory and free radicalscavenging constituents from Premna tomentosaLinn.

Sridhar Rao Ayinampudi a,b,*aDepartment of Chemistry, Mahatma Gandhi University, Nalgonda, Andhra Pradesh 508 254, Indiab Indian Institute of Chemical Technology, Hyderabad 500 007, Andhra Pradesh, India

a r t i c l e i n f o

Article history:

Received 11 July 2013

Accepted 11 August 2013

Available online 28 August 2013

Keywords:

Premna tomentosa

Verbenaceae

Phytochemicals

a-Glucosidase inhibition and Free

radical scavengers

* Department of Chemistry, Mahatma GanE-mail addresses: asriict@gmail.com, ved

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.08.013

a b s t r a c t

A bioassay-guided fractionation and chemical investigation of the whole plant of Premna

tomentosa resulted in the isolation and characterization of premnalin (1), along with the

known compounds coniferaldehyde (2), syrangaldehyde (3), acetoxy syrangaldehyde (4),

lupeol (5), betulin (6), 2-(4-methoxyphenyl)-2-butanone (7), icetexatriene-1 (8), icetexatriene-

2 (9). Their structures were established on the basis of extensive spectroscopic such as (IR,

MS, 2D NMR) data analysis and by comparison with the spectroscopic data reported in the

literature, premnalin (IC50: 12.11 mg/mL) & (SC50: 20.58 mg/mL), acetoxy syrangaldehyde (IC50:

18.41 mg/mL) & (SC50: 20.83 mg/mL) displayed potent a-glucosidase inhibition and free radical

scavengers (DPPH).

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction pharmacological uses of P. tomentosa extracts, little is known

The genus Premna (Verbenaceae) comprises a group of more

than 200 different trees, distributed in tropical and subtrop-

ical areas of the world. Premna tomentosa (Verbenaceae) is a

well known medicinal plant used extensively for the treat-

ment of various ailments. In Indian system of medicine, all

parts of P. tomentosa have been employed for the treatment of

various disorders.1 Its bark extract is claimed to have a

lasting cure for hepatic disorders2 Extracts from P. tomentosa

leaves are known to have diuretic, hepatoprotective, anti-

oxidant, lipid-lowering, immunomodulatory activities, and

protective against acetaminophen-induced mitochondrial

dysfunction properties.3e8 In spite of the various

dhi University, Nalgondasreedhar@gmail.com.2013, JPR Solutions; Publi

about the chemical constituents. Previous studies on this

species have resulted in the isolation of various compounds,

including flavonoids, triterpenoids, and steroids,9 as part of

our continuing efforts directed towards the discovery of the

structurally interesting and biologically active compounds

from the Indian medicinal plants.10,11

The a-glucosidase inhibitors present broad-spectrum

therapeutic applications.12 Recently, mammalian intestinal

a-glucosidase inhibitors in particular, have found potential

therapeutic application in treatment of postprandial diabetes

and have been observed to play important role in mitigating

several diabetic complications.13 These observations have

spurred aggressive efforts to synthesize14 as well as isolate

, Andhra Pradesh 508 254, India. Tel.: þ91 8978987990 (mobile).

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 8 9 3e8 9 6894

and identify a-glucosidase inhibitors from traditional medic-

inal plants15 for development of new therapeutics. Post-

prandial hyperglycemia is also reported to induce oxidative

stress by overt generation of free radicals16 that further

aggravate diabetic complications17 Therefore, combination of

a-glucosidase inhibitory and free radical scavenging proper-

ties in a therapy appears to become an exciting therapeutic

strategy for the management of postprandial hyperglycemia

as well as attenuation of resultant oxidative stress. In the

course of our study on traditional medicinal plants, we have

reported several phytochemicals possessing these activities.18

In the course of our search for the modulators of dietary car-

bohydrates digestion for the management of postprandial

hyperglycemia in diabetes, we encountered potent a-glucosi-

dase inhibitory and free radical scavenging active compounds

in P. tomentosa, which find wide usage in Indian medical sys-

tem, Ayurveda. Herein, we are reporting the isolation and

structural elucidation of phytochemicals as a potential a-

glucosidase inhibition and free radical scavengers.

2. Materials and methods

2.1. Plant material

The whole plant material P. tomentosa were collected from the

forest ofTirumala inChitoorDist. (Andhra Pradesh, India) in the

month of January, 2005 and identificationwasmade by Prof. Dr.

K. Madhava Chetty, Department of Botany, Sri Venkateshwara

University, Tirupathi. Voucher specimens (PT-01e05) of the

plants are deposited at the herbarium of the S. V. University.

2.2. General

Column chromatography was performed on silica gel (60e120

mesh). Melting points were recorded on Fisher Johns appa-

ratus and were uncorrected. FABMS was recorded on VG Auto

spec-M instrument. IR spectra were recorded on Nicolet

spectrometer. 1H NMR and 13C NMR spectra obtained on var-

ian 200, 400 MHz and Bruker 300 MHz spectrometers using

TMS as internal standard. HMBC, HSQC, NOSEY and DQCOSY

experiments were done on Oxford 500 MHz spectrometer.

2.3. Isolation of secondary metabolites from whole plantof P. tomentosa

Thedriedplantmaterial (2kg)waspowderedandextractedwith

n-hexanes in a Soxhlet apparatus for 24 h. The solvent was

evaporated under reduced pressure in a rotary evaporator to

obtain a residue (15 g). The residue was adsorbed on silica gel

and subjected to column chromatography over silica gel and

eluted with n-hexanes first followed by mixture containing

increasingamounts of ethyl acetate. The fraction elutedat 2, 4, 6

& 10% were collected separately concentrated and rechroma-

tographed using silica gel (60e120 mesh, 100 g) to obtain com-

pound 6& 7 (0.012 g & 0.02 g), compound 1& 2 (0.026 g & 0.03) in

pure form. After completing petroleumether extract, powdered

plant material was extracted with chloroform to obtain 20 g of

residue. The residuewas adsorbed on silica gel and subjected to

column chromatography over silica gel (60e120mesh, 80 g) and

eluted with n-hexanes first followed by mixture containing

increasing percentages of ethyl acetate. The fractions eluted at

12, 14, 16, 18 and 20% were collected separately, concentrated

and rechromatographed over silica gel (60e120 mesh, 30 g) to

obtain compound 3, 4 & 5 (0.06 g, 0.009 g & 0.010 g) and com-

pound 8 & 9 (0.01 g & 0.023 g) in pure form.

2.4. Premnalin

(1): mp 215e216 �C. IR(KBr)nmax: 3412, 2357 & 1617 cm�1, 1H

NMR (200MHz, CDCl3) d: 9.80 (1H, s, H-7), 7.05 (2H, s, H-2, 6), 5.80

(1H, OH), 3.98 (6H, H-3, 5-OMe), 3.0 (2H, t, H-8), 1.2e2.20 (10H,

m), 2.35 (3H, s, 4-H) and 0.91 (3H, t, 14). 13CNMR (50MHz, CDCl3)

(d): 191.5 (C-7), 158.0 (C-8), 148.0 (C-3, 5), 107.0 (C-4, 1), 106.0 (2,

6), 56.5 (C-3, 5-OMe), 32.5 (C-8), 29.4e30.2 (C-9, 10, 11, 12, 13),

15.5 (C-14). HRESIMS: m/z [M]þ 294.1668 (calcd: 294.1675).

2.5. Bioassay procedures

2.5.1. a-Glucosidase inhibitory activityEstimation of intestinal a-glucosidase inhibitory activity was

carried out as reported earlier.19 Rat intestinal acetone powder

(Sigma Chemicals, USA) in normal saline (100:1, w/v) was soni-

cated properly and supernatant was treated as crude intestinal

a-glucosidase after centrifugation at 3000 rpm� 30min. 10 ml of

test samples dissolved inDMSO (5mg/mL solution)weremixed

and incubated with 50 ml of enzyme in a 96-well microplate for

5 min. Reaction mixture was further incubated for an

other10 min with 50 mL substrate [5 mM, p-nitrophenyl-a-D-

glucopyranoside, prepared in 100 mM phosphate buffer (pH

6.8)]. Absorbance at 405 nmwas recorded at room temperature

(26-28 �C). Percent aeglucosidase inhibition was calculated as

(1 � B/A) � 100, where A was the absorbance of reactants

without test compound and B was the absorbance of reactants

with test samples. All the samples were run in triplicate and

acarbose was taken as standard reference compound. Several

dilutions of primary solution (5 mg/mL DMSO) were made and

assayed accordingly to obtain concentration of the sample

required to inhibit 50% activity (IC50) of the enzyme applying

suitable regression analysis.

2.5.2. Free radical scavenging activityFree radical (DPPH) scavenging activity assay procedure was

adopted from previous report.20 In a 96-well microplates, 25-

mL-test sample dissolved in dimethyl sulfoxide (1 mg/mL

DMSO), 125 mL of 0.1 M triseHCl buffer (pH 7.4) and 125 mL of

0.5 mM DPPH (1, 1-diphenyl-2-picrylhydrazyl, Sigma Chem-

icals, USA, dissolved in absolute ethyl alcohol) were mixed

and shaken well. After incubating 20 min in dark, absorbance

was recorded spectrophotometrically (SPECTRA MAx PLUS384,

Molecular Devices, USA) at 517 nm. The free radical scav-

enging potential was determined as the percent decoloriza-

tion of DPPH due to the test samples and calculated as (1 � B/

A) � 100, where A is absorbance of DPPH control with solvent

and B is absorbance of decolorized DPPH in the presence of

test compound. All the analysis was done in duplicate; Trolox

was taken as reference compound. Several dilutions of pri-

mary solution (1 mg/mL DMSO) were made and assayed

accordingly to obtain concentration of the sample required to

Fig. 2 e Isolated compounds from Premna tomentosa Linn.

Fig. 1 e Biosynthetic pathway of premnalin.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 8 9 3e8 9 6 895

Table 1 e IC50 and SC50 values of compounds for their intestinal a-glucosidase inhibitory and DPPH scavenging activityrespectively.

Compound a-Glucosidase inhibition(IC50%)

Free radical scavenging activity(SC50, % of inhibition mg/ml)

1. Premnalin 12.11 � 0.61 20.58 � 0.88

2. Acetoxy syranzaldehyde 18.41 � 1.31 20.83 � 0.21

3. Trolox 15.94 � 0.04

4. 1-Deoxynojirimycin 50.00 � 0.91

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 8 9 3e8 9 6896

scavenge 50% (SC50) of DPPH free radical applying suitable

regression analysis.

3. Results/discussion

Compound 1 was obtained as an optically inactive light or-

ange solid, and the molecular formula was established as

C16H22O5 by HREIMS, m/z 294.1668. The1 H and 13C NMR

spectral analysis clearly indicates the presence of 16 protons

and 22 carbons respectively. The 1H NMR displayed a peak at

d 9.80 (1H) indicating the presence of an aldehyde proton, a

peak at d 3.98 (6H, s) indicates the presence of two aromatic

methoxyl groups. In addition, a signal at d 6.02 integrated for

one proton due to presence of aromatic moiety. Moreover, a

peak at d 3.0 integrating for two protons as a triplet is due to a

benzylic methylene and a peak appearing at d 0.9 as a triplet

integrating for three protons is due to terminal methyl of an

aliphatic chain. 13C NMR and other spectral data supporting

the title compound is related to syranzaldehyde derivative.

Based on its spectral characteristics, compound 1 (Fig. 2) is

identified as 2-pentyl-3, 5-dimethoxy-4-acetoxy benzalde-

hyde, a new syrangaldehyde derivative and named as pre-

mnalin. In biosynthesis of premnalin (1) follows combination

of shikimic acid as well as acetate-mevalonate pathways, the

complete synthesis showed in Fig. 1

The isolated compounds were screened for rat intestinal a-

glucosidase inhibitory and free radical (DPPH) scavenging

potentials. The results of primary screening are presented in

(Table 1).

4. Conclusion

In conclusion, whole plant of P. tomentosa exhibited certain

important phytochemicals, antioxidant and free radical scav-

enging activity in significant amount. This plant has been in

use for years to treat various ailments. Natural antioxidants of

plant origin have greater application and they can also be used

as nutraceuticals and phytoceuticals as they have significant

impact on the status of human health and disease prevention.

This investigation thus provides a scientific basis for the use of

the plant extracts in home-made remedies and their potential

use in the treatmentof cytotoxic elements.Westrongly believe

P. tomentosa is oneof the best plant to cure thevariousdiseases.

Conflicts of interest

The author has none to declare.

Acknowledgments

Authors thank Prof. K. N. Reddy, Vice-Chancellor, Mahatma

Gandhi University, Nalgonda and Director, IICT, Hyderabad,

India.

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