Evaluation of In Vitro Antioxidant Activity of stem-bark and stem-wood of Premna serratifolia Lin., (Verbenaceae)

Rekha Rajendran*, N Saleem Basha and S Ruby

Mohamed Sathak A. J. College of Pharmacy, Medavakkam, Chennai - 600 119, India.

ABSTRACT

Premna serratifolia Lin., is widely used in Ayurvedic system medicine for the treatment of cardiovascular disorders, arthritis, inflammation etc. The stem-bark and stem-wood were extracted with 95% ethanol and double distilled water and these extracts were screened for their in-vitro antioxidant potential. Inhibition of oxygen-derived free radicals, viz., assays for free radical scavenging by DPPH, reducing power ability and nitric oxide scavenging were performed. All the antioxidant activities were compared with standard antioxidant such as ascorbic acid. Both the extracts of this plant showed effective free radical scavenging activity, reducing power and nitric oxide scavenging activity. All these antioxidant properties were concentration dependent. The highest antioxidant activity was observed with ethanol extracts. Preliminary phytochemical screening revealed the presence of flavonoids, steroids, alkaloids, glycosides and phenolic compounds in the extracts and the results obtained from the current study indicate that Premna serratifolia Lin., is a potential source of natural antioxidants and the extracts have constituents which were capable of showing anti-oxidant activity and the said in-vitro anti-oxidant activity may also be due to the presence of anti-oxidant principles present in the extracts like flavonoids and phenolic compounds. These findings confirm the great interest of the Premna serratifolia whose phytochemistry and phytopharmacology should be investigated further in order to detect possible phytotherapeutic uses in the prevention of ageing related diseases, cardiovascular disorders and Alzheimer disease.

Keywords: Premna serratifolia Lin., DPPH radical, reducing power determination, scavenging of nitric oxide.

INTRODUCTION

Antioxidants are important for food stability, human health and nutrition. They play a major role in the genesis of various diseases such as atherosclerosis, cancer, ageing, rheumatoid arthritis and inflammation. Plants provide a rich source of antioxidants which includes tocopherols, vitamin C, carotenoids and phenolic compounds. There are several other antioxidants namely olive oil phenolics, tocotrienols, oryzanol, squalene, sesame lignans, pycnogenol, flavonoids, isoflavones, resverator, alpha lipoic acid etc. which have been shown to possess health benefits. However, BHA (tert-butyl-4-hydroxy-anisol) and BHT (tert-butyl-4-hydroxy-toluene) are most commonly used synthetic antioxidants but both are suspected to cause liver damage. Therefore, it is important to search for the safe and effective natural antioxidants by using various screening methods.

Many medicinal plants have become major candidates of research for identifying their chemical constituents rich in antioxidant potential capable of protecting cellular damage induced especially due to free radical formation and reactive oxygen species (ROS). Total anti-oxidants assays are used to compare the antioxidant activities of different molecules which used absorption spectroscopy. Many such assays have been employed for antioxidant characterization. The activity of anti-oxidants can be followed by the loss of absorbance at a specified wavelength. Antioxidants are the compounds that inhibits or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reaction.

Premna serratifolia Lin., is one of the most widespread in the forests of India, usually occurring in deciduous forests. The whole plant possesses medicinal properties useful in the treatment of cardiovascular disease, skin diseases, inflammatory diseases, rheumatism, anorexia and jaundice. It’s an important ayurvedic medicinal herb and its synonym is Premna integrifolia Lin., It is popularly known as “Munney” in Tamil, “Agnimantha” in Ayurvedic system of medicine. Root forms an ingredient in well known Ayurvedic formulation “Dasamula” for variety of affections¹. Phenolic substances from the root-bark showed anti-bacterial activity against Staphylococcus aureus, Bacillus subtilis and Streptococcus haemolytics ². Decoction of the pant, exhibited marked anti-inflammatory and anti-arthritic activity against acute, sub-acute and chronic inflammation induced in both immunological and non-immunological experimental models ³.

Ethanolic (50% v/v) root extract, exhibited anti-hyperglycaemic activity and the extract was found to produce marked reduction in blood glucose concentration and justified the use of the roots of the plant for treating diabetes as suggested in folklore remedies ⁴ and this plant also possess anti-parasitic activity ⁵.

From the leaves of Premna corymbosa var. obtusifolia, Premcoryoside, a Verbascoside iridoid glycoside conjugate, was isolated along with Verbascoside and three monoacyl 6-O-alpha-L-rhamnopyranosylcatalpols. Their structures were determined mainly by NMR spectroscopy ⁶. the major compound present in this plant is flavonoid and alkaloid ⁷. The volatile constituents of the flower buds of Premna serratifolia were isolated by vacuum distillation from a hexane concrete and the major components are 1-octen-3-ol (16.9%), (Z)-3-hexenol (10.2%), 2-phenylethyl alcohol (8.9%), (E,Z)-2,4-nonadienal (6.2%), (E,Z)-2,6-nonadienal (5.0%) and linalool (4.4%) ⁸.

However, a detailed pharmacological screening of the Premna serratifolia Lin., stem-bark and stem-wood extracts have not been reported. Literature survey revealed that there was no work has been done on the antioxidant activity of Premna serratifolia Lin., against radical scavenging. This plant has been traditionally used in the treatment of cardiovascular disease, arthritis and inflammation. This aroused the curiosity to screen for its in-vitro anti-oxidant activity. The present study was thought worthwhile to carry out the free radical scavenging activity of Premna serratifolia Lin., stem-bark and stem-wood ethanol and aqueous extracts.

MATERIALS AND METHODS:

Collection of Plant Material:

Fresh stem-bark and stem-wood of Premna serratifolia Lin., was collected from The Indian Medical Practitioners Co-operative Pharmacy and Stores garden, Chennai, Tamil Nadu. Plant material was identified⁹and authenticated (PARC / 2007 / 71) by a Botanist, Dr. P. Jayaraman, Plant Anatomical Research Centre, Chennai. Materials were cleaned with water and dried in the shade until a constant weight was obtained.

Preparation of Extracts:

The materials were extracted with 95% ethanol and double distilled water in a soxhlet extractor. Extracts were concentrated; the percentage yield for ethanol and aqueous extracts were 7.90% and 7.5%w/w and these extracts used for the assessment of in-vitro anti-oxidant screening and preliminary phytochemical screening.

Preliminary Phytochemical Screening:

The extracts were then subjected to preliminary phytochemical screening^10,11 to detect the presence of polyphenolic compounds. The qualitative chemical test performed were Shinado test, Ammonia fuming test, lead acetate test, boric acid test for flavonoids and ferric chloride test, nitric acid test, ammonia hydroxide-potassium ferricyanide test, lead acetate test for tannins. All the tests confirmed the presence of flavonoids and tannins in ethanol extract.

Chemicals and Instruments:

1,1-diphenyl-2-picrylhydrazyl (DPPH) (Aldrich), Naphthylethylenediamine dihydrochloride (Loba chemie), Thiobarbituric acid (TBA) and Trichloro acetic acid (Sd fine chemicals Ltd). All other reagents used were of analytical grade. UV spectra were recorded in Shimadzu 1601 UV-Visible spectrophotometer.

EVALUATION OF ANTIOXIDANT ACTIVITY:

Scavenging Of DPPH Radical:

This assay ¹²based on the measurement of the scavenging ability of anti-oxidant test extracts towards the stable radical. The free radical scavenging activity of the ethanol and aqueous extracts of stem-bark and stem-wood of Premna serratifolia Lin., were examined in-vitro using DPPH [1,1-Diphenyl, 2-picryl-hydrazyl] radical. The test extracts were treated with different concentrations from a maximum of 250µg/ml to minimum of 4µg/ml. The reaction mixture consisted of 1ml of 0.1mM DPPH in ethanol, 0.95ml of 0.05 M Tris-HCl buffer (pH - 7.4), 1ml of ethanol and 0.05ml of the herbal extract. The absorbance of the mixture was measured at 517nm exactly 30 sec after adding extracts. The experiment was performed in triplicate and percentage of scavenging activity was calculated using the formula 100 - [100/blank absorbance × sample absorbance]. The blank was also carried out in similar manner, using distilled water in the place of extracts. The activity was compared with ascorbic acid, which was used as a standard anti-oxidant.

Reducing Power Determination:

Different amounts of the extracts in ethanol and in aqueous solutions ¹³ were mixed with 2.5ml of (pH 6.6) 0.2M phosphate buffer and 2.5ml of 1%potassium ferricyanide [K₃Fe(CN)₆]. The mixture was incubated at 50˚C for 20 minutes. 2.5ml of 10% trichloroacetic acid was added to the mixture, which was then centrifuged for 10 minutes at 1000rpm. 2.5ml upper layer of solution was mixed with 2.5ml of distilled water and 0.5ml of 0.1% ferric chloride. The absorbance was measured at 700nm. The blank was also carried out in similar manner, using distilled water in the place of extracts. Increase in the absorbance of the reaction mixture indicated the increase in the reducing power. The activity was compared with ascorbic acid, which was used as a standard anti-oxidant.

Scavenging Of Nitric Oxide:

Sodium nitroprusside (5µM) in standard phosphate buffer solution ¹⁴was incubated with different concentration of the ethanol and aqueous extracts dissolved in standard 0.025M phosphate buffer (pH 7.4) and the tubes were

Table – 1 In-Vitro Free Radical Scavenging Effect of Premna serratifolia Lin., By DPPH Method.

	Percentage Scavenging (Mean ±SEM) of triplicates
	4 µg/ml	8 µg/ml	15 µg/ml	30 µg/ml	60 µg/ml	125 µg/ml	250 µg/ml	IC ₅₀ µg/ml
Ethanol extract	25.02 ±0.002	25.86 ±0.002	27.85 ±0.001	31.3 ±0.001	42.44 ±0.001	44.03 ±0.002	52.12 ±0.002	203
Aqueous extract	21.35 ± 0.002	22.54 ±0.001	23.34 ±0.001	30.77 ±0.001	37.40 ±0.001	45.22 ±0.002	50.13 ±0.002	213
	0.1µg/ml	0.2µg/ml	0.4µg/ml	0.6µg/ml	0.8µg/ml	1 µg/ml
Ascorbic acid	5.90 ±0.002	13.36 ±0.001	31.51 ±0.001	46.18 ±0.003	62.15 ±0.001	78.12 ±0.001		0.65

Table – 2 In-Vitro Free Radical Scavenging Effect Of Premna serratifolia Lin., By Reducing Power Determination.

	Absorbance (Mean±SEM) of triplicates
	4µg/ml	8µg/ml	15µg/ml	30 µg/ml	60µg/ml	125µg/ml	250µg/ml	500µg/ml
Ethanol extract	0. 088 ±0.002	0.162 ±0.002	0.272 ±0.001	0.388 ±0.001	0.434 ±0.001	0.596 ±0.002	0.705 ±0.002	0.898 ±0.002
Aqueous extract	0.054 ±0.002	0.117 ±0.001	0.156 ±0.001	0.232 ±0.001	0.300 ±0.001	0.372 ±0.002	0.425 ±0.002	0.511 ±0.002
	0.1µg/ml	0.2µg/ml	0.4µg/ml	0.6µg/ml	0.8µg/ml	1µg/ml
Ascorbic acid	0.056 ±0.002	0.180 ±0.001	0.242 ±0.001	0.492 ±0.003	0.805 ±0.001	0.968 ±0.001

Table – 3 In-Vitro Free Radical Scavenging Effect Of Premna serratifolia Lin., By Nitric Oxide Scavenging Method.

	Percentage Scavenging (Mean±SEM) of triplicates
	4µg/ml	8µg/ml	15µg/ml	30µg/ml	60µg/ml	125µg/ml	250µg/ml	IC₅₀ µg/ml
Ethanol extract	42.6 ± 0.002	42.71 ± 0.002	42.90 ± 0.001	43.19 ± 0.001	43.77 ± 0.001	46.28 ± 0.002	47.92 ± 0.002	325
Aqueous extract	1.13 ± 0.002	6.02 ± 0.001	6.49 ± 0.003	7.53 ± 0.003	9.03 ± 0.004	14.68 ± 0.001	22.22 ± 0.002	614
	0.1µg/ml	0.2µg/ml	0.4µg/ml	0.6µg/ml	0.8µg/ml	1µg/ml
Ascorbic acid	3.14 ± 0.001	13.54 ± 0.002	29.28 ± 0.001	40.33 ± 0.001	61.47 ± 0.004	75.23 ± 0.001		0.67

incubated at 25°C for 5 hours. After 5 hours, 0.5ml of incubation solution was removed and diluted with 0.5ml of Griess reagent (prepared by mixing equal volume of 1% sulphanilamide in 2% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in water). The absorbance of chromophore formed was read at 546nm.

RESULTS:

DPPH Scavenging:

The ethanol and aqueous extracts of stem-bark and stem-wood of Premna serratifolia Lin., showed promising free radical scavenging effect of DPPH in a concentration dependant manner up to a concentration of 250 µg/ml. Ethanol extract showed more scavenging activity than the aqueous extract. The reference standard ascorbic acid also showed a significant radical scavenging potential in the concentration of 1µg/ml. The DPPH radical inhibition of aqueous, ethanol and ascorbic acid was 50.13%, 52.12% and 78.12% respectively [Table - 1].

Reducing power determination:

As the concentration of the extracts increased, the absorbance was also increased correspondingly, indicating that both the ethanol and aqueous extracts have potent anti-oxidant activity by reducing power ability. Ethanol extract showed more reducing power ability than the aqueous extract, when compared with that of the standard anti-oxidant ascorbic acid [Table - 2].

Nitric oxide scavenging:

Ethanol extract of stem-bark and stem-wood of Premna serratifolia Lin., showed significant free radical scavenging action against nitric oxide (NO) induced release of free radicals at the concentration 250 µg/ml, the percentage of scavenging was found to be 47.92% than aqueous extract as 22.22% of NO inhibition, respectively. Ascorbic acid was used as the reference standard and its percentage of inhibition was 75.23% [Table - 3].

DISCUSSION:

In Indian system of medicine, certain herbs are claimed to cure various pathological conditions. The claimed therapeutic reputation has to be verified in a scientific manner. In the present study one such drug Premna serratifolia Lin., stem-bark and stem-wood was taken for the study. The extracts of stem-bark and stem-wood of Premna serratifolia Lin., possess significant anti-oxidant activity. Reactive Oxygen species (ROS) generated endogenously or exogenously are associated with the pathogenesis of various diseases such as atherosclerosis, diabetes, cancer, arthritis and aging process^15,16. Thus antioxidants which can scavenge ROS are expected to improve these disorders.

The free radical scavenging activity of the extracts was evaluated based on the ability to scavenge the synthetic DPPH. This assay provided useful information on the reactivity of the compounds with stable free radicals, because of the odd number of electrons. DPPH shows a strong absorption band at 517nm in visible spectrum (deep violet colour). As the electron became paired of in the presence of free radical scavenging, the absorption vanishes and the resulting discoloration stoichiometrically coincides with respect to the number of electrons taken up. The bleaching of DPPH absorption is representative of the capacity of the ethanol and aqueous extracts to scavenge free radicals independently.

The reducing properties are generally associated with the presence of reductones, which have been shown to exert anti-oxidant action by breaking the free radical chain by donating a hydrogen atom. Reductones are also reported to react with certain precursors of peroxide, thus preventing peroxide formation. Increase in the absorbance of the extracts indicated the anti-oxidant activity.

Nitric oxide is an essential gas required for several normal physiological processes like neural signal transmission, immune response and control of blood pressure. However the elevation of nitric oxide level was found in several pathological conditions including cardiovascular disease, diabetes etc. Sodium nitroprusside serves as a chief source of free radicals. The absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with napthylethylene diamine is used as the marker for nitric oxide scavenging activity¹⁷. The chromophore formation was not complete in the presence of extracts of stem-bark and stem-wood of Premna serratifolia Lin., (ethanol and aqueous extracts), which scavenges the NO thus formed from the sodium nitroprusside and hence the absorbance decreases as the concentration of the extracts increases in a dose dependent manner.

The preliminary phytochemical screening showed the presence of flavonoids and tannins in the ethanol and these flavonoids have been reported to possess various biological properties, related to anti-oxidant mechanism. The ethanol of stem-bark and stem-wood of Premna serratifolia Lin., have potent inhibition of DPPH radical scavenging, nitric oxide radical generation and reducing power ability than the aqueous extract. The ethanol extract have cardiotonic effect¹⁸ and the observed in-vitro anti-oxidant activity, further confirms the anti-oxidant principles responsible for its cardiotonic and anti-oxidant activity.

It can be concluded that the extracts have constituents which were capable of showing anti-oxidant activity and the said in-vitro anti-oxidant activity may also be due to the presence of anti-oxidant principles present in the extracts like flavonoids and phenolic compounds.

The results of present study with Premna serratifolia Lin., stem-bark and stem-wood extracts suggested the potential antioxidant activity which has good correlations with the therapeutic use in the treatment of cardiovascular disorders by practitioners of Ayurvedic system of medicine. Plants which belong to Verbenaceae family are rich in flavonoids and bio flavonoids are known for their cardiotonic and anti-oxidant activities. Further research is in progress to identify the biomolecules responsible for the antioxidant activity.

ACKNOWLEDGEMENT:

This work is carried at the Department of Pharmacognosy, Madras Medical College, Park Town, Chennai 600 003. Authors are highly thankful to the Dean and the Vice Principal for providing the necessary facilities. The authors would like to thank Director Dr. P. Jayaraman, Plant Anatomical Research Centre (PARC), Tambaram, Chennai, for identifying and authenticating the plant and also Dr. V. R. Seshadri, Secretary, IMCOPS, Chennai, for helping in collecting the plant material.

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Received on 02.03.2009

Accepted on 10.05.2009

Research Journal of Pharmacognosy and Phytochemistry. 1(1): July.-Aug. 2009, 11-14