IN VITRO ASSESSMENT OF ANTIOXIDANT AND ANTIPROLIFERATIVE EFFICACY OF ANDROGRAPHIS PANICULATA

IN-VITRO ASSESSMENT OF ANTIOXIDANT AND ANTIPROLIFERATIVE EFFICACY OF ANDROGRAPHIS PANICULATA

G. Deora

Department of Human Genetics, Guru Nanak Dev University, Amritsar - 143005, Punjab, India.

ABSTRACT: The present study investigated the antioxidant and antiproliferative efficacy of Andrographis paniculata, a well-known medicinal plant, using in-vitro systems. The dried leaves of A. paniculata were powdered and subjected to successive solvent extraction taking from polar to non-polar solvents (descending polarity) and simultaneously from non-polar to polar solvents (ascending polarity). Solvents used for extraction were hexane, chloroform, ethyl acetate, acetone, methanol, and water. All the extracts were screened for the presence or absence of various secondary metabolites. The antioxidant activity of all extracts at 100 μg/ml was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. Further DPPH assay and reducing power assay were used to check the free radical scavenging capacity of methanolic and water extracts at their lower concentrations. The methanolic and water extracts of A. paniculata exhibited maximum DPPH inhibition of 20.75% and 17.71% respectively, at 100 μg/ml. A positive correlation between reducing power and concentration was found. Thin Layer Chromatography (TLC) of methanolic and water extracts of A. paniculata was performed. The antiproliferative effect of different concentrations of methanolic and water extracts on C6 glioma cells was investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The methanolic extract exhibited 50% inhibition of proliferation of C6 glioma cells at much lower concentration (12.5 μg/ml) than did the water extract (200 μg/ml). This study is a novel approach towards the antioxidant and antiproliferative role of methanolic and water extracts of A. paniculata which suggests its anticancer potential.

Andrographis paniculata, Antioxidant activity, DPPH, Reducing power, Antiproliferative, MTT

INTRODUCTION: Medicinal plants are being used in traditional system of medicine from hundreds of years in many countries of the world due to their therapeutic properties ¹, which are very useful in healing various diseases and the advantage of these plant-based traditional medicines is their being 100% natural ².

Andrographis paniculata, highly reputed as “Kalmegh” and “King of Bitters”, is one of the important herbs comprising of several medicinal properties. A. paniculata is well known traditional medicinal plant species with a bright economic horizon, belonging to Acanthaceae family, found throughout Southeast Asia ³.

The bitterness of A. paniculata is related to its various pharmacological properties ⁴. Diterpenoids and flavonoids are the main chemical constituents of A. paniculata, which are believed to be responsible for most of the biological activities of this plant ⁵.

Free radicals, also known as reactive oxygen species (ROS), play a role in the etiology of several major diseases, including cancer. Reactive oxygen species (ROS) cause DNA strand breakage, sister chromatid exchanges and DNA–DNA and DNA–protein cross-links, in addition to base modifications, which have been associated with carcinogenesis ⁶. Since free radicals play a key role in the pathology of diseases, the supply of antioxidants, via the food chain, is of great importance for a healthy life ⁷.

The present study aimed to elucidate whether leaf extracts of A. paniculata has antioxidant and antiproliferative activity. As many of the diseased conditions (like cancer, atherosclerosis, cardio-vascular diseases, and degenerative disorders) are due to oxidative stress, the antioxidant potential of leaf extracts of A. paniculata was studied by performing DPPH and reducing power assays.

This study was undertaken because the leaves of A. paniculata are well known for their antioxidant property ⁸. C6 glioma cell line was used, and MTT assay was done to study the antiproliferative effect of methanolic and water leaf extracts of A. paniculata.

MATERIALS AND METHODS:

Materials: 2,2-diphenyl-1-picrylhydrazyl (DPPH) from Sigma Aldrich, USA. All other chemicals like ninhydrin reagent, ammonia, pyridine, sodium nitroprusside, ether, lead acetate, acetic acid, sodium hydroxide, potassium hydroxide, dilute hydrochloric acid, dilute acetic acid, concentrated sulphuric acid, ferric chloride, acetic anhydride, L-ascorbic acid, sodium hydroxide, trichloroacetic acid, phosphate buffer, potassium ferricyanide, ferric chloride, and other solvents were of analytical grade from CDH.

DMEM (Dulbecco’s Modified Eagle’s Medium), Hank’s balanced salt solution (HBSS) and FBS (foetal bovine serum) from HIMEDIA, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) from Life Technologies.

Methods:

Preparation of Different Extracts of A. paniculata: Extraction method involved separation of medicinally active fractions of plant tissue from inactive/inert components by using selective solvents and extraction technology. Solvents diffuse into the solid plant tissues and solubilize compounds of similar polarity ⁹.

The dried leaves of A. paniculata were powdered and subjected to successive solvent extraction taking from polar to non-polar solvents (descending polarity) and simultaneously from non-polar to polar solvents (ascending polarity). Solvents used were water, methanol, acetone, ethyl acetate, chloroform, and hexane.

For extraction according to ascending polarity, 25 gram of powered plant leaf material was dissolved in 100 ml of hexane and was placed in orbit shaker at 130 rpm for 24 h at room temperature. The extract was filtered with the help of muslin cloth, and the filtrate was then subjected to centrifugation at 12000 rpm for 30 min at 5 ºC.

The residue obtained was then dissolved in chloroform and was subjected to the same processing as shown in Flow chart 1. Similarly, extraction was performed in descending polarity from polar to non-polar solvents as described in Flow chart 2.

Estimation of the Concentration of Extracts: An empty Petri plate was weighed. 1ml of descending water extract was added to it. The Petri plate was kept in the incubator overnight at 37 ºC and again weighed. The concentration was calculated using the formula:

Conc. =  y – x  =  a g/ ml

Y = weight of Petri plate after incubation

X = weight of empty Petri plate

The concentration of all extracts was calculated similarly.

Phytochemical Screening of Plant Extracts: Standard screening tests of extracts were carried out for various plant bioactive constituents. All the extracts were screened for the presence or absence of various secondary metabolites like amino acids, anthraquinones, glycosides, steroids, flavonoids, phytosterols, saponins, tannins, and triterpenoids.

FLOW CHART 1: EXTRACTION BY MACERATION OF LEAF POWDER OF A. PANICULATA BY INCREASING ORDER OF SOLVENT POLARITY

FLOW CHART 2: EXTRACTION BY MACERATION OF LEAF POWDER OF A. PANICULATA BY DECREASING ORDER OF SOLVENT POLARITY

Antioxidant Activity: Antioxidant activity of the extracts was analyzed by two methods:

DPPH Assay: The DPPH radical scavenging assay ¹⁰, an easy, rapid, and sensitive method was used to determine the antioxidant activity. The radical scavenging activities of the plant extracts were determined using UV spectrophotometer at 550 nm. DPPH (0.1 mM) was prepared in methanol. 2 ml of DPPH was added to 300 µl of 100 µg/ml concentration of each extract. Blanks were prepared to contain 2 ml of DPPH and 300 µl of solvent in which extract was prepared. Then the absorbance was measured at 550 nm at zero and twenty minutes. A decrease in the absorbance of the reaction mixture after twenty minutes indicates free radical scavenging activity. The further assay was performed on different concentrations of ascending polarity methanolic and descending polarity water extracts (chosen on the basis of the DPPH assay results and presence of maximum phytochemicals) starting from a concentration of 10 to 100 µg/ml. Vitamins C (Ascorbic acid) was used as the antioxidant standard. The radical scavenging activity was calculated as % inhibition of DPPH.

% Inhibition = A control – A test × 100 / A control

A control is the absorbance of the blank and

A test is the absorbance of the extract.

Reducing Power Assay: Antioxidant activity was also determined by Ferric ion Reducing Antioxidant Power (FRAP) assay ¹¹. Different concentrations (10 to 190 µg/ml) of the ascending polarity methanolic and descending polarity water extracts were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide (2.5 ml, 1% w/v). The mixture was incubated at  50 ºC for 20 min. Trichloroacetic acid (2.5 ml, 10%) was added to each mixture, which was then centrifuged at 1036 rpm for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride solution (2.5 ml, 0.1%). The absorbance was measured at 700 nm. A blank was prepared without adding extract. Increased absorbance of the reaction mixture indicates an increase in reducing power.

Thin-layer Chromatography: Thin-layer chromatography (TLC) was employed in this study to analyze the compounds present in the ascending polarity methanolic and descending polarity water extracts. Normal phase silica gel pre-coated TLC plates were used. Thin Layer Chromatography (TLC) of methanolic and water extract of A. paniculata was performed on chloroform: methanol (191:9) and chloroform: methanol (7:3) solvent systems respectively. The distances of bands from origin were measured, and R_f (retention factor) values were calculated.

Antiproliferative Assay: Antiproliferative activity of ascending polarity methanolic and descending polarity water extract was tested on C6 glioma cells using MTT test ¹². Undifferentiated cells were subcultured by trypsinization (0.1% trypsin) when they reached 70-80% confluency. The experiment was performed in 96 well plates. Two replicates were used for the analysis of each concentration. C6 glioma cell line (40000 cells/ml) was treated with serially diluted concentrations of extracts for 24 h. The optic absorbance of each sample was measured at a wavelength of 595 nm using ELISA Reader.

% Viability = Mean Absorbance of Sample × 100 / Mean Absorbance of Control

The relationship between the concentration of extracts of A. paniculata and % viability was determined.

RESULTS AND DISCUSSION: Although A. paniculata has long served as traditional medicine, very few authentic studies evaluating its properties are available. In-vitro studies on A. paniculata have shown the presence of many bioactive constituents ¹³ having pharmacological as well as medicinal properties  which include its activities as anti-snakebite ^{14, 15}, anti-diabetic ^{16, 17, 18, 19, 20}, anti-cancer ^{16, 21, 22, 23, 24, 25, 26, 27, 28}, cytochrome inhibitory ^{29, 30}, anti-viral ^{16, 31}, anti-protozoal ³², anti-malarial ³³, cardio-protective ³⁴, anti-inflammatory ^{16,  35,  36,  37, 38, 39, 40}, antioxidant ^{41, 42, 43, 44}, cerebro-protective ^{45, 46}, hepato-protective ^{47, 48}, anti-bacterial ^{16, 42, 44, 49, 50} and anti-human immunodeficiency virus (HIV) ⁵¹.

The concentration of ascending polarity hexane, chloroform, ethyl acetate, acetone, methanol, and water extract was 6, 5, 3, 2, 3, and 7 mg/ml, respectively. The concentration of descending polarity water, methanol, acetone, ethyl acetate, chloroform, and hexane extract was 18, 4, 6, 4, 3, and 6 mg/ml, respectively. The maximum concentration was that of descending polarity water extract (18 mg/ml).

The phytochemical screening of the plant extracts showed the presence of amino acids, flavonoids, glycosides, saponins, steroids, tannins, and triterpenoids as elaborated in Table 1 and 2. Most of the bioactive components were showed to be present in ascending polarity methanolic and descending polarity water extract. These results are consistent with the findings on aqueous extract of A. paniculata, which show the presence of glycosides, saponins, tannins, and alkaloids, but not of anthraquinones ⁵². Diterpenoids and flavonoids are the main chemical constituents of A. paniculata, which are believed to be responsible for the most biological activities of this plant ⁵.

Phenolics, alkaloids, terpenoids, and cardiac glycosides detected in the extracts are compounds that have been documented to possess medicinal properties and health-promoting effects ^{53, 54, 55, 56}.

TABLE 1: PHYTOCHEMICAL CONSTITUENTS OF ASCENDING POLARITY EXTRACTS OF A. PANICULATA

(+) present, (-) absent

TABLE 2: PHYTOCHEMICAL CONSTITUENTS OF DESCENDING POLARITY EXTRACTS OF A. PANICULATA

(+) present, (-) absent

All extracts except ascending polarity hexane showed a decrease in absorbance, but significant decrease in absorbance was showed by ascending polarity methanolic and descending polarity water extract as depicted in Fig. 1 and 2. Further assays were performed on different concentration of water and methanol extract starting from 10 to 100 µg/ml. All concentrations significantly lowered the DPPH free radical scavenging activity. The ascending polarity methanolic extract of A. paniculata had maximum inhibition of 20.75% at 100 µg/ml. The descending polarity water extract of A. paniculata appeared to have maximum inhibition of 17.7% at 100 µg/ml, as shown in Fig. 3 and 4. These results are consistent with the research done on leaf, fruit, and stem extracts of A. paniculata ⁵⁷. However, DPPH scavenging assay is not capable of detecting all anti-oxidants, and therefore, there is a need to use several assays to demonstrate antioxidant activities.

The results of the reducing power assay on ascending polarity methanolic and descending polarity water extracts showed a linear increase in the intensity of blue color as the concentration of extracts increases as depicted in Fig. 5.

An idea about the polarity of various chemical constituents is also obtained while performing TLC analysis. Compound showing high R_f value in the less polar solvent system has low polarity and with less R_f value have high polarity.

Six bands were obtained on staining the TLC plates with iodine vapors for ascending polarity methanolic extract. The distances of bands 1 to 6 from origin were 1.1, 2.1, 2.5, 3.5, 4 and 4.7 respectively.

The solvent front was 7.9 cm, as shown in Fig. 6. The R_f values of bands 1 to 6 were 0.13, 0.26, 0.31, 0.44, 0.50, 0.59 respectively. Five bands were obtained for descending polarity water extract. The distances of bands 1 to 5 from origin were 1.2, 2, 3.4, 4.3 and 5.1 respectively. The solvent front was 7.9 cm, as shown in Fig. 7. The R_f values of bands 1 to 5 were 0.15, 0.25, 0.43, 0.54, and 0.64 respectively.

FIG. 6: THIN LAYER CHROMATOGRAPHY OF ASCENDING POLARITY METHANOLIC EXTRACT OF A. PANICULATA

FIG. 7: THIN LAYER CHROMATOGRAPHY OF DESCENDING POLARITY WATER EXTRACT OF A. PANICULATA

The minimum viability (16.83%) was at the highest concentration of the ascending methanolic extract (200 µg/ml), and maximum viability (99.13%) was at the lowest concentration of extract (1.562 µg/ml). Similarly, the minimum viability (55.17%) was at the highest concentration of the descending polarity water extract (200 µg/ml) and maximum viability (98.11%) was at the lowest concentration of extract (1.562 µg/ml). The ascending polarity methanolic extract exhibited 50% inhibition of proliferation of C6 glioma cells at much lower concentration (12.5 μg/ml) than did the descending polarity water extract (200 μg/ml) as shown in Fig. 8, 9 and 10.

FIG. 10: EFFECT OF ASCENDING POLARITY METHANOLIC AND DESCENDING POLARITY WATER EXTRACTS OF A. PANICULATA ON PROLIFERATION OF C6 GLIOMA CELL LINE ASSESSED BY MTT ASSAY

CONCLUSION: Although A. paniculata is most commonly used in Indian Ayurvedic medicine, but the mechanistic aspects of its effects and isolation of active components are still not explored. The results of the present study provide the evidence of the antioxidant and antiproliferative effect of ascending polarity methanolic and descending polarity water leaf extracts of A. paniculata.

 The presence of various bioactive constituents indicates the therapeutic potentials of A. paniculata. Thus this study is a novel approach towards assessing the cytoprotective and anticancerous role of leaf extracts of A. paniculata. It shows great potential as a safe and effective antineoplastic agent. Further characterization of the A. paniculata leaf extracts to identify the active antiproliferative components will be of immense therapeutical value.

ACKNOWLEDGEMENT: Author is highly thankful to Dr. Vasudha Sambyal, Professor, Department of Human Genetics, Guru Nanak Dev University, Amritsar, Punjab for her valuable guidance and support in writing this paper.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Oubre AY, Carlson TJ, King SR and Reaven EM: From plant to the patient, an ethnomedical approach to the identification of new drugs for the treatment of non-insulin dependent diabetes mellitus. Diabetologia 1997; 40: 614-17.
2. Calixto JB: Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (Phytotherapeutic agents). Brazilian Journal of Medical and Biological Research 2000; 33: 179-89.
3. Valdiani A, Kadir MA, Tan SG, Talei D, Puad MA and Nikzad S: Nain-e Havandi (Andrographis paniculata) present yesterday, absent today: A plenary review on the underutilized herb of Iran’s pharmaceutical plants. Molecular Biology Reports 2012; 39: 5409-24.
4. Coon JT and Ernst E: Andrographis paniculata in the treatment of upper respiratory tract infections: a systematic review of safety and efficacy. Planta Medica 2004; 70: 293-98.
5. Tang W and Eisenbrand G: Chinese drugs of plant origin, chemistry, pharmacology and use in traditional and modern medicine. Springer Verlag, Berlin 1992; 97-103.
6. Cadenas E and Davies KJA: Mitochondrial free radical generation, oxidative stress, and aging. Free Radical Biology and Medicine 2000; 29: 222-30.
7. Scalbert A and Williamson G: Dietary intake and bioavailability of polyphenols. Journal of Nutrition 2000; 130: 2073-85.
8. Trivedi N and Rawal UM: Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice. Indian Journal of Experimental Biology 2001; 39: 41-46.
9. Green RJ: Antioxidant Activity of Peanut Plant Tissues. Master’s Thesis. North Carolina State University. USA 2004.
10. Blois MS: Antioxidant determinations by the use of a stable free radical. Nature 1958; 26: 1199-00.
11. Oyaizu M: Studies on product of browning reaction prepared from glucose amine. Japanese Journal of Nutrition 1986; 44: 307-15.
12. Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 1983; 65:55-63.
13. Chao WW and Lin BF: Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian). Chinese Medicine 2010; 5: 1-17.
14. Samy RP, Thwin MM, Gopalakrishnakone P and Ignacimuthu S: Ethnobotanical survey of folk plants for the treatment of snake bites in the southern part of Tamilnadu, India. Journal of Ethnopharmacology 2008; 115: 302-12.
15. Meenatchisundaram S, Parameswari G and Michael A: Studies on antivenom activity of Andrographis paniculata and Aristolochia indica plant extracts against Daboia russelli venom by in-vivo and in-vitro Indian Journal of Science and Technology 2009; 2: 76-79.
16. Aromdee C: Modifications of andrographolide to increase some biological activities: a patent review (2006–2011). Expert Opinion on Therapeutic Patents 2012; 22: 169-80.
17. Subramanian R, Asmawi MZ and Sadikun A: In-vitro alpha-glucosidase and alpha-amylase enzyme inhibitory effects of Andrographis paniculata extract and andrographolide. Acta Biochimica Polonica 2008; 55: 391-98.
18. Wibudi A, Kiranadi B, Manalu W, Winarto A and Suyono S: The traditional plant, Andrographis paniculata (Sambiloto), exhibits insulin-releasing actions in vitro. Acta Medica Indonesia 2008; 40: 63-68.
19. Nugroho AE, Andrie M, Warditiani NK, Siswanto E, Pramono S and Lukitaningsih E: Antidiabetic and antihyperlipidemic effect of Andrographis paniculata (Burm. f.) Nees and andrographolide in high-fructose-fat-fed rats. Indian Journal of Pharmacology 2012; 44: 377-81.
20. Xu J, Li Z, Cao M, Zhang H, Sun J, Zhao J, Zhou Q, Wu Z and Yang L: Synergetic effect of Andrographis paniculata polysaccharide on diabetic nephropathy with andrographolide. International Journal Biological Macromolecules 2012; 51: 738-42.
21. Verma N and Vinayak M: Antioxidant action of Andrographis paniculata on lymphoma. Molecular Biology Reports 2008; 35: 535-40.
22. Zhao F, He EQ, Wang L and Liu K: Anti-tumor activities of andrographolide, a diterpene from Andrographis paniculata, by inducing apoptosis and inhibiting VEGF level. Journal of Asian Natural Products Research 2008; 10: 467-73.
23. Chun JY, Tummala R, Nadiminty N, Lou W, Liu C, Yang J, Evans CP, Zhou Q and Gao AC: Andrographolide, an herbal medicine, inhibits interleukin-6 expression and suppresses prostate cancer cell growth. Genes and Cancer 2010; 1: 868-76.
24. Lee MJ, Rao YK, Chen K, Lee YC, Chung YS and Tzeng YM: Andrographolide and 14-deoxy-11,12-didehydroa-ndrographolide from Andrographis paniculata attenuate high glucose-induced fibrosis and apoptosis in murine renal mesangeal cell lines. Journal of Ethnopharmacology 2010; 132: 497-05.
25. Yang S, Evens AM, Prachand S, Singh AT, Bhalla S, David K and Gordon LI: Mitochondrialmediated apoptosis in lymphoma cells by the diterpenoid lactone andrographolide, the active component of Andrographis paniculata. Clinical Cancer Research 2010; 16: 4755-68.
26. Mulukuri NVLS, Monda NB, Prasad MR, Renuka S, Ramakrishna K: Isolation of diterpenoid lactones from the leaves of Andrographis paniculata and its anticancer activity. International Journal of Pharmacognosy Phytochemical Research 2011; 3: 39-42.
27. Cheung MT, Ramalingam R, Lau KK, Chiang WL, and Chiu SK: Cell type-dependent effects of andrographolide on human cancer cell lines. Life Sci 2012; 91: 751- 60.
28. Zhang ZR, Zaharna MA, Wong MMK, Chiu SK and Cheung HY: Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation. PLoS ONE 2013; 8: e54577.
29. Pekthong D, Martin H, Abadie C, Bonet A, Heyd B, Mantion G and Richert L: Differential inhibition of rat and human cytochrome P450 by Andrographis paniculata extract and andrographolide. Journal of Ethnopharmacology 2008; 115: 432-40.
30. Ooi JP, Kuroyanagi M, Sulaiman SF, Muhammad TST and Tan LM: Andrographolide and 14-Deoxy-11,12-Didehydroandrographolide inhibit cytochrome P450s in HepG2 hepatoma cells. Life Sciences 2011; 88: 447-54.
31. Chen JX, Xue HJ, Ye WC, Fang BH, Liu YH, Yuan SH, Yu P and Wang YQ: Activity of andrographolide and its derivatives against influenza virus in-vivo and in-vitro. Biological and Pharmaceutical Bulletin 2009; 32: 1385-91.
32. Dua VK, Verma G and Dash AP: In-vitro antiprotozoal activity of some xanthones isolated from the roots of paniculata. Phytotherapy Research 2009; 23: 126-28.
33. Mishra K, Dash AP, Swain BK and Dey N: Anti-malarial activities of Andrographis paniculata and Hedyotis corymbosa extracts and their combination with curcumin. Malaria Journal 2009; 8: 26-35.
34. Ojha SK, Nandave M, Kumari S and Arya DS: Antioxidant Activity of Andrographis paniculata in Ischemic Myocardium of Rats. Global Journal of Pharmacology 2009; 3: 154-57.
35. Chao WW, Kuo YH, Li WC and Lin BF: The production of nitric oxide and prostaglandin E2 in peritoneal macrophages is inhibited by Andrographis paniculata, Angelica sinensis and Morus alba ethyl acetate fractions. Journal of Ethnopharmacology 2009; 122: 68-75.
36. Chao WW, Kuo YH and Lin BF: Anti-inflammatory Activity of New Compounds from Andrographis paniculata by NF-κB Trans-Activation inhibition. Journal of Agricultural and Food Chemistry 2010; 58: 2505-12.
37. Hsieh CY, Hsu MJ, Hsiao G, Wang YH, Huang CW, Chen SW, Jayakumar T, Chiu PT, Chiu YH and Sheu JR: Andrographolide enhances nuclear factor-?B subunit p65 Ser536 dephosphorylation through activation of protein phosphatase 2A in vascular smooth muscle cells. Journal of Biological Chemistry 2011; 286: 5942-55.
38. Lu WJ, Lee JJ, Chou DS, Jayakumar T, Fong TH, Hsaio G and Sheu JR: A novel role of andrographolide, an NF-kappa B inhibitor, on inhibition of platelet activation: the pivotal mechanisms of endothelial nitric oxide synthase/cyclic GMP. Journal of Molecular Medicine 2011; 89: 1261-73.
39. Lu WJ, Lin KH, Hsu MJ, Chou DS, Hsaio G and Sheu JR: Suppression of NF?B signaling by andrographolide with a novel mechanism in human platelets: regulatory roles of the p38 MAPK-hydroxyl radical-ERK2 cascade. Biochemical Pharmacology 2012; 84: 914-24.
40. Tzeng YM, Lee YC, Cheng WT, Shih HN, Wang HC, Rao YK and Lee MJ: Effects of andrographolide and 14-deoxy-11,12-didehydroandrographolide on cultured primary astrocytes and PC12 cells. Life Sciences 2012; 90: 257-66.
41. Lin FL, Wu SJ, Lee SC and Ng LT: Antioxidant, antioedema and analgesic activities of Andrographis paniculata extracts and their active constituent andrographolide. Phytotherapy Research 2009; 23: 958-64.
42. Premanath R and Devi NL: Antibacterial, antifungal and antioxidant activities of Andrographis paniculata leaves. International Journal Pharmaceutical Science Research 2011; 2: 2091-99.
43. Wasman SQ, Mahmood AA, Chua LS, Alshawah MA and Hamdan S: Antioxidant and gastroprotective activities of Andrographis paniculata (Hempedu Bumi) in Sprague Dawley rats. Indian Journal of Experimental Biology 2011; 49: 767-72.
44. Malahubban M, Alimon AR, Sazili AQ, Fakurazi S and Zakry FA. Phytochemical analysis of Andrographis paniculata and Orthosiphon stamineus leaf extracts for their antibacterial and antioxidant potential. Tropical Biomedicine 2013; 30: 467-80.
45. Das S, Gautam N, Dey SK, Maiti T and Roy S: Oxidative stress in the brain of nicotine-induced toxicity: protective role of Andrographis paniculata Nees and vitamin E. Applied Physiology, Nutrition and Metabolism 2009; 34: 124-35.
46. Radhika P, Annapurna A and Rao SN: Immunostimulant, cerebroprotective & nootropic activities of Andrographis paniculata leaves extract in normal & type 2 diabetic rats. Indian Journal of Medical Research 2012; 135: 636-41.
47. Devaraj S, Jegathambigai R, Kumar P, Sivaramakrishnan S: A study on the hepatoprotective effect of Andrographis paniculata (Burm. F) Nees on mice. Journal of Phytology 2010; 2: 25-30.
48. Roy DN, Mandal S, Sen G, Mukhopadhyay S and Biswas T: 14- Deoxyandrographolide desensitizes hepatocytes to tumor necrosis factor-alpha-induced apoptosis through calcium-dependent tumor necrosis factor receptor superfamily member 1A release via the NO/cGMP pathway. British Journal of Pharmacology 2010; 160: 1823-43.
49. Roy S, Rao K, Bhuvaneswari C, Giri A and Mangamoori LN: Phytochemical analysis of Andrographis paniculata extract and its antimicrobial activity. World Journal of Microbiology and Biotechnology 2010; 26: 85-91.
50. Sule A, Ahmed QU, Samah OA and Omar MN: Screening for antibacterial activity of Andrographis paniculata used in Malaysian folkloric medicine: A possible alternative for the treatment of skin infections. Ethnobotanical Leaflets 2010; 14: 445-56.
51. Tang C, Liu Y, Wang B, Gu G, Yang L, Zheng Y, Qian H and Huang W: Synthesis and Biological Evaluation of Andrographolide Derivatives as Potent Anti-HIV Agents. Archives of Pharmaceutical and Medicinal Chemistry 2012; 345: 647-56.
52. Sunday JA, Obiageri OO, Uford SI, Mujitaba SA and Magaji G: Quality Control Tests on Andrographis paniculata Nees (Family: Acanthaceae) -an Indian ‘Wonder’ Plant Grown in Nigeria. Tropical Journal of Pharmaceutical Research 2010; 9: 387-94.
53. Salah W, Miller NJ, Pagauga G, Bolwell GP, Rice E and Evans C: Polyphenolic flavonoids as a scavenger of aqueous phase radicals and chain-breaking antioxidants. Archives of Biochemistry and Biology 1995; 2: 339-46.
54. Del-Rio A, Obdulio BG, Castillo J, Marin RR and Ortuno A: Uses and properties of citrus flavonoids. Journal of Agricultural and Food Chemistry 1977; 45: 4505-15.
55. Liu RH: Potential synergy of phytochemicals in cancer prevention: mechanism of action. Journal of Nutrition 2004; 134: 3479-85.
56. Okwu DE: Phytochemical and vitamin content of indigenous spices of South Eastern Nigeria. Journal of Sustainable Agriculture and the Environment 2004; 6: 30-34.
57. Rafat A, Koshy P and Sekaran M: Antioxidant potential and content of phenolic compounds in ethanolic extracts of selected parts of Andrographis paniculata. Journal of Medicinal Plants Research 2010; 4: 197-02.
    

    ---

    How to cite this article:

    Deora G: In-vitro assessment of antioxidant and antiproliferative efficacy of Andrographis paniculata. Int J Pharm Sci & Res 2014; 5(10): 4456-66. doi: 10.13040/IJPSR.0975-8232.5(10).4456-66.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---