Ethnobotanical Leaflets 13: 791-800, 2009.
Role of Phenolics in Anti-Atherosclerotic Property of
Thuja occidentalis Linn.
S. K. Dubey* and A. Batra1
* Dept. of Pharmacognosy,
Corresponding author: E-mail : email@example.com
The present study was carried out to evaluate the Lipid peroxidation activity and related hypolipidaemic
activity of an (EFTO) ethanol fraction of extract of aerial part of Thuja occidentalis Linn. (Cupressaceae). Lipid peroxidation
activity was carried out to evaluate the antioxidant potential, and hypolipidaemic activity on cholesterol fed rats. The
antioxidant activity of ethanol fraction was increased in a concentration
dependent manner. About 100, 150, 200, 250 & 300 µg EFTO (ethanol fraction of extract
of aerial part of Thuja occidentalis) inhibited the FeSO4
induced lipid peroxidation in a dose dependent
manner and showed IC50 value 195.60µg/ml. in hypolipidaemic
activity EFTO at the dose of 200 mg and 400mg/kg body weight significantly
reduced serum cholesterol (77 and 92%),
Keywords: Thuja occidentalis; Antioxidant; hypolipidaemic activity.
Thuja occidentalis, commonly known as Arbor vitae or white
cedar, is indigenous to
Materials and Methods
Fresh aerial part (twigs) of Thuja occidentalis
were collected from Jaipur,
Preparation of extract
Shade dried and powdered twigs (40-mesh size, 1kg) were soxhlet extracted with 90% EtOH (Dept. of Botany), the solvent was removed and the residue was triturated with hot (650C) petroleum ether (60-800C). Solvent was evaporated from the petroleum ether soluble portion and the residue dissolved in ethanol. On removal of the ethanol by evaporation, a semi solid reddish brown mass (12.76g) was obtained. Phytochemical investigations showed the presence of flavonoids (quercetin, kaempherol), tannic acids, polysaccharides and proteins.
albino rats of either sex, weighing 175-220 g, provided by the Sanjeevan
acid was obtained from
Determination of Anti oxidant activity
Assay of lipid per oxidation
The extent of lipid peroxidation in goat liver homogenate was measured in vitro in terms of formation of thiobarbituric acid reactive substances (TBARS) by using standard method (5) with minor modifications (6) with the help of spectrophotometer (Shimadzu model 1601).
Goat liver was purchased from local slutter house. Its lobes were dried between blotting papers (to remove excess blood) and were cut into small pieces with a heavy-duty blade. They were then homogenized in glass-Teflon homogenizing tubes in cold phosphate buffer saline (pH 7.4). It was centrifuged at 2000rpm for 10 min, and supernatant was diluted with phosphate buffer saline up to final concentration of protein 0.8-1.5 mg/0.1ml. protein concentration was measured by using standard method of Lowery et.al. (7) . To study the comparative response, the experiments was divided into nine groups. Liver homogenates (5%, 3ml) was aliquoted to nine different 35mm glass Petri dishes. The first two groups were treated as control and standard where buffer and Vit. E were added. In the third to seventh group, different concentrations of EFTO were added. Lipid peroxidation was initiated by adding 100µl of 15mM ferrous sulphate solution to 3 ml of liver homogenate (8). After 30 min, 100µl of this reaction mixture was taken in a tube containing 1.5ml of 10% trichloro acetic acid. After 10 min, tubes were centrifuged and supernatant was separated and mixed with 1.5ml of 0.67% thio barbituric acid. The mixture was heated in a water bath at 850C for 30 min, and in boiling water bath to complete the reaction. The intensity of pink colored complex formed was measured at 535 nm.
The percentage of inhibition of lipid peroxidation was calculated by compring the results of the test with those of controls as per the following formula i.e. Eq. (1);
Inhibition(%) = (Control Absorbance- Test Absorbance) X 100/Control
Determination of total phenolic compound
Total soluble phenolic compound in the EFTO were determined with the Folin-Ciocalteu reagent according to the method of Slinkard and Singleton (9). A 0.1 ml aliquot of a suspension of 1mg of EFTO in water was totally transferred to a 100ml Erlenmeyer flask and the final volume was adjusted to 46 ml by the addition of distilled water. Next 1 ml Folin – Ciocalteu reagent was added to this mixture, followed by 3 ml 2% Na2CO3 3 min latter. Subsequently, the mixture was shaken for 2h at room temperature and absorbance was measured at 760 nm. All tests were performed in triplicates. The concentration of total phenolic compounds in EFTO was determined as µg pyrocatechol equivalents using the following equation obtained from a standard pyrocatechol graph;
Determination of hypolipidaemic activity
Acute toxicity was determined for EFTO and minimum and maximum cut off dose, which found, is respectively 200 and 400 mg /kg body weight (10).
Twenty four wistar rats were divided in following 04 groups of 06each.
Group I (-ve Control):
Group II (+veControl): High Fat Diet (
The compositions of the two diets were as follows:
Control Diet (
Wheat flour 100g
Hydrogenated vegetable oil 5ml
Salt mixture(NaCl, KCl, CaCl2) 1.5g
Citric acid 0.5ml
Vitamin B complex composition
High fat Diet
Wheat flour 100g
Hydrogenated vegetable oil 10ml
Salt mixture (NaCl, KCl, CaCl2): 1.5g
Cholesterol (dried egg yolk) 0.5g
Citric acid 0.5ml
Vitamin B complex composition
Group I served as normal control and was given normal saline only along
with normal diet. Group II-IV received high fat diet plus cholesterol for
induction of hyperlipidaemia. In, addition to this,
Body weight of each animal was registered at the beginning and at the
end of the experiment. During the whole period, the animals have free access
to food and water. Twenty hours prior the end of the experiment, food was
withdrawn and blood samples were taken by retro orbital puncture. The blood
samples were centrifuged for 2 min at 16 000 g. Serum Total
These parameters were estimated by using Span Diagnostic & Erba Diagnostic Kits.
index = (
All data are expressed as mean ±
Assay of lipid peroxidation
The results presented in Table-1 showed that the ethanol fraction of the Thuja occidentalis inhibited FeSO4 induced lipid peroxidation in a dose dependent manner. The extract at 300µg/ml exhibited maximum inhibition (61.516±0.131 %) of lipid peroxidation nearly equal to the inhibition produced by Vit.E. The IC50 value was found to be 195.60µg/ml.The inhibition could be caused by the absence of ferryl-perferryl complex or by changing the ratio of Fe3+/Fe2+ or by reducing the rate of conversion of ferrous to ferric or by changing the iron itself or combination thereof (12).
Table 1. Effect of Ethanol Fraction of Thuja occidentalis (EFTO) on Lipid peroxidation.
Values are mean ±
Amount of total phenolic compound
Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl groups. In the EFTO (1.0 mg) 123µg/ml, pyrocatechol equivalents to phenols were detected. The phenolic compound may contribute directly to the anti oxidative action (13). The results indicate a strong association between antioxidant activities and phenolic compounds are probably responsible for the ant oxidative property of T. occidentalis. Phenolic compounds are also effective hydrogen donors, which makes them good antioxidants (14). Similarly, Shahidi and Naczk (15) reported that naturally occurring phenolic compound exhibit antioxidant activities. Thus, the therapeutic property of T. occidentalis may be possibly attributed to the phenolic compounds present.
A significant (p<0.05 and p<0.001) reduction in body weight of
rats was observed in EFTO treated Groups
A 65% increase in serum total cholesterol
(TC) was noticed in rats fed high fat diet plus cholesterol (Group II) in
compared to rats fed normal diet (group I). Administration of EFTO lowered
the serum total cholesterol by 77 to 92% in Group
Table 2. Body weight of high fat diet / Thuja occidentalis fraction treated rats.
(Values are Mean ± SE from 06 animals in each groups).
A significant reduction in
Table 3. Lipid profile of high fat diet /Thuja occidentalis fraction treated rats
Values are Mean ± SE from 06 animals in each group.
Lipids are widely
involved in oxidation reactions and these reactions, can be induced by so
called Reactive Oxygen Species (
A rise in
In the present study, ethanolic extract of Thuja occidentalis resulted in significant
reduction in total cholesterol and
A significant fall in
The decrease in serum TG level and reduction in atherogenic index in plant extract treated groups is an important finding of this experiment. Most of the hypolipidaemic drugs do not decrease serum triglycerides level but Thuja occidentalis extract showed it significantly. Reverse back of atherogenic index provides strong additional benefits in the prevention and treatment of atherosclerosis.
It can be concluded from the present study that the potent free radicle scavenging effect of Thuja occidentalis extract interfere in adsorption, degradation and excretion of cholesterol. However, this possibility remains to be investigated in detail.
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