PART I .

NATURAL AND SYNTHETIC ANTIOXIDANTS FuR EDIBLE FATS,

2

Introduction

Many constituents of foods sucn as fats, vitamins

and colouring and flavouring materials react with atmospheric

oxygen leading to the development of rancidity. This causes

discolouration, loss of natural and fresh aroma and nutritive

value,due to the destruction of vitamins and essential fatty

acids,^ It has been shown by many workers that autoxidised

fats or fatty acid esters have deleterious effects and may

2sometimes even cause death when fed to experimental animals.

Attempts have been made to overcome this, and Machhin and

CO-workers^ successfully demonstrated that these deleterious

effects can either be eliminated or reduced by the addition

of an inhibitor like tocopherol in the diet.

All fats which contain unsaturated fatty acids are

subject to oxidation as a result of their contact with even

traces of oxygen. It is now well established that this

oxidative process proceeds through a free radical chain

reaction involving three stages namely the initiation,

propagation and termination stages*

Initiation takes place through activation of the

unsaturated fatty acids by heat, ultraviolet light or traces

of pro-oxidants, resulting in the formation of free radicals,

presumably by abstraction of a hydrogen atom from the active

methylene group adjacent to the double bond. The free radical

so formed, could undergo 1-3 shifts to form new free radicals.

All of them react, with oxygen giving rise to peroxy radicals

3

which subsequently abstract auiother proton from nearby

unsaturated fatty acids moiety to form the hydroperoxides,

and another free radical.^*^ This is the propagation stage,

It is illustrated in figure I with reference to the

autoxidation of methyl oleate.

CH^-(CH2)^-CH2-CH . CH-CH2-(CH2)^-C00CH3

Initiation-four free radicals.

-CH*-CH=CH-CH2-

-CH »CH-CH*-CH2-

-CHg-CHaCH-CH*.

n-CH -CH*-CH«CH.

2

Propagation-two stages

(1) -CH^-CHaCH-CHg- + 0 2 —^ -CH-CH=iCH-CH2-

O-O’t'

0 - 0*

—CH—CHs^H—CH2”

O-OH

Figure I ; Autoxidation of methyl oleate

The hydroperoxides decompose to carbonyl compounds,

acids, and alcohols, for example

CH2(CH2)6-CH-CH»CH-(CH2)7C00CH^

O-OH

CH^(CH2)^CH0 ♦ OCH(CH2)gCOOCH3

CH^(CH2)-7CH=CH-CH-{CH2)^C00CH3

O-OH

CH^(CH2)yCH,CH-CH0 ♦ H0(CH2>^C00CH^

The exact nature of the decomposition of hydroperoxides

is not understood but it is known that the presence of

hydroperoxides, though they have not necessarily a flavour

themselves, is very detrimental to the keeping properties of

an edible oil. The typical odours and flavours of rancid

fats are due to the secondary products of oxidation and not

the peroxides. According to Lea and Swoboda^, the aliphatic

aldehydes from Cg to detectable organoleptically at

-S -9weight concentrations as low as 10“ to 10 •

Termination of the chain reaction is probably effected

by mutual destruction of chain carriers to give dimeric

products suid inert substances which no longer contribute x,o the

reaction chain.

5

7Moureau first employed the term

antioxygens to define those substances which prevent the

oxidation of unsaturated glycerides until they themselves

have been destroyed. It has been postulated that the

effectiveness of the antioxidants or inhibitols lies in their

ability to break chain reactions involved in tne oxidative

rancidity, in the following way: The oxidant A unites with

oxygen to form A02* The next stage involves the simultaneous

oxidation of the antioxidant B by the peroxide and the

transformation of the oxidised oxidant to a lower oxide,

AO, according to the scheme AOg + B-» AO + BO. It is

presumed that these two oxides are mutually antogonistic,

hence they react with each other to regenerate the three

original molecules in the following manner; AO + BO—►A + B+O2 .

The chain reaction is thus broken and the development of

rancidity is temporarily forestalled. The antioxidants

are gradually destroyed or are transformed to inert products.

During tne induction period the anti oxidants break up the

chain reactions almost as soon as they are started. When they

became exhausted, as is true at the end of induction period,

the oxidative reactions proceed at a normal but greatly

accelerated pace.

An antioxidant for successful use in foods,

in addition to producing a useful degree of stabilisation,

and being non-toxic both before and after oxidation, must be

adequately dispersible in the medium and must not impart any

foreign odour, colour or taste to the food either on heating or

on prolonged storage.

Chemically^phenols constitute the largest and most

important cl^ss of antioxidants. Practically any phenolic

substance xith two or more hydroxyl groups (at least one of

which must be free) in the ortho or para position to each other,

or suiy naphthol is likely to show some antioxidant property,

although activity varies widely with the nature and positions of

the substituent groups. The phenolic antioxidants completely

loose their activity if the hydroxyl groups are converted to

' ethers or esters#

In the naphthalene series, only one hydroxyl group

is enough to render it an effective antioxidant. Thus

-naphthol is a powerful antioxidant, whereas ^-naphthol is

much less effective, though both of them are toxic.

Similarly, though certain amines and aminophenols are quite

effective in retarding oxidation, they have found little

application in edible fats because of their toxic nature.

At present all primary antioxidants permitted for use in edible

Qfats are phenols only .

Phenolic anti oxidants are usually more effective in

retarding oxidation of animal fats than of vegetable fats,

because most of the vegetable oils contain considerable amount

of tocopherols or other natural antioxidants and further

addition of primary antioxidants are relatively less effective

than similar addition to animal fats in which the content of

natural antioxidants is usually very low or none at all.

During the refining of fats a part of the natural antioxidant

is lost*

Many acidic substances such as citric, ascorbic,

phosphoric acids etc. though not effective antioxidants

by themselves, enhance the activity of the phenolic

compounds considerably and are usually called as ^nergists.

Some of these, especially citric acid, also work as

metal deactivators by complexing with traces of copper and

iron which act as prooxidants.

The ascorbic acid structurally related to o-diphenols,

and possessing reducing properties, may perhaps be able to

act as primary antioxidant under some conditions. It can

also reduce quinones to inols and it is this property

which might be helping it to increase the antioxidant

properties of phenolic compounds by regenerating the phenols

from tneir oxidation products. On the other hand, in the

presence of copper, the ascorbic acid itself is oxidised

probably by free radical mechanism and then acts as a powerful

Qprooxidant for fats.

A few naturally occurring substances have been proposed

from time to time as preservatives for fats. Some of these

include cereal and oilseed flours; tannins and tannic acid,

gallic acid, wheat germ oil, sesamol from sesame oil, and

gossypol from cottonseed o il.^^

The antioxidant activity of wheat germ oil is due to

high percentage of tocopherols present in it*^ The various

forms of tocopherols differ in their activity depending upon

8

the number of methyl groups present on the nucleus.

Thus S -tocopherol is the most active followed by y8- and

12and then oC-tocopherols . However, recently Lea has

demonstrated that for polyunsaturated systems the antioxidant

activity of tocopherols was found to increase with nuclear

raethylation of tocol^, though y-tocopherol was effective

Tocopherol

oC - 5,7,Srtrimethyl tocol

5,3-dimethyltocol

y - 7,S-dimethyltocol

& - S,-methyl tocol,

in all systems and at all temperatures^^. From the study

of parent cnromans, Golumbic^^ has shown that besides the

phenolic group, the oxygen in the heterocyclic ring

(position 1) is also essential for the antioxidant activity

of tocopherols,

Flavones, isoflavones, anthocyanins and a few other

naturally occurring phenolic colouring matters have been

found to stabilize fats to varying extents. Thus

kamala dye^^ and curcumin^^ have been shown to be antioxidants

for butter fat and vegetable oils, Quercetin has been shown

to stabilize cottonseed oil and also a mixture of lard and

17cod-liver oil , Lea and Swoboda found that gossypetin

and 1-epigallocatechin gallate (from tea leaves) are very

effective antioxidants amongst the naturally occurring

flavonols , Mehta and Sheshadri demonstrated that out of

the 27 different flavonols tested for antioxidant activity,

19robinetin and gossypetin are the most effective

Catechin and aca-aatechin have been shown to be effective

20antioxidants for groundnut oil *

21Heimann and Reiff studying the relationship between

the structure and activity of flavanols found that

(1) o(-P -unsaturated ketone system and the free

hydroxyl group in the position of the pyrone ring and

(2) the free diphenolic 3*i4' grouping in the B ring

all contribute to the antioxidant activity, whereas the

m-diphenolic 5 :7 grouping in the A ring tends to reduce the

activity. Simpson and Uri examined thirty different

flavones and cam^ to the conclusion that a hydroxyl group in

the 3 position imparts a high antioxidant activity which

is enhanced by quinol structures in the 2-phenyl and

benzenoid portions of the molecule. The p-quinol strictures

appear to impart considerably higher activity than does

the o-quinol. Hydroxyl groups meta to one another make

no contribution to the activity and may even exert a

22prooxidant effect •

Quercetin II Gossypetin I I I

;1()

Flavones are also known to form complexes with

metals, chelation occurring at 3-hydroxy, 4-keto grouping

or perhaps at 4-keto, 5 hydroxy grouping when present^^.

0-Diphenolic grouping can also show some metal chelating

properties^^.

25Lundberg e t . ^ , first reported the antioxidant

properties of nordihydroguaiaretic acid (N .D .G .A ,) and

it has since been used commercially for the presei^vation

of dry fats. It is also a good stabilljser for

carotene and vitamin A in o i l s ^ * Gallic acid and its

27esters , specially propyl and octyl gallates are widely

used for the preservation of edible fats#

Butylated hydroxy anisole (B .H .A .) and butylated

hydroxy toluene (B .H .T .) both synthetic products are being

used (within specified limits) commercially for the

preservation of fats and fatty foods, B.H.A* has been

shown to be an effective antioxidant for lard, com and

groundnut oils and possesses very good carry over

properties*

With synthetic antioxidants like B .H .A . and B.H.T*

or compounds like N .D .G .A . obtained from non-edible sources,

the question of toxicity is an important one. Some

deleterious effects may be noticed only after prolonged

usage even with a substance which has been passed on as

non-toxic on feeding tests of comparatively shorter duration,

It became imperative therefore to discover new antioxidants

from materials which are being used for edible purposes for

11

2Qcenturies. Thus Kaloyeareas^ found that the alcoholic

extract of anise seeds was a good stabilizer for olive oil

but the mustard seed extract was ineffective. Maveety

observed that powdered exhausted cloves increased the

stability of lard and cookies made from it^^.

Hense and Quackenbush^^ showed the antioxidant properties

of tomato lipids on lard. This according to them is due

to the presence of tocopherols and phosphatides in it .

The fruit of osage orange tree and its vsurious extracts

and isolates have been shown to possess good antioxidant

activity^^.

Evaluation and isolation of antioxidant principles

present in spices

There has been no systematic study of the

antioxidant principles or attempt at evaluating various

edible spices for their antioxidant properties. Hence

the present study was undertaken. The stabilising power

of common Indian spices was investigated and wherever

possible the ingredients responsible for this activity

were isolated. The main reasons for selecting the spices

for this investigation were (1) to isolate an effective

antioxidant that would be essentially nontoxic;

(2) to investigate the correctness of the practice prevelent

in certain parts of India^ of adding betel leaf or chilli

powder in small amounts, to butterfat (ghee) in order to

make it stay fresh for longer periods; and (3) to find

out whether spices are responsible for exceptional

keeping qualities of pickles etc*

}

Results and discussion

Initially the stabilizing effect of twenty-five

spices (listed in Table I) on refined groundnut, mustard,

sesame, safflower, coconut oils and butterfat was studied.

After the preliminary screening work, only groundnut oil

and lard were used as substrates*

The screening test consisted of addition of finely

powdered spices or finely minced green substances like

betel leaf, green chilli, onions etc. to the oils in

three different concentrations and heating them for five

minutes to 270-75°C in the case of groundnut oil and

220-25°C, with other oils, immediate cooling and filtering.

The oils so treated along with the untreated samples were

then subjected to "swift stability test"^^ in which

air at the rate of 2.33 cc. per second is passed for 5 hours

through 20 cc. of substrate maintained at 97»7®C,

The substrates were then chilled and their peroxide and

acid values determined*

These tests revealed that betel leaf, clove, cinnamon

leaf, nutmeg fruit, tunneric, chilli, black pepper, onions,

and ginger were very effective; while cardamom^mustard seed

and cubeb had very poor activity and the rest had fair

activity for stabilisation of fats^^*^ (Tables I and II),

It was also noticed that the activity of each spice varied

with the substrate, possibly because of the difference in

their chemical compositions. Lea has also observed

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17

that the relative activitiesi^different antioxidants vary

considerably with the fatty acid composition of the

substrate and viith temp, and level of oxidation at

37which the measiirements are made*

The results obtained in the preliminary screening

tests point out that (1) the antioxidant principles of the

spices are quite soluble in oils and are easily

extractable if they are heated even for short durations with

the substrates; (2) the active ingredients are stable to

short heat treatments and (3) the initial heating of the

oils also decomposes some of the prooxidants present in

them; this is in agreement with the observations of

Triebold?^

The publication of these preliminary results of

the present investigation^^, drew the attention of

3Qvarious workers in this field . Thus Chipault et .a l.^

woricing on thirty-two different spices observed that

although almost all the ground spices had some antioxidant

effect on prime steam lard, rosemary and sage exhibited

particularly pronounced effect. Antioxidant activity of

the extracts of a number of spices including cinnamon,

allspice, thyme, clove, rosemary, sage etc. on linoleic

acid was subsequently confirmed by Bgli et.al^^

Dhar reported the antioxidant activity of petrol ether

soluble portions of alcoholic extracts of chilli, garlic and

onion on butter fat^^. Sahasrabudhe and Bhatia came to

the conclusion that the mixtures of some of the spices gaveI O

enhanced activity with groundnut oil •

18

Lundberg and his colleagues have also demonstrated

that out of the thirty-two spices studied, cloves, turmeric,

allspice, and rosemary (in the decreasing order) protect

a simple oil-in-vgater emulsion against oxygen absorption*

The spices found relatively more effective in the

preliminary screening tests were selected for further

intensive work and the rest discarded. These more active

spices were finely powdered and extracted with various

solvents and wherever possible their active principles wereI

also isolated. These were then tested for their

antioxidant activity by the Active Oxygen Method^

using groundnut oil and lard as the substrates. This

test consists of passing air at the rate of 2.33 cc ./sec .f

through 20 cc. of the substrate maintained at 97,8°C

till a prespecified peroxide value is reached.

Red chilli (Capscium annum) was finely powdered

and extracted with ether, petrol ether, and alcohol,

capsaicin the pungent principle of chilli, was also isolated4 5

by the method of Lapworth and Royle , but it did not show

any antioxidant activity, rather it was a prooxidant.

The alcoholic extract of chilli showed some antioxidant

activity. This extract on keeping gave a solid portion

which on further examination was found to be crude ascorbic

acid and It showed a good activity with groundnut oil.

(Table I I I ) . No other extract or fraction showed any

a n t i oxidant activity. Thus the anti oxidant activity of

Table I I I

A.O.M»Tests with red chillies and its extracts on

refined groundnut o il .

(Antioxidant added to oil by wt.)

i n

Antioxidant

*Powdered whole chillies (0.!

Powdered whole chillies (0,5%)

Ethyl ether extract (0 .05% )**’*'

Petroleum ether extract (0 .1 ^ )***

Alcoholic extract (0 .05% )***

Alcoholic extract (0.1?i>)**

Solid fraction from alcoholic extract[ 0 . 1 % ) ^ *

Liquid fraction from alcoholic extract( 0 . 1 % ) * *

Capsaicin (O.OS^o)***

Ascorbic acid (0 .03% )***

Ascorbyl palmitate (0 .06% )***

Ascorbyl palmitate (0 .12^ )***

Capsaicin (0 .25% )***

Capsaicin (0.005%) + ascorbic acid (0 .03% )***

Capsaicin (0.05%) + ascorbyl palmitate (0.06%)

Stability*_^index._^

1.20

2.101.00

1.15

1.302.29

3.20

1.30

1.60

4.00

4.20 5.00

1.45

3.60

2.70

♦ Oil shaken with the antioxidant for 6 hr^and filtered.

Oil heated with the antioxidauit to 2 0 0 ° C . / ^ mm. for 5 rain,

♦’S'* Oil mixed with the antioxidant.

Stability Index

Time (in ho,ursj t ^ e n to reach a p 20 m .e.q ./Kg .by fat containing th'

eroxide value o: e antioxidant.

Time (in hours) taken to reach the same peroxide value by the control.

n30

chilli is mainly due to ascorbic acid, the presence of which

46in this spice has been reported earlier. This works as a

synergist and activates the minor amounts of natural

anti oxidants present even in refined vegetable oils.

In the case of lard or other animal fats which are void

of any’natural antioxidant, ascorbic acid does not show any

activity. This has been further proved by adding ascorbyl

palmitate, the fat soluble derivative of ascorbic acid to lard

and subjecting it to A*0,M, test, when no antioxidant effect

was noticed. (Table VIII)

Clove (Syzygium aromaticum) was extracted with alcohol

and benzene. The main constituents of its essential oil viz,

eugenol and caryophyllene were separately tested for their fat

stabilizing power. Isoeugenol was also tested for the sake

of comparison.

The results obtained (Tables IV & V) show that

isoeugenol is much more active than eugenol both for groundnut

oil and lard. It is well known that the essential oil ofI rt

cloves contains eugenol. This probably undergoes

very very slow conversion to isoeugenol under the influence of

heat or light. This premise has been supported by the

spectrophotometric examination of an authentic sample of

eugenol after refliixing it for 5 hours in nitrogen atmosphere.

Thus the ultraviolet absorption curves of eugenol and heat treated

eugenol samples showed ^ msoc at 279 and 274 respectively,

According to Vespe and Boltz^^, the amount of eugenol and

isoeugenol in a mixture can be calculated by taking the molar

absorbancy indices at 254 and 282 nj|u. The values obtained on

Table IV

A«Q«M, Tests with cloves and its extracts on refined

groundnut oil.

(Antioxidant added to oil by wt.)♦

2L

Antioxidant Stability*Index

Powdered cloves (0 .5%)*

Powdered cloves (0 .5^ )**

Powdered cloves (1 .5% )**

Alcoholic extract (0 .1% )***

Benzene extract (0 .03% )***

Eugenol (0 .05% )***

Caryophyllene (0 .03% )***

Iso-eugenol (0 .03% )***

Iso-eugenol (0.03%) + ascorbic acid (0.03%)

Iso-eugenol (0.03%) + ascorbyl palmitate

(0 .06% )***

1.2

1 .9

2 .4

1.3

1 .4

1.2

1.0

2 .4

5.6

5.6

* Oil shal<en with antioxidant for 6 hrs. and filtered.

** Oil heated with the antioxidant.*** Oil mixed with the antioxidant.

Time (in hours) taken to reach a peroxide „ value of 20 m .e .q ./Kg . by fat containing the* Stability Index =___________________ aritioXidant______________________

Time (in hours) taken to reach the same peroxide value by the control.

2 ^

Splcea

A«Q»M« te s ts with various fra c t io n s o f sp ices o f re fin ed la r d ,

Table V,

F raction and i t s wt. S ta b ility *Index,

1. Cinnamon le a f

Benzene ex tra ct 0 . 1 )E ssen tia l o i l (0 .1 )E ssentia l o i l , phenolic p ortion (0 .05) E ssen tia l o i l . phenolic portion (0 .1 ) Sugenol (0 .05 )Eugenol (0 .1 )Iso-eugen ol 0.03%

2.05.63.06.0 3.5 6.0

32.0

2. Turmeric ((

3. Dried ginger

4 . Nutmeg f r u i t

( Curcumin (0,005) 2 ,7( Curcumin (0 .05) 7 .2( Curcumin (0 ,1 ) 10.3

Curcumin (0 .1 ) + c i t r i c acid (0 .1 ) 11,0Curcumin (0 ,05) + methionine (0 .0 5 ) 13.4

( Curcumin (0 .1 ) + methionine (0 .0 5 ) 16.2( Benzene ex tra ct (0 ,1 ) 5 .0( Benzene ex tra ct washed with hexane

(0 .1 ) 5.5/ Hexane e x tra ct (0 .1 ) 3.0/ Benzene ex tra ct (0 .1 ) 3 .0V A lcoh ol ex tra ct (0 .1 ) 3 .0( A lcoh ol e x tra c t , phenolic p ortion (0 .05 ) 2.3( A lcoh ol ex tra ct , phen olic p ortion (0 .1 ) 5*0( O leoresin (O .l) 3 .0# A lcohol is o la te o f benzene e x tra c t (0 .1 ) 6 .5) A lcohol is o la te from hexane so lu b le ; p ortion o f benzene ex tra ct (0 ,1 ) 6 .0/ A lcohol ex tra ct , l iq u id portion (0 .1 ) 6 .0( A lcoh ol e x tra c t , s o lid p ortion (0 .1 ) 1 .5

5. Onion

Acetone ex tra ct a fte r ex tractin g withbenzene (0 .05 ) 2,5Ether so lub le portion o f acetone ex tra ctobtained a ft e r ex tra ction with benzene(0 .0 5 ) 24.0Ether so lu b le portion o f acetone ex tra ct obtained a fte r ex tra ction withhexane (0 .05 ) 15.0Ether is o la te from the residue obtaineda fte r ex tra ctin g o f f the hexauie so lu b lep ortion from the acetone ex tra ct (0 ,0 1 ) 4 .0Ether is o la te from the residue obtaineda fte r ex tra ctin g o f f the hexane so lu b lep ortion from the acetone ex tra ct (0 ,05 ) 24.0

n* Time ( in hours) taken to reach a peroxide value o f 20 m.eq.

S t a b il it y /Kg, o f lard contain ing the an tiox ida n t._____________________Index. Time ( in hours) taken to reach the same peroxide value by

lard alone.

S J

calculation according to their formula showed the presence of

8,65% isoeugenol in the heat treated eugenol,

A circumstantial evidence in favour of this

inference came also from the measurements of refractive indices.

3 0 ®The n£ for heat treated eugenol was four».ci to be 1,5632;

whereas eugenol and isoeugenol (cis and trans) were

1.537S, 1.5694, 1.5750 respectively.

When whole spice was used for stabilizing a fat,

it was found necessary to add the finely powdered spice to

the fat and heat it for a short period, to achieve the

maximum activity. Thus, in the case of chilli and cloves

when the powdered spice (0.5%) was added to'groundnut oil,

shaken mechanically for 6 hours at room temp* filtered and

tested, it was observed that it showed better keeping

qualities (12 A,0»M. hours) than the untreated groundnut

oil (10 A.O.M . hours). However, when the oil samples

containing the same amount of these spices were heated

(under reflux) for 5 min. at 200°/5-6 mm. Hg. pressure,

cooled and filtered, the antioxidant effect was greatly

enhanced, e .g . 21 and 19 A*0.M. hours for chilli and

clove respectively. (Tables I I I and IV ) . This clearly shows the

ease with which the antioxidant principles can be extracted

by the oil, under the influence of heat. Many investigators

have shown that heat treatment of some biological materials,

either before or after addition to a fat produces fat

stabilizing substances. However, it is not likely that

heating the oils for such short durations under reduced

2i

pressure, as mentioned above, would have such effects,

if at all, to any appreciable extent.

Various extracts of cinnamon leaf when tested showed

that the phenolic fraction of its essential oil

possessed the maximum activity (Table V ) , It is known thatI d

this oil is also rich in e\igenol. Hence the antioxidants

activities of clove and cinnamon leaf is due to eugenol

or to isoeugenol formed by very slow isomerisation of the eugenol

present in these essential oils*

Finely powdered turmeric (Curcuma longa) was

extracted with benzene which was then extracted with

boiling hexane to remove the essential oil. The two

extracts and also curcumin the colouring principle of

turmeric were then individually tested for antioxidant

activity on refined lard (Table V ) , The antioxidant

activity of curcumin confirms the earlier findings of Ramaswamy

and Banerjee,^^

The yield of curcumin is usually from 0,5 to 0.8%

when extracted from turmeric by the methods reported

in l i t e r a t u r e , H e n c e it was decided to develop a new

method for improting its yields.

Curcumin IV*

Freshly powdered turmeric on repeated extraction

with alcohol (in a soxhlet) ’gave a red coloured semi­

solid (115& yield) on removsil of the solvent. This was

then extracted with boiling hexane to remove the essential

oil 7 a pale yellow coloured liquid having a typical odour

of turmeric (2.85^ on turmeric). The essential oil free

material was dried and dissolved in a 20^ solution of sodium

carbonate, filtered and acidified with d il . sulphuric acid

and allowed to stand at room temperature* On fiIteration,

washing and drying,crude curcumin (4.55^ on the weight of

turmeric) was obtained which was further purified to give fehe

curcumin and two of its isomers in the ratio of approx.

3 :1 :1 . The curcumin so obtained analysed correctly anoJ

was as effective for the detection of boron as the one

obtained by any other method*

Activities of various extracts of dry ginger

(Zingiber officiaale) . nutmeg fruit (Myristica frgrans)

and onions (Allium cepa) on lard are given in Table V,

But for the yellow solids obtained by ether extraction of

the acetone extract of onion, no other extract gave any

pronounced a c t i v i t y . T h i s yellow solid which melted

at 245-$0®C. (with decomposition) on repeated

crystallisations from dil. alcohol and drying melted at

310-12®C (with decomposition). It gave a pentacetyl

derivative melting at 191-2®C. From the properties and

analysts these compounds were found to be quercetin (II)

and its pentacetyl derivative. The presence of

52quercetin in onions has been reported. Hence the

<r-

2{)

antioxidant activity of onion is due to the presence of

quercetin* Lewis and Watts^^ have also observed the

antioxidant and copper chelating properties of the

juices of onions and garlic and the extracts from their

skins. This has been attributed to the presence of some

flavanoid compounds by them.

Hasselstrom has shown that the activity of pepper

54is due to the presence of tocopherols in i t . Hence, no

further »ork was done in this spice.

The work on betel leaf (Piper betle) consisted of

heating the chopped leaves with groundnut oil at 200°/5»6 mm.

for 5 min. and of obtaining the essential oil by solvent

extraction as well as by steam distillation. The essential

oil was sepeur-ated into phenolic and non-phenolic constituents*

The former on careful distillation under vacuum gave solid

and liquid fractions* The solid fraction on recrystallisation

from benzene-petrol ether melted at 4S*5°C. and gave a

dibenzoyl derivative melting at 72°C. On further

examination, it was found to be identical to 4-allyl

catechol (hydroxy-chavicol), the presence of which in Java

betel leaf oil had been reported earlier by Schimmel^and

its presence in Indian betel leaf oil has sincee been

confirmed by Dutt^^ also. The liquid phenols consist of

a mixture of eugenol, carvacrol, chavicol, and chavibetol*

The antioxidant activity of the non-phenolic portion, the

liquid phenols, and hydroxychavicol was determined with

groundnut oil. The results show that though both the liquid

phenols and hydroxy-chavicol are antioxygenic, the latter is

2 /

by far the most active compound tested in the present

investigation. Table V III .

Hydroxychavicol is colourless, freely soluble in

oils, and imparts no odour or taste to the substrate in the

amounts used for antioxidant p u r p o s e s . B e i n g present in betel

leaf, it is apparently non-toxic because for centuries

this leaf is being eaten in India and other Asian countries

with no deleterious effects whatsoever. The presence of ascorbic

acid also, in betel leaf.has been reported by Banerjee and

Pain*^^

To make a comparative study of the antioxidant

activity of hydroxychavicol with some of the well known and

commercially used antioxidsuits, these were tested under

identical conditions, for stabilization of groundnut oil and

lard. The results (Tables VII and V III) show that this new

antioxidant from betel leaf is more effective, than N .D .G .A .

and propylgallate* Like the other phenolic antioxidants, its

activity is greatly enhanced by the addition of acid synergists.

B, Synthesis and modification of structure of certain

anti oxidants

As hydroxychavicol was found to be an ideal

antioxidant it was decided to synthesise it . Tarious attempts

have been made earlier to prepare allyl ethers of catechol which

readily undergo Clais^en’ s rearrangement at 180-220®C. to give

57allyl catechols*^* These methods when followed always gave

28

Table vi

A.P.M . Tests with betel leaves and its extracts on

refined groundnut oil,

(Antioxidant added to oil by wt.)n

Antioxidant Stability * Index.

Fresh leaves (2,0%)^ 2.2

Fresh leaves (5.0$^)* 3,6

Phenolic fraction (B) from alcohol extract(0.005%)** 2 .5

Phenolic fraction (B) from alcohol extract{0,0n>)** 3 .0

Phenolic fraction (B) from alcohol extract(0,05% )** 4 .0

Phenolic fraction (B) (0,025%) + ascorbyl palmitate (0 ,06% )** 6 .1

Phenolic fraction (B) (0.025%) + citric acid(0 .05% )** 5 ,0

Non-phenolic fraction (C) of alcoholicextract (0 . 03^ )* * 1 .6

Liquid phenols (B^) (0 ,05% )** 2 ,9

Liquid phenols (Bl) (0,10?^)** 3 .1

Hydroxychavicol (6 3 ) (0,005%)** 2,7

Hydroxychavicol (B2 ) (0,01iio)** 3 .1

Hydroxychavicol (B2 ) (0 .025%)** 3.3

Hydroxychavicol (B2 ) (0.025%) + citric acid(0 ,05^ )** 6 .3

Hydroxychavicol (B2) (0,025%) +ascorbylpalmitate (0.06?i»)** 7*6

* Oil heated with chopped fresh leaves.

** Oil iiixed with the anti oxidant.

* Time (in hours) taken to reach a peroxide value ofStability 20 m .»q./Kg. of fat containing antioxidant.________

index* ' Time (in hours) taken to reach the same peroxidevalue by the control.

2;)

Table V I I .

A .P.M . Tests with some knovin antioxidants on refined

groundnut oil,

(Antioxidant added to oil by wt.)

Antioxidant Stability^Index

Propyl gallate (O.Ol/b) 4 .1

Propyl gallate {0.03%) 5 .0

Propyl gallate (0,03^) + ascorbyl ^, palraitate ifi'06 5 .4

N .D .G .A . iO .05%1 2 .7

N .D .G .A .(0 . 05%) + ascorbyl palmitate 5.0

(0.06fi)

B .H.A. iO.Oljfo) 2.6

B .H .A . [O.O370) 3.S

B .H .A . i0.05‘ji>) 4 .0

B .H .A . (0.03?^) + ascorbyl palmitate (0.06^) 5.8

Time (in hours) taken to reach a peroxide value of ;jt Stability 20 m .eq./Kg. of fat, containing anti oxidant._________

Index, Time (in hours) taken to reach the saoje peroxidevalue by the control.

A.O.M.Tests with some antioxidants on refined lard.

(Antioxidant added to lard by wt,) .

Antioxidant Stability Index *

N .D .a .A . (O.Olfi,) 32

N .D .G .A . (0.03^) 45

N .D .a .A . {0.01?b) + citric acid (0.02^) 42

Propyl gallate (0.05?^) 50

Propyl gallate (0.05:^) + ascorbyl palmitate {0,06%) 73

Ascorbyl palmitate (0.06:^) 1

Iso-eugenol (0.03%) 32

Iso-eugenol (0.03fo) + ascorbyl palmitate (0.06^«) 45

Hydroxy chaVi col (0.005/^) 22

Hydroxychavicol (0.01^) 35

Hydroxychavicol (0,02^o) 42

Hydroxychavicol (0.03/^) 46

Hydroxychavicol (0.005)») + citric acid (0.05fo) 33

Hydroxychavicol (0.025^) + citric acid (0.01^) 48

Hydroxychavicol {0,03^) + citric acid (0.01?b) 58

Hydroxychavicol (0,025%) + ascorbyl palmitate{0 .0 6 U 52

Hydroxychavicol (0.03%) + ascorbyl palmitate (0.06%) 60

Hydroxychavicol (0.03%) + methionine (0.03^) 80

TABLE VIII

« Time (in hours) taken to reach a peroxide valueStability Index, of 20 m .eg./Kg. of fat containing antioxidant

Time (in hours) taken to reach the same peroxide value by the control.

3 1

a certain amount (upto 45^) of disdlyl ether of catechol.

The diallyl ether on Claisjien's rearrangement gave 3 ,6 diallyl-

catechol (V ) , and the mono allyl ether gave both 3-ally1-

catechol (VI) and 4-allyl-catechol (I I I )*

CH2=CHCH2

OH

\^^CH2CH=CH2

,0HOH

Diallyl-catechol

^^V^CH2CH = CH2

VI

3-allyl-catechol

CH2CH=CH2

VII

4-allyl-c atechol

The antioxidant activity of these three compounds

was found to be in the ratio of 0 * 7 :0 .3 5 :1 .0 . The antioxidant

properties of these compounds and their mixture on refined lard

are:i presented in Table K . As it is only the hydroxychavicol

that is present in betel leaf, the other two cannot be taken as

pharmacologically safe, unless proved to be so* A new method

developed during the present investigation completely

eliminates the formation of the diallyl ether. It consists

of slowly adding allyl bromide to a large excess of catechol

dissolved in dry acetone in the presence of anhydrous potassium

carbonate and refluxing the mixture for a few hours. The overall

yields of the mono allyl ether by this method were 35-90^

and the unreacted catechol was recovered. The mono allyl

ether on rearrangement, however, gave as usual a mixture of

3-allyl-catechol and 4-allyl-catechol. It is rather

interesting to note that under the Clais$en's rearrangement

monoallyl ether of catechol should give both 3- and 4-allyl-

+>c6

CO

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IIx: x: IIo o IIcrt OS IIO' 9-‘ IIu t II

IIo O II4J II

■P IIc c d IIQ) ro <D II

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"H IIm +j CO IIu c II

OS IIo o IIx; M x: II

c IIC -H d II•H d •H II

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tJ >H <v Itd o rH II3 M x: o n

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IIIIIIIIIIIInIIIIIIIIIIIfII

catechols, because normally para rearrangement of an allyl-aryl

ether occurs only when no ortho position is free*

Mechanism of Clais^en* s rearrangement

It is now well established that the ortho Clais^en^s

rearrangement of an allyl aryl ether occurs by an intramolecular

cyclic mechanism, thus

'CH2

■ ^CH CO OH

4 j CH=CH2

The unstable ketonic form of o-allyl phenol is produced as an

intermediate, wi:iich spontaneously rearranges to the stable

phenolic form,^^*^^

With allyl aryl ethers having both ortho positions

occupied the re/action starts in the same way as the migration

to the ortho position:

0 C H 2 -C H = C H 2 0 0

CH3

OHCH3^^/'^j,CH3

> <H CH2 CH=CH2

X

CH2CH=CH2XI

Thus the ether V III has been shown to give first

the intermediate IX , This cyclohexadienone cannot be stabilized

by passing over to a phenolic system and hence the allyl group

continues to move to the para position with the foraation of the

new cyclohexadienone X, which immediately goes over to the

stable phenol X I , This is the para Claisen’ s rearrangement. *

3 1

A few compounds are known which rearrange with

some migration of the allyl group to the para position, although

a free ortho position is available. It may be significant

to note that all such compounds viz.^.T-^iiJnstjhyl allyl-2-methoxy-

phenyl ether, allyl 2-hydroxyphenyl ether, and allyl 2,3-

methylenedioxy phenyl ether are derivatives of polyhydroxy-

benzenes.^^ No explanation is available in the literature

for this anamolous behaviour.

As analogous to the ortho rearrangement, the rearrangement

of catechol fiiallyl ether to 3 ,6-diallyl-catechol can be

explained as shown in the equation, (a)

HC^ 0 0CH2-CH=CH2 |i

0CH2~CH = CH2_______^ H

CH2=CH-CH2

H2C —CH=CH2

( a )

However, in the case of mono allyl ether of catechol,

this rearrangement must be taking an entirely different course,

because, as mentioned earlier, a mixture of 3-allyl-catechol

and 4-allyl-catechol is always obtained although normally

only the former would be expected. This perhaps can be

explained by presuming that a part of the rearrangement

takes place by the ortho mechanism as shown in equation, (b)

35

0CH2CH = CH2 OH

0 CH

ch2

OH --------- >

CH2CH=CH2 A OH

> <H CH2CH = CH2

OH

(b)

(c)

CH2CH = CH2

It may logically be assumed that, simultaneously,

under the activating influence of the hydroxyl group a parallel

rearrangement also occurs in the counter direction i .e . by first

forming an intermediate with ortho hydroxyl group, thus giving

rise to the cyclohexadienone which rearreuiges to the new

cyclohexadienone intermediate and finally to 1 ,2 dihydroxy-4-ally1-

benzene as shown in equation (c ) . In other words part of the

rearrangement takes place by ortho mechanism and the rest by

para mechanism.

Kharasch et.al^^ have pointed out that under the

influence of ultraviolet light and in the presence of solvents

3 h

like isopropanol, the phenyl allyl ether and the phenyl

benzyl ether rearrange to give p-allyl-phenol and p-benzyl-

phenol respectively, although under the ibhermal rearrangement

only the ortho isomers are obtained, Schmid and Schmid^^

have shown that in the photochemical rearrangements, the

reaction takes place by dissociation and recombination

mechanism. With a view to study the photochemical behaviour

of catechol mono-allyl ether, it was dissolved in isopropanol

and irradiated with ultraviolet light upto 120 hours. On

working up and distillation 4-ally1-catechol in a maximum

yield of 25?b (together with 3-allyl-catechol^ nearly the same

amount)was obtained. The remaining portion was unreacted

catechol mono-allyl ether. In order to find out the presence

of any other substance^various fractions obtained were subjected

to gas liquid chromatographic analysis.

The gas liquid chromatogram of catechol mono allyl

ether revealed a sharp peak with retention time (tj^) of 9 .3 mts.

On the other hand authentic sample of 3-allyl-catechol under

identical sets of operating parameters emerged out of the

column in a sharp peak with tj value of 10,3 minutes. The

retention data of these two compounds reveal them to behave

nearly the same on the column and actually when their mixture

(1 :1 ) is chromatographed, it is interesting to note that these

two products emerge out as a single peak with tj value of 9*3 mts,

This clearly shows that due to their closely similar retention

character, these two products cannot be resolved under

this set of conditions#

3 7

When the chromatogram of the three fractions of

photosensitised (120 hours) catechol mono allyl ether wfire run

under identical conditions it was observed that fraction 1 and

2 revealed a single peak which is evidently due to catechol

mono-allyl ether and 3-ally1-catechol. The third fraction

which was identified as 4-allyl-catechol by its melting

point, dibenzoyl derivative and infrared absorption curve,

when subjected to gas liquid chromatography gave a sharp single

peak with tj value of 12,8 mts* showing the complete homogenity

of the sample. From the retention data compiled in

Table X it is apparent that the retention behaviour of

4-isomer is remarkably different than Uiat of the other

isomer. Therefore it is feasible to resolve a mixture

containing the two.

T^ e X

Specimen Retention time (tp) in minutes.

Catechol monoallyl ether 9 .3

3-Allyl-catechol 10.3

3-Allyl-catechol + catechol

mono-allyl ether (1 :1 ) 9«S

Photo-sensitised catechol

mono-allyl ether

Fraction 1

Fraction 2 10.3

Fraction 3 1 2 .S

Further work on the total separation of the two isomers

could not be done because of the V,P.C.apparatus going out of

order.

38

F i g y l l

Gas liquid chromatograms of catechol monoallyl ether,

3-allylcatechol, 4-allylcatechol and tne three fractions

obtained on ultraviolet irradiation and distillation of

catechol monoallyl ether. Arrow indicates the point of

injection of the sample. (S) denotes the air peak.

Retention time (tfj) is given on the peak*

3 .)

It has been pointed out that polyphosphoric acid

though an effective dehydrating agent is a mild condensing

i*eagent. Generally it does not undergo a violent reaction with

hydroxylic corapounds*^^ Because of these properties it

67has been used for acylations of phenols by Nakazawa

and phenol ethers by Sukh DeV| the acyl group going

preferentially in the para position to the hydroxyl group.

Hence, it was decided to condense acrylic acid with

catechol using polyphosphoric acid to get acryloyl-catechol,

and then to preferentially reduce the carbonyl group, without

affecting the double bond by some of the latest reducing

agents. Though acryloyl-catechol was obtained (25% yield)

all attempts to reduce it to 4-ally1-catechol were

unsuccessful.

OH

0 = C - C H = CH2

Modification of structure;

A study of the Tables IV and V shows that isoeugfenol

is much more active than eugenol both with groundnut oil

and lard. This shows that a double bond in conjugation with

the benzene nucleus is responsible for the enhsuiced activity.

Attempts to isomerise 4-allyl-catechol to 4-propenyl-catechol

under conditions similar to those used for isomerisation of

eugenol were unsuccessful as each time a viscous resinous mass

was obtained. It was then decided to demethylate eugenol

4 0

and isoeugenol so as to get 4-allyl-catechol and 4-propenyl-

catechol in one step. But reactions with hydriodic acid,

pyridine hydrochloride, or anhydrous aluminium chloride all

failed, in each case either the starting material was

recovered as such or a resinous product and catechol were

obtained.

CurcuiBin (diferuloyl methane) IV has two unsubstituted

positions ortho- to the hydroxyl groups. As analogous to

butylated-hydroxy-anisole or butylated-hydroxy-toluene, if

two tert. butyl groups could be introduced in the said

positions,the antioxidant activity of curcumin, it was expected,

would be enhanced. This was attempted by taking a solution

of curcumin in t-butyl alcohol and adding to it BF^-

ethereate, when an exothermic reaction was observed.

On working up, it was noticed that though t-butylation had

taken place the resulting product had formed a stable boron

complex which could not be broken up.

In order to correlate the effect of the presence of

various groups on the antioxidant activity, a few compounds

were synthesised. These are(1 and 2) propyl and allyl esters

of protocatechuic acid ( 3 :4-dihydroxy-benzoic acid)

X III & ilV j;(3 and k) 4-propionyl-and 4-lauroyl-catechols

XV and XVI prepared by condensing propionic acid and

lauric acid with catechol in the presence of zinc chloride and

phosphorous oxychloride. These two compounds were used as such

and (6 and 7) also after subjecting them to Clemmensen’ s

reduction, so as to give 4-propyl- and 4-d©decyl-catechols

XVII and XVIII.

-,-1

4 1

The antioxidant activities of all these compounds, as well as

of catechol, protocatechuic acid, dihydroeugenol,

pyrogallol and propyl gallate on refined lard,at O'OS/, cone,

Table X I , Fig. U1 •

The following conclusions can be drawn based on the

present study:

1 . Presance of an ester, acid or a keto group

on the nucleus reduces the activity considerably, this is

clear from the fact that protocatechuic acid, its esters,

and the propionyl-and lauroyl-catechols all are less effective

than catechol itself*

2. C-alkyl derivatives are more active than the

esters or the corresponding o-alkyl derivatives*

3* A double bond in conjugation with an aromatic ring

leads to maximum activity, followed by the double bond at the

-positions, A double bond farther away does not

have any effect on the anti oxidant activity of the compound.

Thus isoeugenol is more active than eugenol, and dihydroeugenol

is a very poor antioxidant. Allyl ester of protocatechuic

acid is a very feeble antioxidant. On the other hand propyl-

catechol is also not a powerful antioxidant. The difference

between the activities of eugenol and 4-ally1-catechol is so

great that it shows clearly that blocking of even a single

hydroxyl group considerably reduces the antioxidant activity.

The results obtained with various compounds are shown

diagramatically in Figure HI. For the sake of comparison,

(MN

CMro

(VI

o

oXoLU

o

_1>-ollJ CJ Q Ul O I- Q < .

_1

2 O0 ^u. ljj

is1

UJH<

ist- o < < (Jo

caQ.

-I01oUiI-<o

in

o(C

ozUJCDDUJoq :QVXQ

into

o2UJCDUJoCrt

oo

00't

CVJin

UJCDZ)UJ

oXoUJI-<o

<Q

COto

OXoUlV-<o

<Ito

oXoUJ

o_J>-

<I

CJ

h-UJCJa:UJ

a

oin

<CD-I>-CLO(Ea

o00<CDOa:>■a.

oCO

<CDQ

CDto

OQ

<D

b£|

Ox:o

Baso

CmO

-P(t5jC-P

o•p

■S3Oa,eoo

O' •V,■P c D OaE O o

><a.'

x :+3

•H OI— I•r-i X X>nJ t3 4J C(0

<L' >. x : 4-> ■P -H

r—( Cm -Ho ^

CtJO•M (0 ■PcT) d' ^ x :

■pax: <0 ■P -H

M CTS iH 3

« e

0. O iH Cm O3 o; C x;

4Ja'

XI so

<D 4) U U 3 3M M

•H «!-♦ttt Ct,

Table XI

Antioxidant activity of some antioxidants on

refined lard.

4J

Compound Cone. taken

A.O.M.'houEB to reach a peroxide val. of 125 m .eq ./ Kg.

StabilityIndex’!'

Pyrogallol

Propylgallate

Catechol

Protocatechu^ic acid {3,4-dihydroxy benzoic acid)

Propyl protocatechuateX III

Allyl protocatechuate XIV

4-P ropi ony1-c ate chol ( 3 ,4-dihydroxy-propio- phenonei XV

4-Lauroyl-catechol ( 3 ,4-Uihydroxy-lauro- phenone) X\'t

4-Propyl-catechol XVII

4-Dodecyl-catechol XVIII

Dihydroeugenol ( 2-methoxy, 4-propy 1- phenol)

Citric acid

Lard alone

0.05%

0.05^

0.05%

0.05%

0.05%

0.05%

0.05%

0.05%

0.05%

0 . 01%

0.05%

0 . 01%

0.05%

0 . 01%

12075

60

33

30

21

15*

19.5

33

15

48

22.5

7.5

2.25

1.5

ao

50

40

22

20

14

10

13

2210

32

15

5

1.5

ss = = s 3 s s s s s :

=<'Stability Index,

Time (in hours) to reach a peroxide value of 125 m .eq./ Kg. by the antioxidant containing fat._____________________

Time (in hours) taken to reach tne same peroxide vftlue by lard alone.

4 1

the antioxidant activity of catechol is taken as unity and

the results obtained with other compounds are given as fractions

or multiples of catechol activity (given inside the nucleus).

Though the compounds are all tested on equal weight

basis, it is clear from the figures that for almost the same

molecular weight the 4-allyl-, 3-allyl- and the 4-propy 1-

catechols, the first one is more active than the second which

is more active than the third, showing thereby that a para

allyl substituent enhances the activity of a parent phenol

to a greater extent than the ortho allyl substituent. However,

if the allyl group is reduced to propyl, the antioxidant activity

drops down considerably*

The difference in activities of 4-propyl-catechol and

4-dodecyl-catechol inspite of the higher molecular weight of

the latter, may perhaps be due to its higher solubility.

The activity of N*D.G,A, is about the same as that of

4-ad.lyl catechol If taken on equal weight basis, but would be

only half as much if considered on equivalent molar basis,

Quercetin has been found to be much less active than

propyl gallate on an equal weight basis. It was quite natural,

23however, that Simpson and Uri found it to be about 30-100?^

of that of propyl gallate on equivalent molsu? basis.

4 5

EX.PERIMSNTAL

•«s

‘ t I J

EXPERIMENTAL

I . Preliminary testing of spices for the antloxidant properties:

Twenty-four common Indian spices and betel leaf

(Piper betle Linn.) were selected for this study. The spices

which were in the dry form were powdered to pass through 100 mesh

sieve. The green materials e .g . onion (Allium cepa),

garlic (Allium sativumj* betel leaf (Piper betle}, green ginger

(Zingiber officinale) and green chilli (Capsicum annum) were cut

into very fine bits. Varying aunounts of these substances were

added to fresh samples of refined groundnut, safflower, sesame

and mustard oils and heated for 5 minutes. In the case of

groundnut oil, the heating temperature was 270-75^C, and with

other oils a temperature of 220-25^^• oils were then filtered

and brought to room temperature within 25 minutes. In each case,

a sample of the oil (control) was also given a similar heat

treatment without the addition of any spice. These samples

together with a sample of the original oil were then subjected

to a modified ’Swift stability test*.^^ The oil sample (20 cc.)

was taken in a test tube 1 in , x 8 in . and immersed into a

thermostat at 208°F . Air, washed with d il, potassium

permanganate solution, was bubbled through each test-tube at the

rate of 2.33 cc ./sec. for 5 hours. The acid and peroxide

values of aerated and unaerated samples were then determined.

(Table I ) . The refined oils were stored at room temperature

before use for some time till they showed appreciable rise in the

peroxide value on being subjected to Swift stability test*

4 /

I I , Preliminary testing of spices for the antioxidant effect on

butter fat aind coconut o il ;

Only those spices which were found effective in

Exp.I were added to refined coconut oil and butter fat (ghee)•

These were heated to 220-25®C, for 5 min, filtered and cooled

immediately to room temperature and subjected to the Swift

stability test as in the last experiment. The results are

presented in Table II*

i) Preparation of substrate; Freshly expressed

groundnut oil was refined with alkali and stored at

approx. -2®C. for further experiments. Fresh lard was also refined

by treatment with a mixture of decolourizing carbon and

activated earth, dried and stored at -2°C. till the time of use.

In further work only these two oils were used.

ii) Evaluation of the anti oxidant activity; by the

active oxygen method: This consists of taking 20 cc. of the

substrate in a test tube (1 in. x 8 in .) maintaining it at

97*6°C. ( t 0.2*^) and passing clean dry air through it at the

rate of 2.33 cc./second {* 10^) till a peroxide value of

20 milli eq ./kg . is reached. The results are expressed in

the terms of stability index which is the ratio of the time

(in hours) taken to reach a peroxide value of 20 mm.eq./kg,

(or 125 mm,eq./kg) by the fat containing the antioxideuit

to the time taken by the fat (blank) to reach the same

peroxide value. Ascorbic acid was converted to its fat

soluble derivative ascorbyl palmitate by the method of Swern

67' and used as synergist wherever mentioned.

48

I I I , Activity of red chilli (Capsicum Annum) and its extracts;-

Powdered red chilli 100 mesh was added to 200 cc.

refined groundnut oil so as to^ive 0.55^ conc. After

thoroughly shaking, it was divided in two parts. First 100 cc,

of it was then stirred mechanically for 6 hours at room temp,

and filtered. Another 100 cc. was taken in a round bottom

flask,fitted with a reflux condenser and heated at 200°C/

5MmMg pressure (by immersing the flask in an oil bath

maintained at 205°C.) for 5 min, and immediately chilled in ice

cold water. The filtered samples were then employed for the

A ,0 ,M , test. The time taken by groundnut oil alone to develop a

peroxide value of 20 m.eq.per kilogram of fat was 10 hours.

The unheated sample (with 0 .5^ chilli) took 12 hours to reach

the same peroxide value, whereas the heat treated sample took

21 hours.

Powdered red chilli was extracted repeatedly with ethyl

ether, petroleum ether (60-80°C) and alcohol at room temp.

The solvents were then recovered and the last traces of solvent

removed undee vacuum. The ether and petrol ether extracts

were dark coloured resinous liquids whereas the alcohol extract

on standing at low temperature gave a pinkish product which

after three crystallisations from dil, alcohol melted at

139-191° (with decomposition), had an optical rotation

o( D ♦ 48® and gave an absorption max. at 245

in the ultraviolet absorption spectrum. This proved that the

substance was ascorbic acid. All these extracts as well as

the non-crystallisable part i . e . the liquid portion of the

alcoholic extract were added to the groundnut oil and tested by

the A.O.M . test.

Capsaicin (m.p.65^) the pungent principle of chilli

was extracted by the method Lapworth and Royle^^ and tested

for its antioxidant behaviour*

The various concentrations used, treatments given and

stability indices obtained with these substances are given lo

Table I I I ,

IV , Activity of cloves; (Syzygium aromaticum) :

Clove powder (100 mesh) was added to groundnut

oil at the concentration of 0.55^, and a part of it

mechanically stirred for 6 hours at room temp, and the other

part given k similar heat treatment as in the last

experiment. Both of them were then used for the A ,0,M ,

test. Clove powder was also extracted separately with benzene

and alcohol at room temperature. As the essential oil of

cloves contains caryophyllene and eugenol, there were tested

at 0.05% conc. To compare the activity of eugenol with its

isomer, isoeugenol was sdso added to groundnut oil and

subjected to the A .O .M .test. As isoeugenol showed more

activity than eugenol a mixture of isoeugenol and ascorbic

acid, as well as isoeugenol and asoorbyl palmitate were also

tested. The concentrations, treatment given and the stability

indices are all given in Table IV,

iV, Isomerisation of eugenol;

Eugenol (16.4 gm, 0 .1 mole) was taken in a 50 cc.

two necked flask. It was refluxed for 5 hours while nitrogen

was slowly bubbled through i t . It was allowed to cool to room

temperature. A part of it was distilled under vacuum.

5jv)

(boiling 92-102®/l mm.) 1,555 mgm. of the distillate was

dissolved in 125 cc, of ethanol and its ultraviolet

absorption spectra taken*

Compound >max Molar absorbancy index254 nyi 282

Eugenol 279 4S0 2650

Heat treatedeugenol 274 2508 3920

Isoeugenol 256 13200 4640

According to the formula of Yespe and Boltz^^

the isoeugenol content of the distillate came to 8.65%*

Refractive IndexD

Original eugenol 1,5378

Heat treated eugenol 1,5632

Isoeugenol (cis) 1,5694

Isoeugenol (trans) 1,5750

V I, Activity of cinnamon leaf (Cinnamomum tamala);

Powdered (100 mesh) dry cinnamon leaf was extracted

with benzene, and concentrated. The essential oil was

obtained by steam distillation of the leaf. It was separated

into non-phenolic and phenolic fractions, by iSfeating it with

NaOH solution, extracting with ether and acidifying the

alkaline solution and extracting it also with ether, and

wortcing up the two extracts. These fractions and eugenol were

then added to lard and tested by A ,0 ,M , test (Table V ),

V II : Activity of turmeric (Curcuma longa) :

Turmeric powder was extracted repeatedly with

benzene at room temperature. The solvent was removed and

5 1

the dark red semi-solid residue -was extracted a number of

times with hexane. The hexane insoluble material and the

total benzene extract both were added to fresh lard at 0*1^

conc. and subjected to A,0*M, test, (Table V ),

V I II , Extraction of Curcumin (IV) by a new method;

600 gms, of turmeric powder (100 mesh) was extracted

with 95^ alcohol in a soxhlet apparatus, till the extract was

colourless. The solvent was then removed to give 66 gms,

(11^) of a dark red viscous residue. This was extracted

thrice with boiling n-hexane while being vigorously stirred.

The n-hexane extracts were combined together and concentrated

to give 17.11 gm. (2.85/^ based on turmeric) of yellow

coloured essential o il. The hexane insoluble material was twice

extracted by stirring with a mixture of 700 cc, of 20^

NagCO^ and 20 cc. of NaOH solutions, at 70-80®C. for one

hour. The alkaline solution was filtered, cooled and

cautiously neutralised with 25^ ^2®^4 5-6 pH

and allowed to stand for 24 hours. On filtration, washing

and drying it gave an orange coloured solid (25.2 ©ns,

If.2^ on the basis of turmeric). It melted between 176-163^0.

Rescrystallisation from benzene gave curcumin (m .p .131-83°)

axid two of its isomers melting 224-226® and 155«-67°

respectively. The main fraction i .e . curcumin IV

(diferuloylmethane) after two crystallisations gave a

sample which analysed C, 6 8 .6 ; H, 5.3%; *^21^20*^6

C, 68 .47 ; H, 5*47^«* It gave a reddish-brown colour with

NaOH solution and light yellow colour with HCl solution. It was

r . 2

effective in the detection of boron in very dilute solutions.

It was tested for its antioxidant activity. Citric acid and

methionine were used as synergists. The results are presented

in Table V,

IX. Activity of ginger (Zingiber officinale)

Alcohol, benzene and hexane extracts of dry ginger

were prepared in the usual way. The alcoholic extract

was digested with 5% alkali, extracted with ether and the

ether extract removed; the alkaline solution was then

acidified and extracted with ether. The two ethereal extracts

were separately washed, dried and concentrated to give the

non-phenolic and phenolic fractions respectively. The oleo-

resin of ginger was obtained by percolating it with ether at

room temperature and the solvent was removed under vacuum.

The residue was dark coloured viscous liquid. All these various

extracts and fractions of ginger were tried for anti-oxidant

activity with lard (Table V , ) ,

X . Activity of Nutmeg fruit (Myristica fragrans);

Benzene extract of powdered nutmeg fruit was

prepared. It consisted mostly of fixed oil, which was

removed by extraction with hfxane. The total benzene extract and

its hexane soluble portion were separately shaken with

alcohol and the isolates obtained by removal of the solvent#

The whole nutmeg fruit was also extracted with alcohol.

After removal of the solvent, the residue on cooling separated

into a mobile liquid and a semi solid fraction which were

separated*

These substances were tested on lard (Table V ) ,

5 a

XI Activity of onions (Allium cepa):

Onion was finely chopped into small pieces and kept

overnight in benzene and filtered. The residue was extracted

with acetone, and the solvent removed. The acetone concentrate

was extracted with ether. The ether soluble portion on removal

of the solvent gave a yellow powder which melted at 245-50°C,

(decomp.). This product was also obtained in a less pure

condition by shaking the onions with hexsme and reextracting the

extract with the help of acetone and ethyl ether.

Alternatively hexane soluble portion was removed from the

total acetone extract of onion, the residue shaken with ethyl

ether and the ether soluble portion was then concentrated.

The antioxidaint index obtained with these extracts and

fractions with lard are given in Table V. The yellow solid

obtained which was found to possess the maximum antioxidant

activity, was recrystallised four times from dil. alcohol.

On drying it melted ot 310-312° (decomposes). It gave a

yellow solution with aq, alkalies and yellow solution with

green fluorescence with conc. HgSO^.

Analysis found C, 59»S; H, 3 , ^

Quercetin requires C, 59*61; H, 3,34%. 0 .5 gm, of

this substance was converted to its acetyl derivative (acetic

anhydride,HgSO^ method) and ciystallised from benzene m .p .191-2°,

d n analysis, it gave C, 53 .9 , H, 4 ,2^

pentaacetyl quercetin ^£5^20^12 53.60, H, 3.93^*

n4

X I I , Activity of betel leaf;

Fresh betel leaves were chopped into very small

pieces and added to a round bottom flask containing groundnut

oil so as to give a conc. of ^0% and on the weight of

oil, fitted a reflux condenser to the flask and heated to

200®C. at 5-6 mm. of Hg pressure, as mentioned in Exp. I I I .

The treated oil was then filtered and subjected to A.O.M. test.

Betel leavowere dried at room temperature for a

period of 10 days, and 100 g. of it was finely powdered and

extracted with alcohol and the solvent removed, last traces

under vacuum* The green coloured viscous residue was extracted

with boiling water and the extract made alkaline after cooling*

It was then transferred to a separating funnel and extracted

thrice with 200 cc. ether to remove the non-phenolic

fraction. The aqueous layer was acidified aind extracted with

ether, this on removal of solvent and distillation under

vacuxim (1 mm. Hg. pressure) separated into two fractions^a

liquid ( b .p ,d8-91°C .) and a solid crystalline substance

(b .p . 105-108°). The latter was re crystallised from benzene-

petrol ether to give colourless needles, m .p.48-48.5°C*

Analysis: Found, C, 71.85, H, ,6 .68%

C9H10O2 requires C, 71 .98, H, 6 .71^ .

Its solution gave a green colour with aq. ferric chloride

solution which changed to brick red on addition of alkali*

A part of it was dissolved in d ll . alkali and treated

with benzoyl chloride (Schotten-Baumann reaction) when a

dlbenzoyl derivative m,p.72®C. was obtained.

f)5

Analysis: Found C, 77 .30 , H, 5.3^

^23^13^4 77 .09, H, 5.03%* Hence the compound

is identical with hydroxychavicol,

5000gras, of chopped betel leaves were steam

distilled to give 65 .0 gm. (1.37<>) of g greenish yellow oil

Hq 1*5052 which had the characteristic odour of the betel

leaves. It was also separated into phenolic and non-

phenolic fractions as mentioned above (yields 605& and 409&).

The phenolic fraction was distilled to give hydroxy chavicol

( .8 gm.) and the liquid phenols (3^ .5 gms.) consisting of a

eugenol, chavicol, chavibetol etc. The various extracts of

betel leaf were tested on groundnut oil for their

antioxidant activity (Table VI)*

X III* Activity of commercieil antioxidants;

For the sake of comparison, hydroxychavicol,

propylgallate, N .D .G .A . and B.H.A , were used in various

conc. with refined groundnut oil and lard. Ascorbyl palmitate

was used as synergist* The results are presented in Tables

V III and IX*

XIV, Synthesis of hydroxychavicol:

It has been synthesised by Kawai, Perkin and

57 58Trikojus and Hurd et .al , « In all these methods catechol

was first converted to its allyl ether by refluxing it with allyl

bromide in the presence of acetone-potassium carbonate.

The resulting catechi mono allyl ether and catechol diallyl

ether were then subjected to Claisen^s rearrangement when

3-allyl catechol, 4-allyl catechol, and 3,6-diallyl catechol

r>b

were obtained. None of these methods was found suitable

in the present investigation as the formation of dieillyl

catechol and 3-allyl catechol were not desirable,

XV« Modified method of synthesis of hydroxychavlcol:

A modified method developed, in the present study

completely eliminated the formation of diallyl catechol.

In a three necked round bottom flask fitted with a mercury

seal stirrer, a reflux condenser carryihg a calcium chloride

gusird tube, and a dropping funnel, were placed catechol

44 gms, (0 .4 moles) dry acetone 125 cc. and freshly fused

anhydrous potassium carbonate 24.5 gms. (0.35 moles).

Freshly distilled allyl bromide 26 cc. or 36.3 gm. (0.3 moles)

was placed in the dropping funnel. Stirring was started and

the flask heated slowly so as to give a gentle refluxing of

acetone. Allyl bromide was added dropwise and refluxing and

stirring continued for 4 hours after the addition of allyl

bromide was over. Solvent acetone was then recovered

(IIS cc . ) , but no allyl bromide could be detected in the

distillate. The contents of the flask were cooled and

filtered under suction. From the residue which consisted of

unreacted catechol, KgCO^ and KBr^ catechol (9 .9 gms.) was

recovered by extraction with ether. The filtrate freed from

solvent was distilled under reduced pressure when 40 g,

catechol monoallyl ether^l;80-S2°/0.8-1.2 mm., n^® 1.5354

was obtained. No other fraction distilled over. Yield 88 .9^ ,

Analysis, Found, C, 72 .05, H, 6 .9 ^ .

^9^10^2 71.98, H,

XVI, Claisen*s rearrangement: Catechol monoallyl ether (30 gms.,

0 ,2 moles) was taken in a two-necked round bottom flask fitted

with a nitrogen inlet and a reflux condenser. It was heated

in an oil bath, while nitrogen was being bubbled slowly and a

vigorous exothermic reaction with brisk refluxing took

place, when the bath temp, had reached about 200-205°.

The colour also changed to red. After 5 mts. at that temp,

the flask was taken out of the oil-bath and immediately

cooled in an ice-bath. On careful fractional distillation

(28 .5 gms.) two fractions were collected#

(1) b.p.llO-112®/3 mm.; 1,5600, 13.5 gms.

(2) b .p ,120-23 /2 mm, 1,5606, 11,8 gms. About 3 gms,

of the substance could not be distilled.

The second fraction on one more vacuum distillation

could be crystallised from benzene-petrol ether. While needles

m .p.4$-48,5° mixed melting point with hydroxychavicol

(from betel leaves) remained undepressed, (reported

values m ,48,5°t b .p .141-44/7 mm., n^^ 1.5600^^ ) .

Analysis Found: C, 71.8 , H, 6 .8% , requires C, 71.98,

H, 6.71> .

The first fraction could not be crystallised.

It is 3-allyl catechol (reported values 1.5595, b .p .132-38°/

9 mm.)^^. Analysis: Found:* C, 71 .8 , H, 6 .5^ ,

requires C, 71 .98, H, 6,7'lfo,

Both these fractions gave deep green colour with

FeCl^ solution.

58

XVII, BF^ catalysed. Condensation of allyl alcohol with catechol

Allyl alcohol (0 ,2 mole, 11,6 gms.) was saturated

with borontrifluoride at 0°C, At the end of the reaction the

temperature was allowed to come to room temperature. 0.01

mole of cLllyl alcohol-BF^ complex was then dissolved in IDOcc.

dry ether and a 0.01 mole of catechol added to it . The flask

was then warmed gently (to 70®C.) and the contents stirred

for two hours. On working up a red coloured viscous mass was

obtained which could not be distilled*

X V III . Preparation of 4~allyl catechol by Ultraviolet irradiation

1, 10 gms. of catechol monoallyl ether dissolved in

150 cc, isopropanol was taken in a 250 cc. quartz conical

flask fitted with a reflux condenser. It was placed about

4” away from a 125 watts, mercury vapour lamp (Mazda MBW/V)

and well covered from all sides. The material was irradiated

for 24 hours, solvent recovered and the residue distilled

under reduced pressure. Only one fraction b .p .S0«g2°/l

1,534S showing that it was unreacted catechol monoallyl

ether^ wo-j ottfcUed,

2, 10 gms. of catechol monoallyl ether dissolved in

100 cc. of isopropanol was taken in a 250 cc. quartz round bottom

flask fitted with a ref lu3</on denser and placed at a distance

1 cm. above the 125 watts. U .V , lamp, in such a way that the lamp

also worked as source of heat. Within a few minutes the

alcohol started refluxing freely. The irradiation was

continued for 100 hrs, after which the solvent was recovered.

The residue was distilled to give 9 gns.(90^) of the catechol

5 ‘,)

monoallyl ether unreacted. The remaining 1 gm. distilled at

97-100°/3»5 mm, 1.5564. On redistillation it gave a low

melting solid which on recrystallisation from benzene-petrol

ether melted at 48 .0°C .

3. 30 gms. catechol monoallyl ether was dissolved

in 50 cc. isopropanol and irradiated and refluxed for 120 hours

and worked up as in the last experiment. The residue was

fractionated to give 14 gms. of unreacted ether, and 7«0 gms.

of 3-allyl catechol and 7 .5 gms. (25%) 4-allyl catechol. The

o 30^latter was crystallised, m.p.48 . n 1.5600, dibenzoyl

0derivative 72 .

On analysis found, C, 7 1 .7» H, 6 .86 , requires

0 , 71 . 9a, H, 6.715^.

No other compound was isolated from the reaction

mixture•

XIX, Gas liquid chromatography of photolysed products;

The Griffin V .P .G . apparatus M .K. II A (Griffin and

George Ltd.) with Katharometer as the detector, was used.

A total length of 6 ft , column and dry nitrogen gas as

carrier was used during this work,

Celite (Johns-Manville 545) was size graded and

waabed by the method of James and Martin*^^* Silicon oil

(May and Baker) was dissolved in ether and impregnated over

celite to provide the stationary phase. The impregnated

celite after removal of solvent was ditied for 6 hours at

90-100°C, and packed in the column. The packing density was

0 .4 g. per cc.

G O

The operating conditions were as follows:

Column temperature 244®

Outlet pressure 10 mm.

Nitrogen flow rate 0 .6 l /hr .

Recorder chart rate 6 in /hr.

Bridge current 65 mA

Sensitivity i

The samples were directly introduced into the

column through the vaccine cap at the top of the column by

means of a hypodermic syringe. The injection of the samples was

made when the recorder maintained a steady base line. Every

sample was run two or three times so as to accurately

determine its retention period.

The authentic sample of catechol monoallyl ether,

3-allyl catechol, and a 1 :1 mixture of these two as well as the

three fractions obtained on distillation of U .V , irradiated

(120 hours) catechol monoallyl ethers were run through the

column# The retention time (in minutes) of these compounds

are given in Table X*

XX. Preparation of acryloyl catechol:

In a three necked flask fitted with mercuiy seal

stirrer, reflux condenser carrying a CaClg guard tube, and a

dropping funnel, 45 gms. of phosphorous pentoxide and 20 cc.

of phosphoric acid were taken and stirred well to get a

homogenous mass. The flask was heated and maintained at llO^C.

5 .5 g . (0 .05 mole) catechol and 3.6 g. or 3.3 cc. (0 .05 mole)

BL

acrylic acid were added to the flask all at once. The

colour immediately changed to red. After 10 mts. of vigorous

stirring, the flask was cooled, ice added to the reaction

mixture, and left overnight. Next day d il . HCl was added

and the reaction mass extracted with ether. On washing and

removal of the solvent, the ethereal extract gave a red

solid which was repeatedly extracted with boiling petroleum

(40-60°) letting the extract stand for 2-3 hours it

deposits crystals of catechol. The supernatant petroleum

extract was removed,^concentrated and distilled. Two fractions

were collected (1) b.p.l0$-110®/l “m. 2 .1 gm. (25% yield)

1.5368 and (2) b.p.l60-70®A mm. n^^ 1.4680. The

first fraction gave a 2 ,4 dinitrophenyl hydrazone m.p.l89®.

All attempts to reduce the carbonyl group without

affecting the adjacent double bond to give the 4-allyl catechol

failed.

XXI. Dftmethylation of eugenol and isoeugenol:

(1) Eugenol 10 cc. was dissolved in 100 cc. petrol

ether ( b.p*100-120°) and 15 gme. of anhydrous aluminium

chloride powder added. It was then refluxed vigorously for

3 hour&, cooled to room temperature, ice amd d il . HCl. added.

•The aqueous part was extracted with ether. The ethereal

extract and the petrol ether solution were mixed, washed and

conc«ntrated to give 8 .2 g. of a viscous liquid. This on

distillation under vacuum gave a solid fraction which sublimed anM

collected in the cooler parts of the apparatus and a

second fraction distilling over at 108-115°/0.3-0.5AM.i

27 5®an orange coloured liquid, n ^ 1 .5300 . The solids obtained

^ 62

on analysis gave C, H, $.33% . Required for

catechol C, 65 .44 , H, 6 .49^ .

(2) Pyridine hydrochloride and eugenol were heated

with vigorous stirring in an oil bath at 150° for 2 hours

and then 100° for 1 hour. The mass was cooled and extracted

with ether. The ethereal solution washed with water and 105&

NaOH solution, the alkaline solution acidified and again

extracted with ether. This ethereal extract was washed,

dried and concentrated (S,3 g.) . On distillation it gave

a viscous liquid 3.S gms. b,p.96- 105°/l mm. methoxylcontent

found 16 .9^ required for eugenol I S .9^. The benzoyl

derivative of the distillate after two crystallisationo r

melted at 69-70 C, the benzoyl derivative of eugenol also

melts at 70°.

(3) Isoeugenol 10 c c ., ether (dry) 150 cc. and 17 gm.

of anhyd. AlCl^ were heated t i l l ether distilled over and then

temperature raised to 120® and maintained for 3 hours. The

product was then woriced up and distilled at 1-2 moi.

pressure. The distillate was a dark brown viscous liquid.

Analysis Found, C, 73.22 and H, ?.31%» Isoeugenol, ^]_o^i2^2

requires C, 73 J4; and H, 7.37f‘>. Hence it is just

polymerised isoeugenol.

X X II. Dihydroeujgenol;

Platinum catalyst was prepared from platinum

chloride. 5 gm. of eugenol in 200 cc. of ailcohol was

hydrogenated in the presence of 0 .06 gm. of Pt-catalyst, at

room temperature. It ..absorbed 765 cc. of hydrogen (750 cc.

H3

being the theoretical) • The solution was charcoaled,

filtered, the filterate concentrated and the residue

distilled. The dihydro-eugenol is a colourless liquid,

boiling point 236-3S°/710 mm. Analysis Found, C, 7 2 .0 , H, S .7^

^10^14*^2 72 .26 , H, S .49^,

X X III , t-Butylation of curcumini

Curcumin (0 .26 gm.) was dissolved in dry tert.

butyl-alcohol (30 cc.) containing 15 cc. of BF^-ethereate.

The colour changed immediately from orange to red. The

flask was left at room temperature for 2 hours and then

refluxed for 12 hours on a xater-bath. A saturated solution of

calcium chloride (200 cc.) was added to the flask sind heated

on a water-bath for half an hour, cooled and filtered.

The filtrate which was a clear red solution was diluted with

water and extracted with ether. The ethereal extracts were

washed with water till free of acid, dried and concentrated.

The residue was crystallised from benzene when dark red

crystals were obtained. It was soluble in ether, alcohol and

benzene giving a reddish solution with green fluorescence.

It gives a deep blue green colour with aq, NagCO^ solution.

It decomposes around 275°C, On ignition it leaves considerable

amount of white residue. Possibly it is the boron complex of

curcumin. Even on boiling the crystals with methanolic HCl

for 4 hours the boron complex could not be hydrolysed,

XXIV. Esters of protocatechulc acid:

(a) 3.16 g, ( .02 moles) of protocatechuic acid

6 1

( 3,4-dihydroxy-benzoic acid) 30 cc, propyl alcohol and 1 cc,

HgSO^ were refluxed for B hours. The mineral acid was

neutralised with potassium bicarbonate, 300 cc. water added

and extracted with 2 x 100 cc. ether. The ethereal solution

was washed and dried, and solvent recovered, last traces

under vacuum. The residue was crystallised from hot benzene.

Light yellow silky needles, on re crystallisation melted

at 117°C. yield 2.75 gm. (63 .0 ^ of theoretical). Its

alcoholic solution gives green colour with FeCl^ solution.

Analysis: Found G, 61 .42 , H, 5.8?b propyl protocatachuate,

^10^12^4 9» 61 .21 ; H, 6.17% .

(b) Similarly using allyl alcohol and protocatechuic

acid, allyl protocatechuate (m .p.100-101°) was obtained.

Found, C, 61 .82 , H, 5.43%, ^io^io°4 requires C, 6 1 .S6, H, 5.15^o.

XXV. Zi.-Dodecvl catechol;

7 gms. catechol, 10 gras, lauric acid, 30 gms. of

freshly fused and powdered zinc chloride and 30 cc. of freshly

distilled POCl^ were mixed and heated at 70® for one and

half hours with mechanical stirring. The reaction mass was

then poured over ice and extracted with 3 x 100 cc. ether.

The ethereal extracts were combined, washed dried over NagSO^

filtered and'concentrated. The residue was crystallised

from pet. ether (60-30°C) when 11,36 g . of glistening

plates - light pink in colour m .p .105-06®were obtained

(83.2% of theoretical). Mixed melting point with catechol

S7-91oC, Alcoholic solution + FeCl^-green colour,changing

to blue on dilution’ with water. Found, C, 73.82, H, 9.6%*

Lauroyl catechol requires C, 73 .93, H, 9.65%.

0.05 gm. of the sample was converted to the 2 ,4-ciinitrophenyl

hydrazone. Bright red needles from alcohol m .p,216°C.

Found, C, 61 .35 , H, 6 .9 5 , N, 11.5%. requires

C, 61 .00 , H, 6 .83 , N, 11.86%.

The 3auroyl catechol was reduced to dodecyl

catechol by the Clemmensen's m ethod.^ 2inc amalgam

(obtained from 33 gm. of 2n wooD.HCl 50 cc ., lauroyl

catechol 10 gm. and xylene 40 cc. were taken in a flask and the

temperature slowly raised to 130-140°. After 3 hours a slow

stream of HCl gas was let in the flask. After a period of 8

hours, a fresh lot of zinc amalgam (from 15 gm. zn) added and

reaction continued for 9 more hours. The reaction mass was

transferred to a separatory funnel the aqueous part removed and

the xylene solution washed three times with water, dried and

concentrated. The residue was distilled under vacuum. A

colourless distillate (8 .0 gms. 84.29% jleid) was obtained which

on crystallisation from ether-petrol-ether melted at 81-2°C.

Found C, 77.86, H, 10.80%. ^13^30^2 " catechol

requires C, 77.65, H, 10.96%.

XXVI 4-Propyl catechol;

Catechol 7 gms propionic acid 4 gms., freshly fused

zinc chloride 30 gms.^and freshly distilled POCl^ 30 cc.

mixed together and stirred at room temperature for 4 hours and

then at 80°C. for half an hour. It was then allowed to stand

overnight. Next day, the product was poured in ice and worked

up as in last experiment. The crude product (7.25 gm; 69.21%

yield) obtained was crystallised from boiling benaene and then

from ether-petrol ether. Colourless needles m .p .145-6°.

7 gms. of propionyl catechol was reduced to propyl catechol

using the Clenunensen*s method as in the last experiment.

Tne product was worked up in the usual way, and distilled.

A colourless liquid b .p .136^3 mm, which solidifies on

standing was obtained. It was crystallised from ether-petrol

ether m.p,60°C (reported in literature m .p .60°,

b .p.l75-S0°/30 mm.). Found C, 70 .9 ; H, 8.2C^.

Propyl catechol requires C, 71 .02 ; and H, 7.95%*

XXVII Antioxidant activities of synthetic compounds.

Pyrogallol, propyIgallate, catechol, protocatechuic

acid, propyl protocatechuate, allyl protocatechuate,

4-propionyl catechol, 4-lauroyl catechol, 4-propyl catechol,

4-dodecyl catechol, dihydroeugenol were all tested for their

antioxidant activity at .05%. conc. on refined lard by the

A .O.M . test. The results are presented in Table X I .

evy

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