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Jun 27, 2014 - Plectranthus amboinicus leaves were investigated by employing ... Chemical compounds which can delay the start or slow the rate of lipid ...

Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND BIO-SCIENCE NEW PERSPECTIVE OF STRESS COMBATING POTENTIAL OF PLECTRANTHUS AMBOINICUS (LOUR) LEAVES – AN INVITRO STUDY DAYANA J, PARAMESWARI CS Post graduate and Research Department of Biochemistry, Bharathi women’s college, Prakasamsalai, Broadway, Chennai – 600108, Tamil Nadu, India. Accepted Date: 26/05/2014; Published Date: 27/06/2014 Abstract: The antioxidant and Lipid Peroxidation (LPO) potential of five different extracts of

Plectranthus amboinicus leaves were investigated by employing established in vitro systems superoxide, nitric oxide, hydroxyl radical scavenging, total antioxidant and reducing power. Hydroalcoholic extract of Plectranthus amboinicus (HAPA), showed significant concentration – dependent inhibition potential than others and was chosen for quantifying the enzymic, non enzymic antioxidants and LPO study. The antioxidant ability suggests that the leaves of PA could be effectively employed as an essential ingredient in diet and medicinal field, to alleviate oxidative stress caused by free radicals. Significant results of enzymic and non-enzymic antioxidants were also obtained. HAPA showed significant inhibition of LPO and damage caused to the hydrophobic core of bio membranes by oxidative stress. Extensive studies are essential to ascertain the in vivo safety of this extract in experimental animal models.

Antioxidants, Phytochemicals, Peroxidation Keywords:

Phenolic Compounds, Free Radicals, Lipid

Corresponding Author: MRS. CS PARAMESWARI Access Online On: www.ijprbs.com How to Cite This Article: PAPER-QR CODE

Dayana J, Parameswari CS; IJPRBS, 2014; Volume 3(3): 452-471

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

INTRODUCTION Metabolic reactions of human body generate many adverse products that may damage the cells and tissues as free radicals. Free radicals are capable of attacking the healthy cells of the body, disrupt their integrity, causing them to lose their structure and function. Antioxidants are available source from nature that may scavenge and protect the integrity of the cell. They are  Nutrients in food that protect the cells from damage by free radicals [1].  Chemical compounds which can delay the start or slow the rate of lipid oxidation reaction in food systems.  Molecule that inhibits oxidation of other molecules. Plants have naturally available antioxidant resource to serve the purpose very best ever since the date of evolution of mankind. Phenols and flavonoids from plant source are potent antioxidants that can serve as secondary metabolites. Plectranthus amboinicus (PA), commonly called as Indian borage or country borage, is a dicotyledonous plant belonging to family Lamiaceae [2]. It is also termed as Coleus aromaticus. It is confined to southern parts of Asia and is distributed throughout India. It is of high medicinal value and it is used to treat malarial fever, epilepsy, common cold, chronic cough, diarrhoea, hiccups, asthma and bronchitis. The antioxidant and antibacterial activity of the leaf extract of P. amboinicus were discussed by Praveena Bhatt and Pradeep Negi [3] in different solvent fractions. The radio protective activity & anticlastogenic nature of Hydroalcoholic extract of Coleus aromaticus (leaves) were studied by Satish Rao et al, [4]. Bhattacharjee et al [5] have reported the antihelmintic nature of ethanolic extract of C. aromaticus leaves. The phytochemical study have revealed the presence of various flavanoids like Quercetin, luteolin, apegenin, Salvigenin, genkwanin and volatile oil in the leaves of C. aromaticus. Allelopathic nature, fungistatic property of P. amboinicus was also reported. The antidiabetic potential of Coleus aromaticus in alloxan induced rats was reported by Subhas candrappa et al6. The Antinociceptive and antipyretic effects of different leaf extracts of Plectranthus amboinicus were reported by Roshan et al [7]. The anti-inflammatory potential of ethanolic and aqueous extracts of leaves was studied by HRBC membrane stabilization as it is similar to that of lysosomal membrane components [8]. The present study focuses on complete antioxidant status survey by five different models namely Reducing power, Nitric oxide scavenging potential, Superoxide scavenging activity, Hydroxyl radical scavenging and Total antioxidant potential in five different extracts which was reported for the first time. Lipid Peroxidation potential of leaves of extract of PA on rat liver homogenate was also done.

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

MATERIALS AND METHODS: COLLECTION OF PLANT MATERIALS: Fresh leaves of Plectranthus amboinicus were collected from local garden in Chennai during the month of August. The leaves were authenticated by Prof. Dr. P. Jayaraman, Director, Plant Anatomy Research Centre (PARC), Chennai, India. A voucher specimen no: PARC/2013/2063 was deposited in the institute. EXTRACTION: The leaves were shade dried for a period of two weeks. The dried leaves were further chapped into small pieces and reduced to fine powder using mechanical grinder. 10gm of leaf powder sample was weighed and soaked with each 500ml of water, hydroalcohol (70:30), ethanol, chloroform and ether solvent (separately) in conical flasks. The flasks were covered with cotton plug and aluminium foil to prevent the solvent from evaporation. The flasks were placed in shaker for 24 hours and filtered using eight layered muslin cloth. The filtrate was concentrated in a rotary evaporator (Roteva) to get crude plant leaf extracts. The extracts were lyophilized, diluted appropriately and stored in air tight containers under refrigeration. These diluted extracts were used for further studies. PHYTOCHEMICAL ASSESSMENT OF LEAF EXTRACTS: Qualitative screening for the presence of various phytochemicals was performed in two different conditions: The fresh leaves and diluted extracts. Fresh leaves were ground and its juice (10 % w/v) with five different solvents incubated 48 hrs and filtered was used for analysis, whereas 10% solutions of diluted extracts were used. Standard tests were performed [9,10]. ANTIOXIDANT ASSAYS: Antioxidants are key scavengers of free radicals generated during adverse conditions and damaging the cells of our body. Assessing the antioxidants property of a plant will reveal the therapeutic efficacy of a plant. Thus antioxidant status of the plant leaves of five different extracts was assessed by five different invitro model systems: Reducing Power: The reducing power of the plant leaf extract was determined according to the method of Oyaizu (1986) [11]. 1 ml of varying concentrations of the extracts was mixed in to the mixture of 2.5 ml of 0.2M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide and incubated at 50oC for 20 min. 2.5 ml of 10% TCA was added and centrifuged at 3000 rpm for 10 min. 2.5 ml of the supernatant was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% FeCl3 and the absorbance was measured at 700 nm against blank. Ascorbic acid was used as the reference standard. Increased absorbance of the reaction mixture indicated the increased reducing power. Nitric oxide scavenging activity: The nitric oxide scavenging activity of leaf extract was assayed by the method of Soler‐Evans et al., 1997 [12]. 0.1 ml of varying concentrations of the extracts mixed with 1.4 ml of 5mM Sodium nitroprusside (0.1M phosphate buffer) and incubated at 25oC for 30 minutes. 0.5ml of the incubated solution was added to 1ml of Griess reagent (Equal volumes of 0.1% Napthylethylene diamine dihydrochloride (Solution A) and 1% Sulphanilamide Available Online at www.ijprbs.com

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

(in 5% Phosphoric acid (Solution B)) and the absorbance was measured at 546nm against blank. Ascorbic acid was used as the reference standard. Scavenging activity was calculated according to the following equation: [(Acontrol ‐ ATest)/Acontrol] X 100

 Equation – 1

Where Acontrol stands for Absorbance of control at 540nm ATest stands for Absorbance of Test sample at 540nm. Superoxide anion scavenging activity: Superoxide anion scavenging activity of the leaf extract was assayed by the method described by Nishimiki, Rao and Yagi, 1972 [13] with slight modification. In this experiment, the superoxide anion radicals were produced in reaction mixture containing 750μl of NBT (300μM), 750μl of NADH (936μM) and 300 μl of extracts at different concentrations and made up to 3 ml with Tris-Hcl buffer (100 mM, pH 7.4). 750μl of PMS (120 μM) was added and incubated at room temperature for a period of 5 min which initiates the colour reaction between superoxide anion radical and NBT. The absorbance was measured at 560 nm against blank. Mixture without sample was used as control and mixture without PMS used as blank. Ascorbic acid was used as the reference standard. The scavenging activity was calculated using equation 1. Hydroxyl radical scavenging activity assay It was determined according to the method reported by Klein et al., 1981[14]. The reaction mixture contains 0.2ml of extracts of various concentrations: 0.1ml of Deoxy-D-ribose (2.8mM), 0.1 ml of FeCl3 (0.1mM), 0.1 ml of EDTA (0.1mM), 0.1 ml of H2O2 (1mM), 0.1 ml of Ascorbic acid (0.1mM), 0.1 ml of KH2PO4-KOH buffer (20mM, pH 7.4) and made up to 1 ml with distilled water and incubated at room temperature for 1 hour.1ml of 1% TBA was added and the mixture was heated to 950C for 20 mins. After cooling to room temperature the absorbance was measured at 532 nm against blank. Ascorbic acid was used as the reference standard. The scavenging activity was calculated using equation 1. Total Antioxidant Capacity: Total Antioxidant capacity of the plant leaf extract was assayed by phosphomolybdenum method of Prieto et al., 1999 [15]. To 0.1 ml of the extract (of different concentrations), 1ml of reagent solution (0.6M sulphuric acid, 28mM sodium phosphate, and 4mM ammonium molybdate) was added. The tubes were capped/covered airtight and incubated at 35 0C for 90 min. The absorbance was measured at 695 nm against blank. Ascorbic acid was used as the reference standard. The scavenging activity was calculated using equation 1. NON-ENZYMIC ANTIOXIDANTS: The non-enzymic antioxidants analyzed were total phenols, total carotenoids and chlorophyll, ascorbic acid, α-tocopherol and reduced glutathione.

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

Determination of Total phenolic content: The phenolic content was determined according to the method of Singleton and Rossi, 1965 [16]. The reaction mixture was prepared by mixing 0.5 ml of varying concentrations of the extracts; 2.5 ml of 10% Folin-Ciocalteu’s reagent dissolved in water and 2.5 ml 7.5% NaHCO3. Blank was concomitantly prepared, containing 0.5 ml solvent instead of extract. The samples were incubated at room temperature for 45 min. The absorbance was measured at 765 nm. Phenol was used as reference standard. The content of phenol in extracts was expressed in terms of µg/mg of extract. Determination of Total Carotenoids and Chlorophylls a and b: [17] Methanolic solutions of plant extracts of the appropriate concentration (1.0 to 4.0 mg/ml) were analyzed under UV/VIS spectrophotometer at 470, 653 and 666 nm. The concentrations of carotenoids and chlorophylls α and b were determined according to the equations reported by Lichtenthaler and Wellburn (1985)[18] as follows: Total carotenoids (mg/L) = 1000 Abs470 – 2.860 Ca – 129.2 Cb/245 Chlorophyll α (mg/L) = 15.65 Abs666 – 7.340 Abs653 Chlorophyll b (mg/L) = 27.05 Abs653 – 11.21 Abs666 Estimation of Ascorbic acid: Ascorbic acid was analyzed by the spectrophotometric method described by Roe and Keuther [19]. Principle: Ascorbate is converted to dehydroascorbate on treatment with activated charcoal, which reacts with 2, 4-dinitrophenyl hydrazine to form osazones. These osazones produce an orange coloured solution when dissolved in sulphuric acid, whose absorbance can be measured spectrophotometrically at 540nm. Extraction of Ascorbic acid: Ascorbate was extracted from homogenization of 1g of the fresh leaf using 4% TCA and the volume was made up to 10ml with the same. The supernatant obtained after centrifugation at 2000rpm for 10 minutes was treated with a pinch of activated charcoal, shaken vigorously using a cyclomixer and kept for 5 minutes. The charcoal particles were removed by centrifugation and aliquots were used for the estimation. Procedure: Standard ascorbate (100 μg / ml in 4% TCA) ranging between 0.2-1.0 ml and 0.5 ml and 1.0 ml of the supernatant of extracts were taken. The volume was made up to 2.0 ml with 4% TCA. 0.5 ml of 2, 4 – dinitrophenyl hydrazine reagent (2% in 9N H2SO4) was added to all the tubes, followed by 2 drops of 10% thiourea solution. The contents were mixed and incubated at 37°C for 3 hours resulting in the formation of osazone crystals. The crystals were dissolved in 2.5 ml of 85% sulphuric acid, in cold. To the blank alone, DNPH reagent and thiourea were added after the addition of sulphuric acid. The tubes were cooled in ice and the absorbance was read at 540 nm in a spectrophotometer. The concentration of ascorbate in the samples were calculated and expressed in terms of mg/g of sample. Available Online at www.ijprbs.com

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

Estimation of Tocopherol: Tocopherol was estimated in the plant samples by the Emmerie Engel reaction as reported by Rosenberg (1992) [20]. Principle: The Emmerie-Engel reaction is based on the reduction of ferric to ferrous ions by tocopherols, which with 2, 2'-dipyridyl, forms a red colour. Tocopherols and carotenes are first extracted with xylene and read at 460 nm to measure carotenes. A correction is made for this, after adding ferric chloride and read at 520 nm. Extraction of tocopherol: The plant sample (2.5 g) was homogenized in 50 ml of 0.1N sulphuric acid and allowed to stand overnight. The contents of the flask were shaken vigorously and filtered through Whatman No.1 filter paper. Aliquots of the filtrate were used for the estimation. Procedure: Into 3 stoppered centrifuge tubes, 1.5 ml of plant extract, 1.5 ml of the standard (D,L-α-tocopherol, 10 mg/L in absolute alcohol) and 1.5 ml of water were pipetted out separately. To all the tubes, 1.5 ml of ethanol (absolute alcohol) and 1.5 ml of xylene were added, mixed well and centrifuged. Xylene (1.0 ml) layer was transferred into another stoppered tube. To each tube, 1.0 ml of 2, 2’- dipyridyl reagent (1.2 g/L in n-propanol) was added and mixed well. The mixture (1.5 ml) was pipetted out into a cuvette and the extinction was read at 460 nm. 0.33 ml of ferric chloride solution (1.2 g/L in ethanol) was added to all the tubes and mixed well. The red colour developed was read exactly after 15 minutes at 520nm in a spectrophotometer. The concentration of tocopherol in the sample was calculated using the formula, Sample A520 – A460 Tocopherols (μg) =

× 0.29 × 0.15 Standard A520

The concentration of tocopherols in the samples were calculated and expressed in terms of μg/g of sample. Estimation of Reduced Glutathione: Reduced glutathione was determined by the method of Halliwell and Gutteridge (1984) [21]. Principle: Reduced glutathione on reaction with DTNB (5, 5'- dithiobis nitro benzoic acid) produces a yellow coloured product that absorbs at 412 nm. Extraction of Glutathione: A homogenate was prepared with 0.5 g of the plant sample with 2.5 ml of 5% TCA. The precipitated protein was centrifuged at 1000 rpm for 10 minutes. The supernatant (0.1 ml) was used for the estimation of GSH.

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

Procedure: The supernatant (0.1 ml) was made up to 1.0 ml with 0.2 M sodium phosphate buffer (pH 8.0). Standard GSH (in 5% TCA) corresponding to concentrations ranging between 2 and 10 nmoles were also prepared. 2 ml of freshly prepared DTNB solution (0.6 mM in 0.2 M phosphate buffer) was added and the intensity of the yellow colour developed was measured in a spectrophotometer at 412 nm after 10 minutes. The values are expressed as nmoles of GSH/g sample. ENZYMIC ANTIOXIDANTS: The Enzymic antioxidants analyzed were Superoxide dismutase, peroxidase and glutathione-s-transferase. Assay of superoxide dismutase (SOD): SOD was assayed according to the method of Kakkar, Das and Vishwanathan (1984) [22]. Principle: The assay of SOD is based on the inhibition of the formation of NADH-PMS-NBT formazon. The colour formed at the end of the reaction can be extracted into butanol and measured at 560nm. Preparation of enzyme extract: The fresh leaves (0.5 g) were ground with 3.0 ml of potassium phosphate buffer (50 mM, pH 6.4), centrifuged at 2000 g for 10 minutes and the supernatants were used for the assay. Assay: The assay mixture contained 1.2 ml of sodium pyrophosphate buffer (0.025 M, pH 8.3), 0.1 ml of PMS (186 µM), 0.3 ml of NBT (300 µM), 0.2 ml of the enzyme preparation and water in a total volume of 2.8 ml. The reaction was initiated by the addition of 0.2 ml of NADH (780 µM) and incubated at 30°C for 90 seconds and arrested by the addition of 1.0 ml of glacial acetic acid. The reaction mixture was then shaken with 4.0 ml of n-butanol, allowed to stand for 10 minutes and centrifuged at 3000 rpm for 5 mins. The intensity of the chromogen in the butanol layer was measured at 560 nm. One unit of enzyme activity is defined as the amount of enzyme that gave 50% inhibition of NBT reduction in one minute. Assay of Peroxidase (POD) [23] Principle: In the presence of the hydrogen donor pyrogallol or dianisidine, peroxidase converts H2O2 to H2O and O2. The oxidation of pyrogallol or dianisidine to a coloured product called purpurogalli can be followed spectrophotometrically at 430 nm. Preparation of enzyme extract: A 20% homogenate was prepared in 0.1M phosphate buffer (pH 6.5) from the various parts of the plant, clarified by centrifugation and the supernatant was used for the assay. Assay: To 3.0 ml of pyrogallol solution (0.05 M in 0.1 M phosphate buffer, pH 6.5), 0.1 ml of the enzyme extract was added and the spectrophotometer was adjusted to read zero at 430 nm. To the test cuvette, 0.5 ml of H2O2 (1% in 0.1M phosphate buffer, pH 6.5) was added and mixed.

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

The change in absorbance was recorded every 30 seconds up to 3 minutes in a spectrophotometer. One unit of peroxidase is defined as the change in absorbance/minute at 430nm. Assay of Glutathione S-Transferase (GST): Glutathione S-transferase was assessed by the method of Rotruck et al.,(1973) [24]. Principle: The enzyme is assayed by its ability to conjugate GSH and CDNB, the extent of conjugation causing a proportionate change in the absorbance at 340 nm. Preparation of enzyme extract: The samples (0.5 g) were homogenized with 5.0 ml of phosphate buffer (0.1 M, pH 6.5). The homogenates were centrifuged at 5000 rpm for 10 minutes and the supernatants were used for the assay. Assay: The activity of the enzyme was determined by observing the change in absorbance at 340 nm. The reaction mixture contained 0.1 ml of GSH (1 mM), 0.1 ml of CDNB (1-chloro-2, 4dinitrobenzene, 1 mM in ethanol) and phosphate buffer in a total volume of 2.9 ml. The reaction was initiated by the addition of 0.1 ml of the enzyme extract. The readings were recorded every 15 seconds at 340 nm against distilled water blank for a minimum of three minutes in a spectrophotometer. The assay mixture without the extract served as the control to monitor nonspecific binding of the substrates. GST activity was calculated using the extinction co-efficient of the product formed (9.6 mM-1cm−1) and was expressed as nmoles of CDNB conjugated/minute. EVALUATION OF THE EFFECTS OF P.amboinicus LEAF EXTRACTS ON LPO IN RAT LIVER HOMOGENATE: Lipid peroxidation (LPO), a well-established mechanism of cellular injury, is used as an indicator of oxidative stress. Principle: Oxidizing agents (ferrous ions and ascorbate, or H2O2) impose a stress on membrane lipids which can be quantified as the extent of thiobarbituric acid reactive substances (TBARS) formed. Homogenate Preparation: Rat liver (Institutional Ethical Committee - Saveetha University, Chennai (009/2010/CPSEA), Control group rat liver was requested after sacrifice and utilized) was procured fresh and washed free of blood using Tris-HCl buffer (40 mM, pH 7.0). A 20% liver homogenate was prepared in the same buffer using a motorized Teflon homogenizer. The homogenate was clarified to remove debris and used as the membrane source for assessing LPO as per the method of Okhawa et al., (1979) [25]. Procedure: The reaction mixture containing 0.1 ml of liver homogenate, 0.1 ml of KCl (30 mM), 0.1 ml of FeSO4 (0.16 mM) and 0.1 ml of ascorbate (0.06 mM) was incubated at 37°C for one hour in the presence (0.1 ml corresponding to 50 mg) and the absence (0.1 ml of KCl) of extracts of P.amboinicus leaves. To 0.4 ml of the reaction mixture, 1.5 ml each of TBA (1%) and Available Online at www.ijprbs.com

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Research Article CODEN: IJPRNK IMPACT FACTOR: 1.862 Parameswari CS, IJPRBS, 2014; Volume 3(3): 452-471

ISSN: 2277-8713 IJPRBS

acetic acid were added and mixed well. The contents were heated in a boiling water bath for 20 minutes. After cooling, 1.0 ml of distilled water and 5.0 ml of n-propanol and pyridine mixture (15:1 v/v) was added. After centrifugation, the pink coloured chromophore obtained was measured at 532 nm in a spectrophotometer. The percentage inhibition of LPO was determined by comparing the results of the control and the test samples. Tocopherol was used as the reference standard. Statistical analysis: The analyses were carried out five times in each extract and the mean values of analysis were used. The Standard deviation and standard error was calculated using SPSS16 software and values are expressed as Mean ± SE or Mean ± SD wherever required. p