Hale and Their Antioxidant Activity

(1)

1018 N o te s

Phenolic Constituents from the Lichen Parmotrema stuppeum (Nyl.)

Hale and Their Antioxidant Activity

Guddadarangavvanahally K. Jayaprakasha* - 3

and Lingamallu Jaganmohan Raob a H um an R esource D evelopm ent

b P lantation Products. Spices and Flavour Technology C en tral Food Technological R esearch Institute, M ysore-570 013, India. Fax: + 9 1 -0 8 2 1 -5 1 7 2 3 3 . E-mail: gkjp@ cscftri.ren.nic.in; gkjp@ yahoo.com

* A u th o r for co rresp o n d en ce and rep rin t requests Z. N aturforsch. 55c, 1018-1022 (2000);

received July 28/A ugust 30, 2000

Phenolic Acids, A n tio x id an t Activity, ß -C arotene- linoleate M odel System

Lichen, Parm otrem a stuppeum (P. stuppeum ) was suc

cessively ex tracted w ith ben zen e and acetone. B oth the extracts w ere fractio n ated on 1% oxalic acid im preg

n a te d silica gel colum n to obtain four phenolic com pounds. T he stru ctu res of com pounds w ere identified by 'H and 13C N M R sp ectra as m ethyl orsenillate, orsenillic acid, a tran o rin and lecanoric acid respectively. A n tio x i

d an t activity o f ben zen e extract, acetone extract and iso

lated com p o u n d s w ere ev alu ated in a ß-carotene-linoleate m odel system . The pure com pounds show ed m o d erate a n tio x id an t activity. This is the first re p o rt on the isolation and ch aracterisatio n of com pounds from the lichen P. stuppeum as well as on th eir antioxidant ac

tivity.

Introduction

Lichens constitute a class of small perennial plants, which are a combination of two organisms- a fungus and an alga-growing together in symbi

otic association. Lichens are widely distributed from the arctic to the tropics and are found on soil, barren rocks and tree trunks. Several lichens possess medicinal properties and a few are con

sumed as delicacies. Certain lichens containing volatile oil were used in perfumery and cosmetic industries. Lichens were formerly used as sources of ferm entable sugars for the production of ethyl alcohol. Parmotrema stuppeum is abundantly growing foliose lichen in South India (The Wealth of India, 1962).

Antioxidants protect the quality of foods by re

tarding oxidative breakdown of the lipid compo

nents (Shahidi et al., 1994). Commercial antioxi

dants are generally synthetic compounds and there

has been a growing interest in replacing them with natural ingredients (Chang et al., 1977). Due to the possible toxicity of synthetic antioxidants there has been an increasing interest in preparing anti

oxidants from natural sources. The use of natural antioxidants in food is limited due to lack of knowledge about their molecular compositions, the content of active com pounds in the raw m ate

rials and the availability of relevant toxicological data. Hence, evaluation of the antioxidative activ

ity of naturally occurring substances has been of interest in recent years (Amarowicz, 1996). This study was carried out to identify the major constit

uents of the lichen P. stuppeum and the antioxi

dant activity of crude extracts and purified com

pounds. This is the first report on the isolation and characterization of com pounds 1 - 4 from P. stup

peum and their antioxidant activity as well.

M aterials and M ethods Materials

All solvents / chemicals used were of analytical grade and obtained from Merck, Mumbai, India.

ß-Carotene, linoleic acid and butylated hydroxya

nisole were obtained from Sigma Chemical Co., (St. Louis, MO, USA). Visible spectra were re

corded using Genesys-5-UV-visible Spectropho

tom eter (Milton Roy, NY, USA). ’H and 13C NM R spectra were recorded at 400 and 100 MHz, respectively, on a B ruker AM X 400 FT instrum ent (Bruker, R heinstetten, Germ any). 13C NM R spectral assignments were given on the basis of spin-echo fourier transform spectra. Tetramethyl silane was used as internal standard.

Source o f lichen

The lichen sample was collected from a local m arket. The species was identified by Interna

tional Mycological Institute (Egham, Surrey, U. K.) as Parmotrema stuppeum (Nyl.) Hale. A voucher specimen was deposited in the reference collection centre (International Mycological Insti

tute, Egham, Surrey, U. K.) (IM I No. 367183).

Extraction and fractionation o f lichen compounds Dried Parmotrema stuppeum was powdered (50 g) and successively extracted in a soxhlet ex

This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution-NoDerivs 3.0 Germany License.

On 01.01.2015 it is planned to change the License Conditions (the removal of the Creative Commons License condition “no derivative works”). This is to allow reuse in the area of future scientific usage.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:

Creative Commons Namensnennung-Keine Bearbeitung 3.0 Deutschland Lizenz.

Zum 01.01.2015 ist eine Anpassung der Lizenzbedingungen (Entfall der Creative Commons Lizenzbedingung „Keine Bearbeitung“) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsformen zu ermöglichen.

(2)

tractor with benzene and acetone for 8 h each. The extracts were filtered and evaporated in vacuum yielded 1.25 and 0.8 g (w/w). TLC of benzene and acetone extracts showed four spots with different concentrations. Hence, both the extracts were mixed and loaded onto 1% oxalic acid impreg

nated silica gel column. Compounds 1 - 4 were eluted with hexane:benzene (3:1 v/v), benzene, 2% , 5% ethylacetate in benzene yielded 200, 150, 650 and 800 mg (w/w) respectively.

Identification o f compounds

The melting points of compounds 1 - 4 were re

corded as 139-40, 174-75, 187-88 and 173-74° C respectively. TLC of purified compounds with an acid-free developing solvent (benzene: EtOAc 95:05 v/v) on silica gel containing 1% oxalic acid was carried out and the compounds are visualised as yellow spots when sprayed with 1 0% sulfuric acid in methanol followed by heating at 110° C.

The R f values of compounds 1 - 4 were found to be 0.65, 0.31, 0.93 and 0.27 respectively. Further, the structures of isolated compounds were con

firm ed by 'H and 13C NMR spectra (Tables I and II).

Antioxidant assay by ß-carotene system

The antioxidant activity of extracts, pure com

pounds was evaluated by the ß-carotene-linoleate m odel system according to Hidalgo et al. (1994) with slight modification (Jaganmohan Rao et al., 1998). 0.2 mg of the ß-carotene in 0.5 ml of chloro

form, 20 mg of linoleic acid and 200 mg of Tween- 40 (polyoxyethylene sorbitan m onopalm itate) were mixed together. The chloroform was re

moved at 40° C under vacuum using a rotary evap

orator. The resulting solution was immediately di

luted with 1 0 ml of triple-distilled w ater and the emulsion was mixed well for 1 min. The emulsion was further diluted with 40 ml of oxygenated w ater before use. 4 ml aliquots of this mixture were transferred into different tubes containing

0 . 2 ml of test extracts and pure com pounds in eth

anol butylated hydroxyanisole was used for com

parative purposes. A control containing 0.2 ml of ethanol and 4 ml of the above m ixture was pre

pared. Optical density (O D ) at 470 nm were taken for the all extracts and pure compounds immedi

ately (t = 0) at 15 min intervals for 1.5 h (t = 90).

The tubes were incubated at 50° C in a water bath.

All determ inations were perform ed in triplicate.

M easurem ent of OD was continued until the col

our of ß-carotene disappeared in the control (Figs 1 and 2). The antioxidant activity (A A ) of the extracts was evaluated in terms of bleaching the ß-carotene using the following formula of H i

dalgo et al. (1994). A A = 100[l-(Ao-A t)/(A °o - A °t)] where A0 and A° 0 are the absorbance values (O D s) m easured at zero time of the incubation for test sample and control, respectively. A t and A°t are the absorbance m easured in the test sample and control, respectively, after incubation for 90 min.

R esults and Discussion

Parmotrema stuppeum was successively ex

tracted using benzene and acetone. Fractionation of benzene and acetone extracts on oxalic acid im

pregnated silica gel column chromatography yielded four crystalline compounds. The com

pounds showed a single spot on TLC. It was no

ticed that without using acidic medium, tailing of spots was observed on the TLC plates and pure com pounds could not be obtained using silica gel column chromatography. Therefore silica gel con

taining 1% oxalic acid was used for TLC and col

umn chromatography. The compounds 1 - 4 were characterised and identified as methyl orsenillate, orsenillic acid, atranorin and lecanoric acid respec

tively, using 'H NM R and 13C NM R spectra (Ta

bles I and II, Scheme I). Chemical shifts of com

pounds were com pared with reported values (W itiak et al., 1967; Devlin et al., 1971; Sundholm and Huneck, 1980, 1981).

The antioxidant activity of benzene extract, ace

tone extract and isolated compounds 1 - 4 at 2 0 0

and 500 ^tg/ml concentrations were com pared with butylated hydroxyanisole is presented in Figs. 1 and 2. It shows the decrease in absorbance of ß- carotene in the presence of lichen extracts/pure com pounds and B H A with the coupled oxidation of ß-carotene and linoleic acid. The addition of extracts compounds 1 - 4 and butylated hydroxya

nisole at 200 and 500 ^g/ml concentrations pre

vents the bleaching of ß-carotene to different de

grees. ß-C arotene in this model system undergoes rapid discoloration in the absence of an antioxi-

(3)

1020 N o te s T ab le I. ]H N M R s p e c tra l d a ta o f c o m p o u n d s 1, 2, 3, a n d 4 (400 M H z).

H

1 3.79 (C O O C H u) 10.11 (C O O H )

2 10.73 (O H ) 13.40 (O H ) 12.50 (O H ) 10.48 (O H )

3 6.15 (H ) 6.12 (H ) d (2.0) - 6.22 (H )

4 9.98 (O H ) 12.13 (O H ) 11.95 (O H ) 10.47 (O H )

5 6.17 (H ) 6.17 (H ) d (1.5) 6.52 (H ) 6.62 (H )

6 2.28 (C H ,) 2.39 (C H ,)

8 2.54 (C H ,) 2.35 (C H 3)

9 10.36 (C H O )

1^' ^- ^-

2' 12.55 (O H ) 10.33 (O H )

3' - 6.60 (H )

4 ' - -

5' 6.40 (H ) 6.61 (H )

6' - -

7' 3.98 (C O O C H 3) 10.01 (C O O H )

2.68 (C H ,) 2.37 (C H 3)

2.09 (C H 3)

C hem ical shifts are follow ed by coupling co n stan ts J (in Hz): values in parentheses.

*: D M S O -d6.

**: CDC13.

d: doublets.

1 2

3

8

4 Schem e I.

C om pounds determ in ed in the Parmotremci ex tract (1) M ethyl orsenillate

(2) O rsenillic acid (3) A tran o rin (4) L ecanoric acid

T able II. 13C N M R spectral data of com pounds 1, 2, 3, and 4 (100 M H z).

c 1* ²* 3** ⁴*

1 104.8 102.9 107.3 108.2

2 164.4 169.1 161.2 160.2

3 100.5 108.7 100.5 100.5

4 100.5 167.5 161.5 161.5

5 161.9 142.9 110.3 109.9

6 140.4 140.4

7 169.6 167.2

8 25.4 167.2

9 193.7 ^-

1' 116.8 116.4

2' 162.9 158.7

3' 110.4 107.4

4' 152.3 152.3

5' 116.0 114.8

6' 139.8 139.6

7' 172.1 170.7

8' 23.8 2 1.0

9' 9.3

C O O C H , 51.8 ^-

C O O H ^- 173.2

C O O C H , 170.3 ^-

c h3 2 2 .1 23.4

* D M S O -d 6.

** CDC13.

dant. This is because of the coupled oxidation of ß-carotene and linoleic acid, which generates free radicals. The linoleic acid free radical formed upon the abstraction of a hydrogen atom from one of

(4)

Table III. A n tio x id an t activity (A A ) o f com pounds 1 - 4 and d ifferen t crude extracts evaluated from pro tectio n of ß -caro ten e at 90 min.

Time (min)

Fig. 1. A n tio x id an t activity of lichen extracts, com p o u n d s an d b u ty lated hydroxyanisole assayed by ß-carotene-li- n e o le a te m odel system at 200 ng/ml concentration.

Q C o n tro l □ B utylated hydroxyanisole B enzene ex tract — Ac e t one extract

— Met hyl orsenillate —I— O rsenillic acid - O - L ecanoric acid A A tra n o rin

Tim e (min)

Fig. 2. A n tio x id an t activity of lichen extracts, com p o u n d s an d b u ty lated hydroxyanisole assayed by ß-carotene-li- n eo lea te m odel system at 500 |a,g/ml co ncentration.

A - C ontrol —B — B utylated hydroxyanisole

—■ — B enzene extract — Ac e t one extract X" M ethyl orsenillate - O - O rsenillic acid

—it— L ecanoric acid

Com pounds/extracts % A A at 200 ng/ml

% A A at 500 ug/ml

Butylated hydroxyanisole 93 96

M ethyl orsenillate (1) 18 40

Orsenillic acid (2) 26 50

A tranorin (3) 14 *

Lecanoric acid (4) 12 36

B enzene extract 30 65

A cetone extract 35 68

* N o t d e term in ed d u e to p recipitation of com pound at higher co n cen tratio n .

its diallylic m ethylene groups attacks the highly unsaturated ß-carotene molecules. As a result, ß- carotene will be oxidised and broken down in part, subsequently the system looses its chromophore and characteristic orange colour, which can be m onitored spectrophotometrically. The extracts and the isolated com pounds 1 -4 can hinder the extent of ß-carotene bleaching by neutralising the linoleate free radical and other free radicals form ed in the system. Extracts and compounds 1 - 4 showed 12-35% and 3 6-68% antioxidant activ

ity at 200 and 500 fxg/ml, respectively. The data (Table III) show that the extracts have a better an

tioxidant activity than the purified compounds 1 - 4. Individual com pounds showed less activity than the acetone extract. Hence, the antioxidant activ

ity of benzene and acetone extracts may be due to a synergistic/cumulative effect of all the com

pounds.

Com pound 2 showed maximum antioxidant ac

tivity at 200 and 500 [ig/ml compared to com

pounds 1, 3 and 4. Com pound 1 showed m oderate antioxidant activity due to the presence of an electron-attracting group (-CO O CH 3). In compounds 3 and 4 the electron-attracting property increases due to the two hydrogen bonds between 2'-O H and l'-C O O C H 3/C O O H groups and 2-OH and 1- C O O - groups and also due to the presence of the electron-attracting property of the COO- group that is conjugated with an aromatic ring. Hence, the antioxidant activity of these compounds decreases (Hong-Yu Zhang, 1999). Also, in com

pound 3 an additional hydrogen bond between the 4-O H and 3-CHO groups and the presence of an electron-attracting group (-CHO ) in ortho posi

tion to -OH has no significant effect on the antiox-

(5)

1022 N o te s

idant activity compared to com pound 4 (Ta

ble III). A similar trend of antioxidant activity of lichen compounds was observed by Hidalgo et al.

(1994).

A m arow icz R., W anasundra U. N , K aram ac M. and Shahidi F. (1996), A ntio x id an t activity of eth an o lic ex

tract of m u stard seed. N ahrung, 40, 2 6 1 -2 6 3 .

C hang S. S., O stric-M atyasevic B., H sieh O. A. L. and H uang C. L. (1977), N atu ral antioxidants from ro se m ary and sage. J. Food Sei. 42, 1102-1106.

D evlin J. P., Falshaw C. P. and Ollis W. D. (1971), P h o to chem ical exam ination of th e lichen Lecanora rupicola (L.) Z ah lb r. J. Chem . Soc. (C ), 1318-1323.

H idalgo M. E., F ernandez E., Q uilhot W. an d Lissi E.

(1994), A n tio x id an t activity of depsides and depsidones. P hytochem istry 37, 1585-1588.

H ong-Y u Z h an g (1999), T heoretical m eth o d s used in elucidating activity differences of p henolic a n tio x i

dants. J. A m er. Oil C hem . Soc. 76, 7 4 5 -7 4 8 .

Jaganm ohan R ao L., Jayaprakasha G. K. and Sakariah K. K. (1998), A n tio x id an t activity of n a tu ra l flavidin.

S ubm itted to P aten t, Intellectual P ro p erty M an ag e

m en t D ivision, C SIR , N ew D elhi. No. NF. 332/98, dt.

23/02/1998.

Shahidi F., W anasundra U. N. and A m arow icz R. (1994), N atu ral antioxidants from low -pungency m u stard flour. Food Res. Intern. 27, 4 8 9 -4 9 3 .

We wish to thank Dr. V. Prakash, D irector and Dr. K. K. Sakariah, Head, Human Resource D e

velopment, Central Food Technological Research Institute, Mysore for their constant encourage

ment.

Acknowledgem ents

Sundholm E .G . and H uneck S. (1980), 13C N M R - S pectra of lichen depsides, depsidones and depsones 1. C o m p o u n d s of the orcinol series. C hem ica Scripta 16, 197-200.

Sundhohn E. G. and H u n eek S. (1981), 13C N M R - Spectra of liehen depsides, depsidones and depsones 2. C o m p o u n d s of the ß-orcinol series. C hem ica Scripta 18 2 3 3 -2 3 6 .

T he W ealth o f India: A D ictionary of Indian Raw M aterial and Industrial Products (T hacker M. S., Lala Shri R., K rishnan M. S., B aini Prashad, C h o p ra R. N , S an tap au H . and Sastri B. N., ed.). Publications and In fo rm atio n D irecto rate, C ouncil of Scientific & In d u strial R esearch , N ew D elhi 1962, 3, p .8 1 -9 0 .

W itiak D. T.. P atel D. B. and Lin Y. (1967), N uclear m ag

netic resonance. Influence of substituents on the long- range spin-spin coupling constant betw een benzylic and ring p ro to n s in the orcinol series. J. A m er. Chem . Soc. 89, 1908-1911.