Free Radical Scavenging Effect Of Phyllanthus Simplex: In Vitro and In Vivo Study




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Free Radical Scavenging Effect Of

Phyllanthus Simplex: In Vitro and In Vivo Study

Shikha Kumara, Nikhil Sachdevac, Mohmad Amira,

Amit Kumarb and Sushil K. Singhc*





تبين أن الخلاصة الكحولية لنبات Phyllanthus simplex (Euphorbiaceae) تحتوي على مركبات فينولية حرة ومترافقة وتم لذلك اختبارها لمعرفة فعاليتها المضادة للأكسدة باستخدام نماذج معملية وأحيائية. وقد أظهرت الخلاصة درجة عالية من التثبيط لفوق أكسدة شحوم الدماغ إلى نسبة 92.01 ± 0.002 % معملياً. كما أظهرت الخلاصة تأثيراً مثبطاً معتمداً على الجرعة لفوق أكسدة الشحوم إلى مدى تركيز 18.7 مكغ/مغ من أنسجة الدماغ. وأظهرت الدراسات الأحيائية باستخدام الجرذان المهقاء أن خلاصة النبات (100 مغ/كغ، فموياً) صدت معنوياً الزيادة في مالونالدهيد المستحثة بمادة رابع كلوريد الكربون (0.5 مل/كغ، فموياً). وقد تبين أن النشاط المضاد للأكسدة للنبات مساوياً لذلك الناتج عن فيتامين هـ (50 مغ/كغ، فموياً) والمستخدم كمادة قياسية.




a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110 062. INDIA. b Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi 110 016.INDIA. c Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi 221 005. INDIA.
*To whom correspondence should be addressed.

E-mail: s_ksingh2000@yahoo.com






The ethanolic extract of Phyllanthus simplex (Euphorbiaceae) was found to contain free and conjugated phenolic compounds and was therefore screened for anti-oxidant activity using in vitro and in vivo experimental models. The extract exhibited a high degree of inhibition on rat brain lipid peroxidation up to 92.01 0.002% by in vitro method. P. simplex extract also exhibited dose dependent inhibitory effects on lipid peroxidation up to the concentration of 18.7 µg/ml of brain tissue. In vivo studies using albino rats showed that the plant extract (100mg extract/kg, p.o.) significantly antagonized CCl4 (0.5ml/kg, p.o.) induced increase in malondialdehyde. The in vivo anti-oxidant activity of P. simplex was found to be comparable to that of Vitamin-E (50mg/kg, p.o.), used as standard.




Keywords: Phyllanthus simplex; Anti-oxidant activity; Lipid peroxidation.


Introduction



The genus Phyllanthus belongs to the family Euphorbiaceae, one of the largest families consisting
of 300 genera and 6000 species (1). Phyllanthus
simplex Retz, commonly known as 'Bhuiaveli in Marathi and ‘Uchchiyusirika’ in Telgu, is a glabrous

perennial herb, having long tap root and compressed branches. Its flowers are solitary and its leaves are distichous. It is widely distributed shrub in tropical and sub-tropical regions (2). Different parts of the plant are used to cure gonorrhoea, jaundice, liver disease and mammary abscess (3). Fresh leaves are used to cure itching in children, and pruritis (4). The plant is also reported to possess astringent, diuretic and cathartic properties (5).

During the course of normal metabolism, re-active oxygen species (ROS) and free radicals are produced, which can induce oxidative damage to any biomolecular component of the cell they encou-nter but lipid, proteins and nucleic acids are par-ticularly important targets. These free radicals, being
the potent initiators of lipid peroxidation in the biological membranes, cause alteration in fluidity, fall in membrane potential, increase in permeability of H + and other ions and eventual rupture leading to release of cell and organelle contents, such as lysosomal hydrolytic enzymes (6). Some of the toxic end products, of metal ion dependent lipid peroxide fragmentation, are malondialdehyde, 4, 5-dihydroxy decenal and 4-hydroxynonenal (7). Lipid peroxides and / or cytotoxic aldehydes derived from them can block macrophage action, inhibit protein synthesis, kill bacteria, inactivate enzymes, cross link proteins, generate thrombin and act as chemotoxins for phagocytes (8). They are responsible for the pathogenesis of a number of diseases such as atherosclerosis, aging, cancer, inflammatory diseases and a variety of other disorders (9-11).

The natural anti-oxidants occur in a variety of fruits, vegetables, leaves and flowers. These include phenolic compounds, flavonoids, terpenoids, hexa-peptides, lignans, essential oils, tocopherols, ascor-bic acid, coumarin, lactones, indoles, plant sterols, carotenoids etc. The antioxidants with free radical scavenging activities could have great importance as prophylactic and therapeutic agents in diseases in which free radicals or oxidants are implicated (12)

The ethanolic extract of P. simplex is rich in free and conjugated phenolics. Therefore, it was analyzed for anti-oxidant activity on the rat brain tissue.
Material and methods
Plant material:

The fresh aerial parts of P.simplex Retz. (Euphorbiaceae) were collected from Banaras Hindu University (BHU) campus and authenticated by Dr. N.K. Dubey (Department of Botany, BHU). An air-dried voucher specimen is deposited in Department of Pharmaceutics, BHU (Specimen No. PCRL 34) for future reference.

The plant material (4kg) was dried, crushed to powder and then successively extracted to exhaust-tion with petroleum ether (60-80oC), acetone and ethanol using cold percolation method. The different extracts were dried under reduced pressure to get the crude extract concentrates (5 g), (5 g) and (4 g) respectively and were analyzed for the presence of phenolic compounds. The presence of phenolic compounds was confirmed by spraying 1% ferric chloride on TLC (benzene: ethylacetate:: 80:20). The ethanolic extract rich in phenolics was screened for the free radical scavenging/anti-oxidant activity in in-vivo and in-vitro models.
Animals:

The study was carried out on male Wistar albino rats (150-200g). The animals were fed with a standard pellet diet (Gold Mohar, Lipton India, Kolkata) and water ad libitum. The rats were kept in standard environmental conditions (temperature 25-28oC) and 12-hour light/dark cycle. Six animals in each group were used in all sets of experiments.



LPO inhibition effect

Preparation of rat brain homogenate:

The rat brains were promptly excised after decapitation. It is then washed with ice-cold KCl solution (1.15% w/v), blotted with filter paper and weighed (750 mg). The 10% tissue homogenate was prepared in ice-cold 1.15% w/v KCl solution using a glass homoginizer equipped with a Teflon pestle. The homogenate was centrifuged at 1500 rpm for 10 minutes to remove nuclear fractions. The supernat-ant was used for the estimation of lipid peroxide level.

The effect of various doses of ethanolic extract (0.1-1 ml, 1000g/ml) on ferric ion / ascorbic acid induced peroxidation of rat brain phospholipids (homogenate) was determined by thiobarbituric acid (TBA) assay. The assay is based on the reaction of TBA with malondialdehyde (MDA), one of the products of lipid peroxidation, where TBA reacts with MDA with/without test samples and the amount of colored MDA-TBA adduct formed on heating under acidic condition was measured at  532 nm (13). MDA is produced, along with other dialdehyde products, from fatty acids containing at least two, or three or more double bonds. A double bond, to the carbon bearing a peroxy function, enables the formation of closed five-membered ring structure prior to decomposition, releasing one molecule of MDA (14).

The rat brain homogenate (0.5 ml) was mixed with sodium lauryl sulphate (8.1%, 0.25 ml). Acetate buffer (pH 3.5, 1.5 ml) and TBA solution (0.8%, pH 7.4, 0.5 ml) were added in succession. Final volume was adjusted to 10 ml with distilled water and the mixture was heated at 85oC for 30 minutes. The products were shaken vigorously with n-butanol: pyridine (15:1, v/v, 5 ml) and centrifuged (1500 rpm) for 10 minutes. The organic layer was taken and absorbance at  532 nm was measured.

An in vitro model was initially standardized to assay the lipid peroxidation reaction. To determine optimal concentration of rat brain homogenate for LPO estimation, increasing volume (0.1 to 1 ml, 75mg/ml) of the rat brain homogenate was mixed with sodium lauryl sulphate (8.1%, 0.25 ml), FeCl3 (0.25 mM, 0.25ml) and ascorbic acid (1 mM, 0.25ml) and incubated at 37oC for 30 minutes. Thereafter, acetate buffer (pH 3.5, 1.5 ml) and TBA solution (0.8%, pH 7.4, 0.5 ml) were added. The volume was adjusted to 10 ml with distilled water and the mixture was heated at 85oC for 30 minutes. The extent of MDA-TBA adduct produced (absor-bance at  532 nm) was plotted against different volumes of rat brain homogenate (Fig. 1). From this experiment the 37.5 mg/ml concentration (corresponding to 0.5 ml volume of the rat brain homogenate) was selected from the straight-line region for the test experiments. The LPO was then determined as described below. The ability of extract to inhibit MDA-TBA adduct formation was calculated using the following equation.


% Inhibition = AUC control – AUC test X 100



AUC control
AUC is area (mm2) under the curve, calculated from the plotting of volume (ml) of rat brain homogenate on X-axis against the absorbance on Y-axis. Where, AUC test and AUC control was calculated with and without adding the plant extract to the homogenate.
In-vivo studies:

For the in vivo studies, the rats were divided into four groups, each group containing 6 animals. Group A received only vehicle (1% polyethylene glycol) in the dose of 2 ml/kg (p.o.) for seven consecutive days. Group B served as an intoxicated control and given CCl4 (toxin) on the fifth day in the dose 0.5 ml/kg (p.o.). Group C was given vitamin E, which was taken as standard, for seven consecutive days in a dose of 50 mg/kg (p.o.). Group D was given ethanolic extract of P. simplex for seven consecutive days in a dose of 100 mg/kg (p.o.). Groups C and D were given toxin (CCl4) on the fifth day in the dose 0.5 ml/kg (p.o.) in addition to vitamin E and the extract, respectively. All the animals were sacrificed on the seventh day i.e. 48 hours after CCl4 challenge, and the brain was immediately harvested for LPO estimation.

The results of the biochemical estimations were reported as mean  S.E.M. The total variation present in a set of data was estimated by ANOVA.

Results and Discussion

The amount of malondialdehyde formed was found to be directly proportional to the concentration of rat brain homogenate. The linearity between the amount of malondialdehyde produced and the con-centration of rat brain homogenate is shown in Fig.1.





Figure 1. The effect of rat brain homogenate content on lipid peroxidation as measured by formation of MDA-TBA adduct.
Table 1: Effect of increasing doses of ethanolic extract of P. simplex on lipid peroxidation inhibition (in vitro).


Dose of ethanolic extract of P. simplex

(in µg/mg of the brain tissue)

% Inhibition of lipid peroxidation

0

0

2.67

9.2  0.005

5.33

11.12  0.008

8.0

23.42  0.003

10.67

44.01  0.006

13.33

63.55  0.006

16.0

79.91  0.006

18.67

87.51  0.009

21.33

92.01  0.011

24.0

92.01  0.007

26.67

92.01  0.002



The data is presented as mean  S.E.M. (n=6)




The ethanolic extract of P. simplex exhibited a high degree of inhibition on the rat brain phospho-lipid peroxidation. From the data (Table 1) it was found that extract of P. simplex gave protection against peroxidation up to 92.01  0.01%. The inhibition was found to be directly dependent on extract dose up to the dose of 18.67 µg/mg of the brain tissue. Above this, a flat region indicates that the inhibitory effect of P. simplex attained a limiting value. The IC50 (concentration for 50% inhibition) of P. simplex extract on the peroxidation inhibition was calculated from graph of % inhibition of lipid peroxidation versus log extract dose (Fig. 2) and was found to be 11.4 µg/mg of brain tissue.


Figure 2. Effect of ethanolic extract of P. simplex on inhibition of lipid peroxidation (in vitro) using 0.5 ml rat brain homogenate.
The results of in vivo study are given in Table 2.

Table 2: Effect of ethanolic extract of P. simplex on lipid peroxidation inhibition (in vivo) and its comparison with Vitamin E against vehicle control and intoxicated control.



Groups
Treatment

Dose

Lipid Peroxide Level (nmol MDA in 100mg brain tissue)

A

Vehicle

2 ml/kg

957.7  2.605

B

CCl4

0.5 ml/kg

2159.14  1.01*

C

Vitamin E+ CCl4

50 mg/kg

1222.68  2.50**

D

Ethanol extract of P. simplex + CCl4

100 mg/kg

1184.81  3.60**,1



Values are expressed as mean  S.E.M. (n=6),

*P<0.001 versus Group A

**P<0.001 versus Group B

1P< 0.05 versus Group C

The normal malondialdehyde (MDA) content, as a measure of lipid peroxidation, in vehicle control rat brain tissue was found to be 957.7 2.605 nmol / 100mg brain tissue. CCl4 administration (0.5 ml/kg, p.o.) produced marked increase in MDA content which was found to be 2159.14 1.01 nmol / 100mg brain tissue and indicated as statistically significant (P<0.05) from the vehicle control. Vitamin E (stand-ard, in a dose of 50 mg/kg, p.o.) showed significant protection (P<0.05) against CCl4 induced augment-ation of MDA. The ethanolic extract of P. simplex, in a dose of 100 mg/kg, p.o., also provided statistically significant (P<0.05) protection against CCl4 induced augmentation of MDA. Moreover, ethanolic extract in a dose of 100 mg/kg, p.o. was significantly better than the vitamin E in a dose of 50 mg/kg, p.o. (P<0.05).

Both results revealed that the ethanolic extract of P. simplex has the capacity to prevent oxidative deterioration of polyunsaturated lipids. The free radical scavenging activity may be due to free and conjugated phenolic compounds viz. rutin, gallic acid, phyllanthin, simplexine (2,15) etc present in the extract. Further, the hepatoprotective activity of the plant and the allied species [such as P.niruri (16)] may be mainly attributed to antioxidant pro-perties of their polyphenolic phytoconstituents as the role of lipid peroxidation induced damage in the pathogenesis of chronic liver diseases has been suggested based on enzymatic studies (17).

Conclusion

The ethanolic extract of P. simplex (rich in phenolics) significantly reduced the malondialde-hyde content, which is a measure of lipid peroxidation and showed anti-oxidant activity in both in-vitro and in-vivo experiments. The results obtained for the extract showed better protection against CCl4 induced peroxidation than Vitamin E, used as the standard. From the above observations it is concluded that the plant possesses active constituents that can be of pharmacological interest. Further investigations are being carried out in the same direction.


Acknowledgement
SK and NS are thankful to U.G.C, New Delhi for the award of research fellowship.

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Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007





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