1 International Food Research Journal 20(1): (2013) Journal homepage: Total phenolic and flavonoid contents, and in vitro antihypertension activity of purified extract of Indonesian cashew leaves (Anacardium occidentale L.) 1* Agung Endro Nugroho, 1,2 Abdul Malik and 3 Suwidjiyo Pramono 1 Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Jogjakarta 55281, Indonesia 2 Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Muslim Indonesia, Makassar 90231, Indonesia 3 Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Jogjakarta 55281, Indonesia Article history Received: 18 April 2012 Received in revised form: 4 June 2012 Accepted:6 June 2012 Keywords Purified extract cashew leaves antihypertension phenolic flavonoid Abstract Cashew tree (family Anacardiaceae) grows widely in many areas of the Southeast Asia countries including Indonesia. Its fruit and nut are used for food, whereas its leaf is one of the traditional antihypertensive medicine in Indonesia. Hypertension affects many people around the world especially in developing countries such as Indonesia. The study aimed to evaluate the in vitro antihypertension effect of purified extract of cashew leaves (PECL) using an isolated organ technique, and determine the total phenolic and flavonoid contents. The results showed that PECL at 0.5 and 1.0 mg/ml was obviously able to inhibit the contraction of isolated rat aorta induced by cumulative addition of phenylephrine. The inhibitory effect of PECL were 25.72±8.19% and 39.60±3.50% (p<0.05), respectively. PECL (1.0 mg/ml) also changed the pd 2 value of phenylephrine from 6.71±0.37 to 5.93±0.33 (p<0.05), and relaxed the isolated-organ mildly by 13.11±0.72%. In addition, PECL contained the total phenolic of 19.78±0.62% and the total flavonoids of 1.97±0.04% which are equivalent to gallic acid and rutin, respectively. All Rights Reserved Introduction Hypertension is a main risk factor of stroke, cardiovascular, and renal failure (August, 2004). This failure was estimated to be 4.5% cause of global disease (WHO and ISH, 2003). Cardiovascular system is affected by arteries that play a role in maintaining the change in peripheral blood flow oscillation. Artery has elastic structures and characteristics (McEniery et al., 2007). Disturbance in its elasticity can influence the vascular resistance, one other than cardiac output directly affecting blood pressure (DiPiro et al., 2005). Hypertension can be treated by traditional medicines (Koffi et al., 2009). Cashew leaf is one of the traditional antihypertensive medicinal plants in Indonesia which is needed to be developed in the form of purified extract to increase the amount of active compounds. Paris et al. (1977) reported that the extract of cashew leaves (ECL) exhibited antihypertensive effect, which was certainly correlated to active chemical compounds contained in the extract. ECL mainly contains phenolic and flavonoid compounds (Paris et al., 1977; Syaharuddin et al., 1998; Razali et al., 2008; Sulaiman et al., 2011; Andarwulan et al., 2012). Torres-Piedra et al. (2011) reported that vasorelaxation effect of traditional medicines were correlated to the content of phenolic and flavonoid compounds. Some epidemiological studies showed inverse correlation between flavonoid consumption and risk of cardiovascular disease (Xu et al., 2011). It suggests that antihypertensive effect of ECL might be related to the present of the compounds. Polyphenolic compounds are widely available in some plants, fruits and vegetables. They are derivatives and/or isomers of flavones, isoflavones, flavonols, catechins and phenolic acids that exhibit cardiovascular protection and improvement of the endothelial function (Han et al., 2007). In the previous study, polyphenol-rich cocoa powder exhibited antihypertensive effect in hypertensive rats, eventhough did not influence the arterial blood pressure in the normotensive rats (Cienfuegos- Jovellanos et al., 2009). In addition, crude extract of Terminalia bellerica fruit that contains flavonoids, sterols and tannins also showed antihypertension *Corresponding author.
2 300 Nugroho et al./ifrj 20(1): effects in anaesthetized-rats (Khan and Gilani, 2008). Development of ECL as antihypertensive medicine was further investigated to obtain a purified extract. The pharmacological effect of purified extract will be more effective because most non-active compounds have been eliminated. Based on the facts, purified extract of cashew leaves (PECL) was investigated for its in vitro antihypertension effects and its total phenolics and flavonoids contents. The in vitro antihypertension effect was evaluated using an isolated organ technique. Phenylephrine was used to contract the rats-isolated aorta. Total phenolics and flavonoids contents in PECL were determined using Folin Ciocalteu reagent and colorimetric assay, respectively. Materials and Methods Materials The following materials were used :phenylephrine-hcl, gallic acid, rutin from Sigma Chemical Co. (St. Louis, MO, USA). The Krebs solution consists of the following composition (mm) : NaCl 118.0; KCl 4.4; CaCl 2 2.5; NaHCO ; MgSO 4 1.1; KH 2 PO 4 1.2; glucose 11.0 from Merck & Co., Inc. (New Jersey, USA). The solution must be made freshly, and continuously bubbled with 95% O2 and 5% CO 2. Animals Male Wistar rats weighing g were obtained from Laboratory of Pharmacology and Toxicology, Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Indonesia. The animal handling protocols of this study were in accordance with the guidelines of the animal care of the Department. Extraction and purification The cashew leaves were collected from the area around Plemburan Sleman, Yogyakarta Indonesia and identified by a botanist at Laboratory of Plant Taxonomy, Faculty of Biology Universitas Gadjah Mada Indonesia. The cashew leaves were then dried and powdered. Total g of powder was macerated by 70% ethanol for 24 h. Subsequently, the residue was remacerated by same solvent four times, and the extracts were mixed into the previous extract. The collected extract was then evaporated under reduced pressure to give of viscous ethanolic extract, then fractionated with n-hexane yielding soluble and insoluble fractions of n-hexane. The insoluble fraction was concentrated by rotary vacuum evaporator to obtain viscous extract, and then dried using freeze drying to eliminate the existence of the remaining traces of water. Determination of total phenolic content Total phenolic content was determined according to the method of Chun et al. (2003). Ten milligrams PECL was included in 10 ml volumetric flask, then added with 0.4 ml reagent of Folin-Ciocalteau and incubated for 4-8 min. Furthermore, the solution was added with 4.0 ml of 7% Na 2 CO 3, then added with distilled water. After 2 h of incubation, the solution absorbance was measured in 750 nm wavelength versus a blank consisting distilled water and Folin- Ciocalteau reagent. Total phenolic content was expressed in gallic acid equivalent (GAE) of each 100 g PECL of dry weight. Determination of total flavonoid content Total flavonoid content was determined according to the procedure of Chang et al. (2002) validated by Mujahid (2011) using rutin as reference standard. Both PECL and rutin (100 mg) were dissolved in 10 ml methanol. Subsequently, the routine solution was diluted to provide a series of concentrations (0.050; 0.075; and mg/ml). The sample solution (0.5 ml) was added with 1.5 ml methanol, 0.1 ml of 10% AlCl 3, 0.1 ml potassium acetate 1M and 2.8 ml distilled water, and then incubated for 30 minutes. Furthermore, absorbance was measured in 415 nm wavelength, and distilled water with 10% AlCl 3 was used as a blank. Total flavonoid content was expressed in gram rutin equivalent (RE) of each 100 gram PECL of dry weight. Preparation of aortic rat The isolated organ was rat aorta that represents a vascular system (Nugroho et al., 2008), obtained from male wistar rats. The animal was sacrificed by cervical dislocation. The aorta was carefully removed, cleaned and cut (Torres-Piedra et al., 2011) into 1 cm length. The strip was mounted in an organ bath containing 20 ml Krebs solution (ph 7.4, 37 C) aerated with 95% O 2 and 5% CO 2. The organ bath was equipped with a level transducer (type 368 HSE, Germany) connected to a bridge amplifier (type 336 HSS, Germany) and a recorder (Kipp & Zonen BBD 41, The Netherlands). The strips was equilibrated for at least 60 min under a resting tension of 1 g. The Krebs solution was replaced with the fresh solution every 15 min. Evaluation of inhibitory effect on the contraction After 60-min equilibration, the isolated organ was contracted with a single concentration of phenylephrine. After the contraction of aorta reached
3 Nugroho et al./ifrj 20(1): a plateau, the organ was washed by fresh Krebs solution for 60 min. Subsequently, the isolated organ was preincubated with various oncentrations of PECL or DMSO for 5 min, and then contracted with cumulative addition of phenylephrine to provide a concentration-response curve. Evaluation of vasodilatation effect In the study, following 60-min equilibration the isolated organ was contracted with a single concentration of phenylephrine. The tissue was washed by the fresh Krebs solution for 60 min with replacement of the Krebs solution every 15 min. The tissue was then contracted with a single concentration of phenylephrine. After the maximun contraction of aorta was reached, cumulative concentrations of PECL (0.1; 0.5 and 1.0 mg/ml) were added into the organ bath to relax the isolated organ. Statistical and analysis of data The data were presented as mean ± the standard error of the mean (SEM). In the in vitro study, the pd 2 value is a negative logaritmic of concentration of agonist which cause half maximal response in the form of either contraction or relaxation (Waldron et al., 1999). That value represents potency of the effect of agonist on the rats isolated-aorta. All data were analyzed statistically using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test. P-values less than 0.05 were considered significant. Results and Discussion Extraction and purification In the study, extraction of g cashew dry leaves with 70% ethanol yielded g viscous ethanolic extract (26.72%) which is accordance with requirements of Farmakope Herbal Indonesia (DepKes RI, 2008). In the purification step, g viscous ethanolic extract was fractionated using n-hexane to yield higher contents of active compounds in the fraction. n-hexane was used in the procedure to eliminate chlorophyll, lipid, and other disturbing materials (Depkes RI, 2008; Konan et al., 2012). Purification step yielded a viscous, brown coloured fraction with a total weight of 4.27 g (28.47% of weight of the ethanolic extract). Total phenolic contents The total phenolic content was determined using Folin-Ciocalteau method. In the study, the absorbances of a series concentrations of gallic acid were plotted to their concentration to yield a linear Figure 1. Linear curve of gallic acid concentration (mg/ml) vs. absorbance for determination of total phenolics content Table 1. Percentage of total phenolic content in the purified extract Concentration (mg/ml) GAE = Gallic Acid Equivalent Absorbance (n) (λ 750 nm) % total fenolic content GAE (b/b) Mean±SEM ± 0.62 calibration curve of gallic acid (y = 106.6x ) with coefficient of correlation (r) value of (Figure 1). In the study, total phenolic content of PECL was 19.78±0.62%. It means that each 100 g dry weight of PECL contained total phenolics that was equivalent to gallic acid of 19.78±0.62 g (Table 1). Purification step yielded higher total phenolics content in comparison to ethanolic extract without purification. Related to this case, Sulaiman et al. (2011) and Andarwulan et al. (2012) reported that ethanolic extract without purification contained the total phenolics content equivalent to gallic acid of 4.36 g/100 g and 0.85 g/100 g dry weight extract, respectively. Total flavonoid content Flavonoids are the most compounds of plant phenolics. In the study, total flavonoid content was determined using a modified method based on the procedure of Chang et al. (2002) validated by Mujahid (2011) using rutin as a reference standard. Principally, the procedure is related to the formation of complex between flavonoid and AlCl 3 that produces a yellow coloured solution. The absorbance is measured spectrophotometrically at maximum wavelength of 415 nm (Mujahid, 2011). The total flavonoid content was equivalent to rutin in milligram per gram dry material of the fraction. The absorbances of a series concentrations of rutin were plotted to their concentration to yield a linear calibration curve of rutind (y = 7.96x ) with coefficient of correlation (r) value of (Figure 2).
4 302 Nugroho et al./ifrj 20(1): Table 2. Percentage of total flavonoid content in the purified extract Concentration (mg/2 ml) Absorbance (n) (λ 415 nm) % total flavonoid content RE (b/b) Mean±SEM 1.97±0.01 RE = Rutin Equivalent Figure 2. Linear curve of rutin concentration (mg/ml) vs. absorbance for determination of total flavonoids content Figure 3. Concentration vs. response curves to Phenylephrine in absence ( O ) or presence ) of DMSO in rats isolated-aorta (Data represent mean±sem, n=4-8) Figure 4. Concentration vs. response curves to Phenylephrine in presence of DMSO (control) ( ), or PECL at 0.1 ( ); 0.5 ( ) and 1.0 mg/ml ( ) in rats isolated-aorta (Data represent mean±sem, n=4-8) Figure 5. Percentage of relaxation responses after incubated by DMSO (10.0, 50.0, and µl) or cumulative addition of PECL (0.1; 0.5 and 1.0 mg/ml) In the study, total flavonoid content of PECL was 1.97±0.01%. It means that each 100 g dry weight of PECL contained total flavonoid equivalent to rutin of 1.97±0.01 g (Table 2). This total flavonoid content was higher than this of ethanolic extract without purification. In previous study, the leaves were extracted without purification yielding total flavonoid content of 0.93 g/100 g dry weight extract (Sulaiman et al., 2011). Inhibitory effects on the contraction of rats isolatedaorta In the study, rat isolated-aorta was preincubated by either the solvent (DMSO) or PECL, and then contracted gradually by a series concentrations of phenylephrine agonist. The values of pd 2 and maximum contraction responses were compared among these groups. These values represent affinity and intrinsic activity (efficacy) of phenylephrine agonist on the target action (Kenakin, 1997). Firstly, DMSO which is a solvent used to dilute the fraction (purified extract) was evaluated for its effect on isolated-aorta. In the study, 100 µl DMSO did not influence the maximum contraction of rat isolated-aorta induced by phenylephrine (P>0.05). The result indicates that DMSO did not influence the intrinsic activity (efficacy) of phenylephrine agonist. The values of pd 2 of phenylephrine in absence and in presence of 100 µl DMSO were 6.71±0.37 and 6.58±0.27, respectively. Statistically, these values were not significant (P>0.05). It indicates that DMSO also did not influence the affinity of phenylephrine. Secondly, PECL was evaluated for its inhibitory effect on phenylephrine-induced contractions. As shown in Figure 4, PECL decreased the maximum contraction of rat isolated-aorta induced by phenylephrine in concentration-dependent manner. PECL at 0.5 and 1.0 mg/ml suppressed the contraction of isolated-aorta by 25.72% and 39.61%, respectively. The result indicates that PECL decreased the intrinsic activity (efficacy) of phenylephrine agonist. In addition (Figure 4), incubation with PECL also decreased the pd 2 value of phenylephrine. At lower concentration (0.1 mg/ml), PECL did not influence the pd 2 value, however, at 0.5 and 1.0 mg/ ml succeeded to shift the pd 2 value of 6.71±0.37 to 6.23±0.24 and 5.93±0.33, respectively. It indicates that PECL was also able to decrease the affinity of phenylephrine. The inhibitory effect of PECL on rat isolated-aorta might be associated with either competitive or non-competitive disruption of the target action of phenylephrine in rat isolated-aorta. Vasodilatation effects on rats isolated-aorta In the study, rat isolated-aorta was precontracted by a single consentration of phenylephrine agonist, and after maximum contraction reached, the tissue
5 Nugroho et al./ifrj 20(1): was relaxed by a series concentrations of PECL. In the study, the solvent (DMSO) did not influence on rats isolated-aorta. PECL at of 0.1; 0.5 and 1.0 mg/ ml relaxed the rat isolated-aorta mildly by 0.1±0.005; 6.64±0.350 and 13.11±0,72, respectively (Fig 5). Indonesia is a second largest biodiversity country that provides many traditional medicines for various diseases. However, the scientific data are still limited. One of traditional medicine that is interesting to be explored is cashew tree. Cashew (Anacardium occidentale L.) is a popular plant in Indonesia due to wide range of applications. Plant parts including leaves, nuts and fruit are used for food, vegetable or traditional medicine. Traditionally, the leaves are used to decrease blood pressure in hypertensive patients. In the study, purified extract of cashew leaves (PECL) that were collected in Indonesia contained total phenolics and flavonoids of 19.78% and 1.97%, respectively. Previously, cashew leaves were also reported containing high amount of phenolic compounds. The alkali- and waterextracts contained gallic acid predominantly, and also protocatechuic, p-hydroxybenzoic, cinnamic, p-coumaric and ferulic acids (Kogel and Zech, 1985). In addition, flavonol glycosides were also identified from the extract of cashew leaves collected in Malaysia. The highest constituent in the extract was kaempferol-3-o-glucoside, followed by kaempferol- 3-O-arabinofuranoside and quercetin-3-o-glucoside (Shukri and Alan, 2010). Similarly as in the leaf, the juice of cashew apples collected from Cote d Ivoire also contained high amounts of total phenolics and total flavonoids. Its bioactive phenolic compounds were such as quercetin, naringenin and phenolic acids (caffeic acid, p-coumaric acid, ferulic acid, gallic acid) (Marc et al., 2012). In addition, cashew nuts extracts also contained flavonoid and phenolic compounds. The ethanolic and ethyl acetate extracts only contained phenolics, however acetonic extract contained both compounds (Kannan et al., 2009). Flavonoid and phenolic compounds have widely been reported as antioxidant agents positively correlated in the treatment of cardiovascular diseases due to their antihypertension properties (Akhlaghi and Bandy, 2009; Xu et al., 2011). Some traditional medicine containing high amounts of flavonoid and phenolic compounds exhibit antihypertension activity. Reportedly, polyphenol-rich cocoa powder obviously produced antihypertensive effect in hypertensive rats, but did not influence the arterial blood pressure in the normotensive rats (Cienfuegos-Jovellanos et al., 2009). In addition, crude extract of Terminalia bellerica fruit containing flavonoids, sterols and tannins markedly decreased the arterial blood pressure of anaesthetized rats. The extract also inhibited the force and rate of atrial contractions in isolated guineapig atria, and also relaxed the phenylephrine and K + - induced contractions in the Ca 2+ -free medium (Khan and Gilani, 2008). Several mechanisms that may contribute to their antihypertension properties are such as the inhibition of phosphodiesterase, reduction of intracelluler Ca 2+, induction of nitric oxide (NO) in smooth muscles (Akhlaghi and Bandy, 2009; Torres- Piedra, 2011). Among them, NO induction from vascular endothelium resulting from antioxidative activity of flavonoid and phenolic compounds have an important contribution in their antihypertension properties. Nowadays, oxidative stress is still an attractive target for cardiovascular prevention and therapy. The fact is related to the abnormal production of reactive oxygen species (ROS) producing decrease in vascular bioavailability of NO that contributes in the pathogenesis of the endothelial dysfunction in the development of hypertension disease (Munzel et al., 2010; Cohen and Tong, 2010). In the production of ROS, many vascular enzymes are involved such as NADPH oxidases, xanthine oxidase, and uncoupled endothelial nitric oxide synthase. Compounds that are potent inhibitors for oxidative stress are considered to be used in hypertension therapy due its vascular function preservation activity (Weseler and Bast, 2010). Polyphenols from fruits, vegetables and beverages can protect the cardiovascular system due to their antioxidative and free radical scavenging activities. The compounds are able to generate the NO from vascular endothelium, and then prevent the endothelial dysfunction that contribute mainly in the pathogenesis of hypertension (Curin and Andriantsitohaina, 2005). Reportedly, acetonic extract of cashew leaves exhibited a poten antioxidative activity determined by DPPH method (Vijayakumar and Kalaichelvan, 2011). In addition, the ethanolic extract also showed antioxidant activities in the DPPH and nitric oxide radical scavenging assay. The high amount of phenolic compounds in the extract of cashew leaves positively contribute to the antioxidative activity (Jaiswal et al., 2010). In the study, purified extract of cashew leaves (PECL) exhibited inhibitory effect on phenylephrineinduced contractions. The potent antioxidative effect of cashew leaves due its flavanoids and phenolics may underlie its antihypertension effect. Further investigation is needed to explain the molecular mechanism of PECL on its antihypertension activities. In this case, phenolic and flavonoid compounds might play main roles in the antihypertension effects needed to be isolated and further investigated for their target
6 304 Nugroho et al./ifrj 20(1): actions. Conclusion Purified extract of cashew leaves which contains higher contents of flavonoid and phenolic compounds exhibited antihypertension effects in rats isolatedaorta. The purified extract is potential to develope as an antihypertension agent. References Akhlaghi, M. and Bandy, B Mechanisms of flavonoid protection against myocardial ischemiareperfusion injury. Journal of Molecular and Cellular Cardiology 46: Andarwulan, N., Kurniasih, D., Apriady, R.A., Rahmat, H., Rotoc., A.V. and Bolling, B.W Polyphenols, carotenoids, and ascorbic acid in underutilized medicinal vegetables. Journal of Functional 4(1): August, P Overview: mechanisms of hypertension: cells, hormones, and the kidney. J Journal of the American Society of Nephrology 15: Chang, C., Yang, M., Wen, H. and Chem, J Estimation of flavonoid total content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analisis 10: Chun, O.K., Kim, D.O. and Lee, C.Y Superoxide radical scavenging activity of the mayor polyphenols in fresh plums. Journal of Agricultural and Food Chemistry 51: Cienfuegos-Jovellanos, E., Quiñones, Mdel., M., Muguerza, B., Moulay, L., Miguel, M. and Aleixandre, A Antihypertensive effect of a polyphenol-rich cocoa powder industrially processed to preserve the original flavonoids of the cocoa beans. Journal of Agricultural and Food Chemistry 57(14): Cohen, R.A. and Tong, X Vascular oxidative stress: the common link in hypertensive and diabetic vascular disease. Journal of Cardiovascular Pharmacology 55(4): Curin, Y. and Andriansitohaina, R Polyphenols as potential therapeutical agents against cardiovascular diseases. Pharmacological Report 57 (suppl): Depkes, R.I Farmakope Herbal Indonesia. Edisi I. Jakarta: Departemen Kesehatan Republik Indonesia. DiPiro, J.T., Talbert, R.L., Yee, G.C., Matzke, G.R., Wells, B.G. and Posey, L.M Pharmacotherapy: A Pathophysiologic Approach, p th edition. New York: The McGraw-Hill Companies, Inc. Han, X., Shen, T. and Lou, H Dietary Polyphenols and Their Biological Significance. International Journal of Molecular Sciences 8: Jaiswal, Y.S, Tatke, P.A., Gabhe, S.Y. and Vaidya, A Chemical Sciences Antioxidant Activity of Various Extracts of Leaves of Anacardium occidentale (Cashew). Research Journal of Pharmaceutical, Biological and Chemical Sciences 1(4): Kannan, V.R., Sumathi, C.S., Balasubramanian, V. and Ramesh, N Elementary Chemical Profiling and Antifungal Properties of Cashew (Anacardium occidentale L.) Nuts. Botany Research International 2 (4): Kenakin, T Molecular Pharmacology : A short course, p USA :The Blackwell Science, Ltd. Khan, A.U. and Gilani, A.H Pharmacodynamic Evaluation of Terminalia bellerica for Its Antihypertensive Effect. Journal of Food and Drug Analysis 16(3): Koffi, N., Marie-Solange, T., Emma, A.A. and Noel, Z.G Ethnobotanical study of plants used to treat arterial hypertension, in traditional medicine, by abbey and krobou populations of agboville (Côte-d Ivoire). European Journal of Scientific Research 35(1): Kogel, I. and Zech, W The phenolic acid content of cashew leaves Anacardium occidentale L.) and of the associated humus layer, Senegal. Geoderma 35(2): Konan, N.A., Lincopan, N., Díaz, I.E.C., Jacysyn, J.F., Tiba, M.M.T., Mendes, J.G.P.A., Bacchi, E.M. and Spira, B Cytotoxicity of cashew flavonoids towards malignant cell lines. Experimental and Toxicologic Pathology. 64(5): Marc, A., Ange, K.D., Achille, T.F. and Georges, A.N Phenolic profile of cashew apple juice (Anacardium occidentale L.) from Yamoussoukro and Korhogo (Côte d Ivoire). Journal of Applied Biosciences 49: McEniery, C.M., Wilkinson, I.B. and Avolio, A.P Frontiers in research review: Arterial function: Age, hypertension and arterial function. Clinical and Experimental Pharmacology and Physiology 34: Mujahid, R Pemilihan metode analisis flavonoid secara spektroskopi UV-Vis serta penerapannya pada seledri (Apium graviolens L.) murbei (Morus alba L.) patikan kebo (Euphorbia hirta L.) dan jeruk nipis (Citrus aurantifolia). Jogjakarta, Indonesia: Faculty of Pharmacy Universitas Gadjah Mada, MSc thesis. Munzel, T., Gori, T., Bruno, R.M. and Taddei, S Is oxidative stress a therapeutic target in cardiovascular disease? European Heart Journal 31(22): Nugroho, A.E., Suhardjono, D., Mulyono and Margono, S.A The vasodilation effects of curcumin and its derivatives on isolated-aortic of rats. Indonesian Journal of Pharmacy 19(2): Paris, R., Plat, M., Barber, P.G., Linhard, J. and Laurens, A Receherce chimique et pharmacologique sur les feuilles d Anacardium occidentale L. (Anacardiacees). Bulletin de la Societe medicale d Afrique noire 22(3): Razali, N., Razab, R., Junit, S.M. and Aziz, A.A Radical scavenging and reducing properties of extracts of cashew shoots (Anacardium occidentale L.), Food Chemistry 111: Shukri, M.A. and Alan, C Analysis of phenolics in Anacardium occidentale shoot extracts
7 using a reversed-phase high performance liquid chromatography tandem mass spectrometry (RP- HPLC-MS). Journal of Tropical Agriculture and Food Science 38(2): Sulaiman, S.F., Sajak, A.A.B., Ooi, K.L., Supriatno and Seow, E.M Effect of solvents in extracting polyphenols and antioxidants of selected raw vegetables. Journal of Food Composition and Analysis 24: Syaharuddin, Padmawinata, K. dan Soetarno, S Isolasi dan penentuan struktur beberapa senyawa kimia dalam daun jambu mete (Anacardium occidentale L., Anacardiaceae). Bandung, Indonesia: School of Pharmacy Institut Teknologi Bandung, MSc thesis. Torres-Piedra, M., Figueroa, M., Hernandez-Abreu, O., Ibarra-Barajas, M., Navarrete-Varquez, G. and Estrada- Soto, S Vasorelaxant effect of flavonoids through calmodulin inhibitor: ex vivo, in vitro, and in silico approaches. Bioorganic and Medicinal Chemistry 19: Vijayakumar, A.D. and Kalaichelvan, P.T Antioxidant and antimicrobial activity using different extracts of Anacardium occidentale L. IJACBT 2(3): 436:443. Waldron, G.J., Ding, H., Lovren, F., Kubes, P., and Triggle, R Acetylcholine-induced relaxation of peripheral arteries isolated from mice lacking endothelial nitric oxide synthase. British Journal of Pharmacology 128(3): Weseler, A.R. and Bast, A Oxidative stress and vascular function: implications for pharmacologic treatments. Current Hypertension Reports 12(3): World Health Organization and International Society of Hypertension Statement on management of hypertension. Journal of Hypertension 21: Xu, Y.J., Kaur, M., Dhillon, R.S., Tappia, P.S. and Dhalla, N.S Health benefits of sea buckthorn for the prevention of cardiovascular diseases. Journal of Functional Foods 3: Nugroho et al./ifrj 20(1):
TECHNICAL REPORT STUDY CHEMICAL PROPERTIES OF RATTAN SHOOT FROM PLANTATION IN THAILAND by Assistant Professor Dr. Noojaree Prasitpan Chemist Analyzer of the ITTO Project on PD 24/00 Rev.1 (I): Promotion
Original Research Article DIFFERENT FRACTIONS OF ANTIOXIDANT ACTIVITY OF SOME MEDICINAL PLANT Baburao Bhukya* Nethaji Institute of Pharmaceutical Sciences, Somidi, Kazipet, Warnagal, Telangana State-506003.
Aidilla Mubarak School of Medicine and Pharmacology and School of Plant Biology Faculty of Medicine, Dentistry and Health Sciences Faculty of Natural and Agricultural Sciences Aidilla completed a Bachelor
Anticancer Effects of and Direction of Research on Hippophae Xu Mingyu (Xiyuan Hospital, Institute of Chinese Medicine, China, Beijing 100091) Seabuckthorn, Hippophae rhamnoides L., belongs to genus Hippophae
Running protein gels and detection of proteins 1. Protein concentration determination using the BIO RAD reagent This assay uses a colour change reaction to give a direct measurement of protein concentration.
ab185915 Protein Sumoylation Assay Ultra Kit Instructions for Use For the measuring in vivo protein sumoylation in various samples This product is for research use only and is not intended for diagnostic
Nuts & Chocolate: The Perfect Pair Penny Kris-Etherton, The Pennsylvania State University Tuesday, April 12, 2016 8:30 a.m. 1. Research has shown positive health benefits for consumption of small amounts
ISLATIN F CAFFEINE FRM TEA Introduction In this experiment, caffeine is isolated from tealeaves. The chief problem with the isolation is that caffeine does not exist alone in the tealeaves, but other natural
TANNIC ACID Prepared at the 39th JECFA (1992), published in FNP Add 1 (1992) superseding specifications prepared at the 35th JECFA (1989), published in FNP 49 (1990) and in FNP 52 (1992). Metals and arsenic
Analysis of Riboflavin in a Vitamin Pill by Fluorescence Spectroscopy** Objectives In this lab, you will use fluorescence spectroscopy to determine the mass and percentage of riboflavin in a vitamin pill.
SODIUM CARBOXYMETHYL CELLULOSE Prepared at the 28th JECFA (1984), published in FNP 31/2 (1984) and in FNP 52 (1992). Metals and arsenic specifications revised at the 55 th JECFA (2000). An ADI not specified
Human serum albumin (HSA) nanoparticles stabilized with intermolecular disulfide bonds Wentan Wang, Yanbin Huang*, Shufang Zhao, Ting Shao and Yi Cheng* Department of Chemical Engineering, Tsinghua University,
99m Tc-Hematoporphyrin linked albumin nanoparticles for lung cancer targeted photodynamic therapy and imaging Su-Geun Yang, Ji-Eun Chang, Byungchul Shin, Sanghyun Park, Kun Na and Chang-Koo Shim* *Corresponding
AAGPs TM Anti-Aging Glyco Peptides Enhancing Cell, Tissue and Organ Integrity Molecular and biological attributes of lead AAGP molecule 1 Acknowledgements This presentation was prepared by Dr. Samer Hussein
International Journal of PharmTech Research CODEN( USA): IJPRIF ISSN : 0974-4304 Vol.1, No.3, pp 870-874, July-Sept 2009 CLINICAL STUDY OF MARKETED AYURVEDIC PREPARATION IN THYROID INDUCED STRESS A. A.
Polyphenols in your diet may regulate food intake Role of dietary polyphenols in food intake Frontier Voice of Nutrition Remarks (May 06, 2013) Nalin Siriwardhana, Ph.D., interviewed Dr. Kiran Panickar,
Fighting the Battles: Conducting a Clinical Assay 6 Vocabulary: In Vitro: studies in biology that are conducted using components of an organism that have been isolated from their usual biological surroundings
D. A. Lewis Anti-Inflammatory Drugs from Plant and Marine Sources 1989 BirkhauserVerlag Basel Boston Berlin TABLE OF CONTENTS Page No. Preface. Acknowledgements..12 SECTION I : INFLAMMATION AND INFLAMMATORY
ISO Standards for Tea Intergovernmental Group- on Tea 10 th Session New Dehli, 12-14 May 2010 Andrew Scott, Chairman ISO TC/34 SC 8 Tea 1 2 Tea Over 3 million tonnes produced In over 30 countries Providing
Genomic DNA Extraction Kit INSTRUCTION MANUAL Table of Contents Introduction 3 Kit Components 3 Storage Conditions 4 Recommended Equipment and Reagents 4 Introduction to the Protocol 4 General Overview
The Physiology of Hyperbaric Oxygen Therapy Free Radicals and Reactive Oxygen Species I. Introduction Definition, Source, function and Purpose A. Definition of free radicals and reactive oxygen species
Chemical analysis service, Turner s Green Technology Group Hourly costs, Academic collaborations leading to co-publications: 627 SEK/hr (excl. VAT) Hourly costs, Industry/Academic collaborations, no publications:
Functional properties of fructans and their potential role in management of intestinal inflammatory pathologies Dr.ssa Valentina Pasqualetti Unità di Ricerca di Scienze degli Alimenti e della Nutrizione
CELLULOSE CHEMISTRY AND TECHNOLOGY TRANSFORMATION OF POLYPHENOLS FROM BIOMASS BY SOME YEAST SPECIES ANCA-ROXANA HAINAL, IOANA IGNAT, IRINA VOLF and VALENTIN I. POPA Gheorghe Asachi Technical University
Introduction Type 2 diabetes mellitus (t2dm) is the most prevalent form of diabetes worldwide. It is characterised by high fasting and high postprandial blood glucose concentrations (hyperglycemia). Chronic
HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for
INTRODUCTION HOW MUCH ASPIRIN, ACETAMINOPHEN, AND CAFFEINE ARE IN YOUR PAIN RELIEVER? USING HPLC TO QUANTITATE SUBSTANCES (Revised: 1-13-93) Headache, sore muscles, arthritis pain... How do you spell relief?
Cautions: 6 M hydrochloric acid is corrosive. Purpose: To colorimetrically determine the mass of iron present in commercial vitamin tablets using a prepared calibration curve. Introduction: Iron is considered
icell Skeletal Myoblasts Application Protocol Introduction Modulating Glucose Uptake in Skeletal Myotubes: Insulin Induction with Bioluminescent Glucose Uptake Analysis The skeletal muscle is one of the
SUCRALOSE Prepared at the 41st JECFA (1993), published in FNP 52 Add 2 (1993). Metals and arsenic specifications revised at the 63rd JECFA (2004). An ADI of 0-15 mg/kg bw was established at the 37th JECFA
EXPERIMENT 11 UV/VIS Spectroscopy and Spectrophotometry: Spectrophotometric Analysis of Potassium Permanganate Solutions. Outcomes After completing this experiment, the student should be able to: 1. Prepare
Units Units Theory of absorbance Light absorption occurs when atoms or molecules take up the energy of a photon of light, thereby reducing the transmission of light as it is passed through a sample. Light
Sex Hormone Testing by Mass Spectrometry Robert L. Fitzgerald, PhD, DABCC Professor of Pathology University of California-San Diego San Diego, CA, 92161 email@example.com Learning Objectives After this
REGULATIONS FOR THE DEGREE OF BACHELOR OF PHARMACY IN CHINESE MEDICINE [PART-TIME] (BPharm[ChinMed]) These regulations apply to students admitted to the first year of study in and after the academic year
Case Study: Factors that Affect Blood Pressure Instructor Version Goal This activity (case study and its associated questions) is designed to be a student-centered learning activity relating to the factors
Asia Pac J Clin Nutr 07;16 (Suppl 1):148-152 148 Original Article Study on the antioxidant activity of tea flowers (Camellia sinensis) Ziying Yang AM, Yi Xu AB, Guoliang Jie AB, Puming He PhD and Youying
Automated Solid Phase Extraction (SPE) of EPA Method 1694 for Pharmaceuticals and Personal Care Products in Large Volume Water Samples Keywords Application Note ENV0212 This collaboration study was performed
A ovel Approach to Quantify Unbound Cisplatin, Carboplatin, and xaliplatin in Human Plasma Ultrafiltrate by Measuring Platinum-DDTC Complex Using LC/M/M Min Meng, Ryan Kuntz, Al Fontanet, and Patrick K.
October 2006 RESTRICTED LUMEFANTRINE Draft proposal for The International Pharmacopoeia (October 2006) DRAFT FOR DISCUSSION World Health Organization 2006 All rights reserved. This draft is intended for
Analysis of Riboflavin in a Vitamin Pill by Fluorescence Spectroscopy Objectives In this lab, you will use fluorescence spectroscopy to determine the mass of riboflavin in a vitamin pill. Riboflavin fluorescence
APPLE BROWNING: A STUDY OF OXIDATION OF FOODS 2005, 1997 by David A. Katz. All rights reserved. Reproduction permitted for education use provided original copyright is included. You are preparing for a
UNIT: Proteins 16tproteins.wpd Task Determination of Total Protein, Albumin and Globulins Objectives Upon completion of this exercise, the student will be able to: 1. Explain the ratio of albumin and globulin
Vol. 27. No. 3. 1983 (151) A Sensitive Assay Method of Creatine Kinase-MB Isoenzyme in Human Serum Using Anti-CK-M Antiserum and Firefly Luciferase* Toshio Imai**, Mitsutaka Yoshida**, Kohichi Suzuki***
LAB 5 PLANT NUTRITION I. General Introduction All living organisms require certain elements for their survival. Plants are known to require carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus
MASS SPECTROMETRIC IDENTIFICATION OF SOME SULPHUR CONTAINING PHENALKYLAMINE DESIGNER DRUGS * Ingrid J. BOSMAN 1, Douwe DE BOER 2, Edwin B. SIDERIUS 1, Lesseps J. A. L. DOS REYS 2, Robert A. A. MAES 1 1
Seahorse XF Cell Mito Stress Test Kit Part # 103015-100 User Guide For use with Seahorse XF e and XF Extracellular Flux Analyzers For Research Use Only 1 Table of Contents Product Description... 2 Introduction...
HIS-Select Nickel Affinity Gel Catalog Number P6611 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description HIS-Select Nickel Affinity Gel is an immobilized metalion affinity chromatography (IMAC)
Antioxidant Products A comprehensive range of ALB BIL FERR GR Ransel Ransod antioxidant TAS testing TIBC kits TF BIL FERR GR Ransel Ransod TAS TIBC TF UA A FERR GR Ransel Ransod TAS TIBC TF UA ALB GR Ransel
Isolation of Caffeine from Tea Introduction A number of interesting, biologically active compounds have been isolated from plants. Isolating some of these natural products, as they are called, can require
NEOS-GR Rapid, Solvent-Free Extraction by Microwave Hydrodiffusion and Gravity (MHG) NEOS-GR Microwave Hydrodiffusion and Gravity (MHG) System The NEOS-GR is the result of a partnership between Milestone
An Investigation into the Structure, Properties and Synthesis of 3-nitrobenzoic Acid. Jane Smith Chemistry 101 Laboratory, Section 43 Instructor: Marie Curie January 2, 2003 Abstract This paper describes
Plant Genomic DNA Extraction using CTAB Introduction The search for a more efficient means of extracting DNA of both higher quality and yield has lead to the development of a variety of protocols, however
BIOLOGICALLY ACTIVE COMPOUNDS IN ROASTED COFFEE Viesturs Kreicbergs, Fredijs Dimins*, Velga Mikelsone, Ingmars Cinkmanis Latvia University of Agriculture, Department of Chemistry, Liela street 2, Jelgava,
Version 1 2015 Module guide International Master Program Cardiovascular Science University of Göttingen Part 1 Theoretical modules Synopsis The Master program Cardiovascular Science contains four theoretical
Free radicals - Enemies of health, beauty and youth Free radicals (FR) are natural molecules produced in the body following biochemical reactions related to the behaviour of singlet oxygen. The human body
Available online www.jocpr.com Journal of Chemical and Pharmaceutical Research, 2012, 4(7):3483-3488 Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5 Simultaneous UV spectrphotometric estimation of
Is Exercise the Best Antioxidant Supplement? Len Kravitz, Ph.D. As an unexpected consequence of the metabolic steps that convert food into energy, the body produces molecules commonly called free radicals.
INDIGO DYEING OF VARIOUS COLORS WITH POLYPHENOLS, AND INVENTING NEW POLYPHENOL SENSOR UTILIZING INDIGO DYEING OF VARIOUS COLORS Dong Eun LEE 1, Seong Hyeon BAE 1, Da Hong KIM 1 1 Busan Jang-An High School-8-16,
DP Chemistry Review Topic 1: Quantitative chemistry 1.1 The mole concept and Avogadro s constant Assessment statement Apply the mole concept to substances. Determine the number of particles and the amount
Rubisco; easy Purification and Immunochemical Determination Ulrich Groß Justus-Liebig-Universität Gießen, Institute of Plant Nutrition, Department of Tissue Culture, Südanlage 6, D-35390 Giessen e-mail:
Purdue-UAB Botanicals Center for Age-Related Disease MS/MS analysis of Polyphenols Jeevan Prasain Ph.D. Pharmacology & Toxicology UAB Polyphenols H Caffeic acid Phenolic acids and derivatives H EGC (Flavanol)
Heart Failure: Diagnosis and Treatment Approximately 5 million people about 2 percent of the U.S. population are affected by heart failure. Diabetes affects 20.8 million Americans and 65 million Americans
Biol3460 Lab 1 1 Biology 3460 - Plant Physiology - Lab Exercise 2 Basic Lab Techniques Objectives: This lab is intended to: (1) provide an introduction to basic techniques used in biology labs (2) provide
Designation: D 4628 02 An American National Standard Standard Test Method for Analysis of Barium, Calcium, Magnesium, and Zinc in Unused Lubricating Oils by Atomic Absorption Spectrometry 1 This standard
BSc in Medical Sciences with PHARMACOLOGY Course Director Dr Christopher John Module Leaders Dr Robert Dickinson (Module 1) Dr Anabel Varela Carver (Module 2) Dr Sohag Saleh (Module 3) Course Administrator
1 Lab 2 Biochemistry Learning Objectives The lab has the following learning objectives. Investigate the role of double bonding in fatty acids, through models. Developing a calibration curve for a Benedict
#3. Acid - Base Titrations 27 EXPERIMENT 3. ACID-BASE TITRATIONS: DETERMINATION OF CARBONATE BY TITRATION WITH HYDROCHLORIC ACID BACKGROUND Carbonate Equilibria In this experiment a solution of hydrochloric
Hydrogen Peroxide Cell-Based Assay Kit Item No. 600050 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION
TRI Reagent For processing tissues, cells cultured in monolayer or cell pellets Catalog Number T9424 Store at room temperature. TECHNICAL BULLETIN Product Description TRI Reagent is a quick and convenient
The work presented in the thesis is focused on the problems related to the prostate gland. Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are the two major problems associated with prostate.
Islet Viability Assessment by Single Cell Flow Cytometry Page 1 of 8 Purpose: To comprehensively assess the viability of the islet cell preparation prior to transplantation. Tissue Samples: A sample containing
LOOK, FEEL AND LIVE BETTER Topical Skin Care Pycnogenol in Topical Skin Care Pycnogenol is widely used in topical and oral applications for various dermatological indications. A unique combination of pharmacological
ISSN: 0973-4945; CODEN ECJHAO E- Chemistry http://www.e-journals.net Vol. 5, No.3, pp. 427-430, July 2008 High Performance Thin Layer Chromatographic Method for Estimation of Cefprozil in Tablet Dosage
INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE SAFETY PHARMACOLOGY STUDIES FOR HUMAN PHARMACEUTICALS
PHYTOCHEMICAL STUDY OF SOME ACTIVE PRINCIPLES WITH ANTIOXIDANT ACTION FROM THE ROSMARINUS OFFICINALIS AND SALVIA OFFICINALIS SPECIES IONELA DACIANA MIERLICI * Keywords: hydroalcoholic extract, sage, rosemary,
Strokes and High Blood Pressure Quick Review from last Week: What is a Stroke Stroke: blood supply to the brain is severely limited or cut off With no blood to carry oxygen to them, brain cells will die
International Journal of Research and Reviews in Pharmacy and Applied science www.ijrrpas.com P.V.V.Satyanarayana*, Alavala Siva Madhavi NEW SPECTROPHOTOMETRIC METHODS FOR THE QUANTITATIVE ESTIMATION OF
Comparative Studies of the Chemical Constituents of Green Tea A Thesis Submitted in Partial Fulfillment of the Requirements for the Master Degree of Science in Chemistry (Analytical Chemistry) By Tharwat
HEXANES Prepared at the 51st JECFA (1998), published in FNP 52 Add 6 (1998) superseding specifications prepared at the 14th JECFA (1970), published in NMRS 48B (1971) and in FNP 52 (1992). ADI "limited
Guide to Reverse Phase SpinColumns Chromatography for Sample Prep www.harvardapparatus.com Contents Introduction...2-3 Modes of Separation...4-6 Spin Column Efficiency...7-8 Fast Protein Analysis...9 Specifications...10
HighPure Maxi Plasmid Kit For purification of high pure plasmid DNA with high yields www.tiangen.com PP120109 HighPure Maxi Plasmid Kit Kit Contents Storage Cat.no. DP116 Contents RNaseA (100 mg/ml) Buffer