RESEARCH ARTICLE Vol. 2, No. 1, pp. 69-74, March 2012
GC-MS studies on Andrographis paniculata (Burm.f.) Wall. ex Nees - A
medicinally important plant
C.S. KALAIVANI1, S. Sahaya SATHISH1, N. JANAKIRAMAN1, M. JOHNSON2* 1
Department of Plant Biology and Plant Biotechnology, St. Joseph’s College (Autonomous), Tir uchirappalli-620 002, Tamil Nadu, India
2
Department of Plant Biology and Plant Biotechnology, St. Xavier’s College (Autonomous), Palayamkottai -627 002, Tamil Nadu, India
*Corresponding Author, Tel: + 91 97 91 81 38 25; Fax: + 91 462 2561 765
Article History: Received 3rdSeptember 2011, Revised 16thFebruary 2012, Accepted 16thFebruary 2012.
Abstract: The present study was aimed to examine the phytochemical constituents present in Andrographis paniculata (Burm.f.) Wall. ex Nees. The preliminary phytochemical constituents were qualitatively analyzed using the method de-scribed by Brindha et al. For GC-MS analysis, 10 g powdered sample is extracted with 30 mL ethanol overnight and fil-tered in ashless filter paper with sodium sulphate (2 g) and the extract is concentrated to 1 mL by bubbling nitrogen into the solution. The compound detection employed the NIST Ver.2.0-Year 2005 library. The biological activities are based
on Dr. Duke’s Phytochemical and Ethnobotanical Databases by Dr. Jim Duke of the Agricultural Research Ser-vice/USDA. The preliminary phytochemical analysis confirmed the presence of various secondary metabolites like stero-ids, alkalostero-ids, phenols, catechine, flavonostero-ids, saponins and tannins. The GC-MS analysis determined the presence of thirteen different phytochemical compounds namely 1,1,3-triethoxy-Propane, Tetradecanoic acid, 3,7,11,15-tetramethly-2-hexadecen-1-ol, n-hexadecanoic acid, 9,12-octadecadienoyl chloride,(Z,Z)-, Phytol, 9,12-Octadecadienoic acid(Z,Z), 9,12,15-Octadecatrienoic acid(Z,Z,Z), 1,2-benzenedicarboxylic acid,diisooctyl ester, Squalene, Retinoic acid methyl es-ter, Androstan-17-one,3-ethyl-3-hydroxy-,(5α) and β-sitosterol. The results of the present study enhance the traditional usage of A. paniculata which possess several known and unknown bioactive compounds. By isolating and identifying these bioactive compounds, new drugs can be formulated to treat various diseases.
Keywords: Andrographis paniculata; GC-MS; Phytochemistry; Herbal chemistry; Medicinal plants.
Introduction
India is endowed with a rich wealth of me-dicinal plants and it is one of the 12 mega biodi-versity centres having 45,000 plant species. In India around 20,000 medicinal plants have been recorded recently, but more than 500 traditional communities use about 800 plant species for curing different diseases. Currently 80% of the world population depends on plant-derived medicine for human alleviation because of its fewer side effects. Medicinal plant based drugs have the added advantage of being simple, ef-fective and offering a broad spectrum of activity with greater emphasis on preventive action (Chin et al. 2006). In the last century, roughly 121 pharmaceutical products were formulated based on the traditional knowledge obtained from various sources (Perumal Samy and Gopa-lakrishnakone 2007). Several phytochemical
screening studies have been carried out in dif-ferent parts of the world (Sangeetha and Vijaya-lakshmi 2011; Vohra and Kaur 2011; Wu et al. 2010). Therefore, characterization of extracts of medicinal plants is necessary, due to its numer-ous benefits to science and society.
Andrographis paniculata (Burm.f.) Wall. ex
Nees commonly known as King of bitters be-longs to the family Acanthaceae. It has been used for centuries as a medicinal herb for the treatment of upper gastrointestinal tract and up-per respiratory infections, fever, herpes and oth-er chronic diseases. It has a broad range of pharmacological effects. The primary medicinal component of A. paniculata is andrographolide, which is a diterpene lactone. Andrographolide has been reported for its anti-cancer (Sheeja and Kuttan 2007), anti-HIV (Calabrese et al. 2000), cardioprotective (Yoopan et al. 2007) and
hepa-Int. J. Med. Arom. Plants GC-MS studies on A. paniculata toprotective (Trivedi et al. 2007) properties
among others. Hence, the present study was aimed to explore the remaining phytochemical constituents of A. paniculata using GC-MS analysis.
Materials and Methods
Collection and preparation of plant material
The leaves of Andrographis paniculata (Burm.f.) Wall. ex Nees were collected from the natural habitats of Tiruchirappalli, Tamil Nadu, India. The leaves were washed thoroughly for 3 times in running tap water to remove soil par-ticles and adhered debris and finally with sterile distilled water. The leaves were cut, shade dried, ground into fine powder and stored in air tight polythene bags until use.
Preliminary phytochemical analysis
Shade dried and powdered plant materials were successively extracted with aqueous, etha-nol, chloroform and petroleum ether with gentle stirring for 72 h separately. The extracts were filtered through Whatmann No. 1 filter paper and concentrated using vacuum distillation. The preliminary phytochemical constituents were qualitatively analyzed using the standard me-thod (Brindha et al. 1981).
GC-MS analysis
10 g powdered sample is extracted with 30 mL ethanol overnight and filtered in ashless fil-ter paper with sodium sulphate (2 g) and the ex-tract is concentrated to 1 mL by bubbling nitro-gen into the solution. The Clarus 500 GC used in the analysis employed a column packed with Elite-1 [100% Dimethyl poly siloxane, 30 nm X 0.25 nm ID X 1 µm df] and the components were separated using Helium (1 mL / min) as the carrier gas. The 2µL sample extract injected into the instrument was detected by the Turbo mass gold mass detector (Perkin Elmer) with the aid of the Turbo Mass 5.1 software. During the 36thminute GC extraction process, the oven was maintained at a temperature of 110ºC with a 2 minutes holding. The injector temperature was
set at 250ºC (Mass analyser). The different pa-rameters involved in the operation of Clarus 500 MS, were also standardized (Inlet line tempera-ture: 200ºC; Source temperatempera-ture: 200ºC; Elec-tron energy: 70eV; Mass scan: (m/z) 45-450). The MS detection was completed in 36 minutes. The relative percentage amount of each compo-nent was calculated by comparing its average peak area to the total areas. The detection em-ployed the NIST (National Institute of Standards and Technology) Ver.2.0-Year 2005 library. The compound prediction is based on Dr.
Duke’s Phytochemical and Ethnobotanical Da-tabases by Dr. Jim Duke of the Agricultural Re-search Service/USDA.
Results
The phytochemical constituents present in the leaves of A. paniculata were reported in Ta-ble 1. GC-MS analysis of ethanolic extract re-vealed the presence of 13 different compounds namely 1,1,3-triethoxy-Propane, Tetradecanoic acid, 3,7,11,15-tetramethly-2-hexadece1-ol, n-hexadecanoic acid, 9,12-octadecadienoyl chlo-ride,(Z,Z)-, Phytol, 9,12-Octadecadienoic ac-id(Z,Z), 9,12,15-Octadecatrienoic acid(Z,Z,Z), 1,2-benzenedicarboxylic acid, diisooctyl ester, Squalene, Retinoic acid methyl ester, Andros-tan-17-one,3-ethyl-3-hydroxy-,(5α) and β -sitosterol. The retention time, molecular weight and the relative percentages of the compounds present in leaves of A. paniculata were recorded in Table 2. The nature of the chemical com-pound and its therapeutic activity is depicted in Table 3. The GC-MS spectrum confirmed the presence of 13 major components with the re-tention time 4.48, 13.36, 14.39, 16.12, 18.05, 18.38, 18.69, 18.81, 24.60, 28.61, 31.54, 32.92 and 37.04 respectively (Figure 1). Interpretation of mass spectrum GC-MS was conducted using the database of National Institute Standard and Technique (NIST08s), WILEY8 and FAME having more patterns. The spectrum of the un-known component was compared with the spec-trum of the known components stored in the NIST08s, WILEY8 and FAME library. The name, molecular weight, molecular formula and structure of the component of the test material were determined.
Int. J. Med. Arom. Plants GC-MS studies on A. paniculata
Table 1: Preliminary Phytochemical Analysis of A. paniculata (Burm.f.) Wall. Ex Nees
Secondary metabolites Aqueous Ethanol Chloroform Petroleum ether
Steroids - + + + Triterpenoids - - - -Sugars - - - -Reducing Sugars - - + + Alkaloids - - + + Phenolic Compounds + - - -Catechine + - - -Flavonoids + - - -Saponins + - + + Tannins + + - -Anthroquinone - - - -Aminoacids + - +
-Table 2: Phytoconstituents present in ethanolic leaf extract of A. paniculata (Burm.f.) Wall. ex Nees using GC-MS
S. No RT Compound MW Peak Area %
1 4.48 1,1,3-triethoxy- Propane 176 4.75 2 13.36 Tetradecanoic acid 228 0.23 3 14.39 3,7,11,15-tetramethly-2-hexadecen-1-ol 296 8.54 4 16.12 n-hexadecanoic acid 256 4.44 5 18.05 9,12-octadecadienoyl chloride,(Z,Z)- 298 0.58 6 18.38 Phytol 296 6.67 7 18.69 9,12-Octadecadienoic acid(Z,Z) 280 0.86 8 18.81 9,12,15-Octadecatrienoic acid (Z,Z,Z) 278 6.05
9 24.60 1,2-benzenedicarboxylic acid,diisooctyl ester 390 1.17
10 28.61 Squalene 410 0.87
11 31.54 Retinoic acid, methyl ester 314 30.04
12 32.92 Androstan-17-one,3-ethyl-3-hydroxy-,(5α) 318 33.39
13 37.04 β-sitosterol 414 2.41
Int. J. Med. Arom. Plants GC-MS studies on A. paniculata
Table 3: Nature of the compounds present in ethanolic leaf extract of A. paniculata (Burm.f.) Wall. ex Nees and its medicinal properties
S. No Compound Compound nature
Therapeutic activity
1 1,1,3-triethoxy- Propane Alkane No activity
2 Tetradecanoic acid Myristic acid Nematicide, Hypocholesterolemic, Antioxidant, Cancer preventive, Lubricant
3 3,7,11,15-tetramethly-2-hexadecen-1-ol
Terpene alcohol Antimicrobial, Anti-inflammatory
4 n-hexadecanoic acid Palmitic acid Lubricant, Antiandrogenic, Flavor, Hemolytic, Antioxidant, Hypocholesterolemic, Nematicide, Pesticide, 5-Alpha re-ductase inhibitor
5 9,12-octadecadienoyl
chloride,(Z,Z)-Linoleoly chlo-ride
Antisecretory Antispermigenic, Antitonsilitic, Antitubercu-lar, Choleretic, Contraceptive
6 Phytol Diterpene Antimicrobial, Anti cancer, Anti-inflammatory 7 9,12-Octadecadienoic
acid(Z,Z)
Linoleic acid Hypocholesterolemic, Nematicide, Anticoronary, Antiarth-ritic, Hepatoprotective, Anti-androgenic, Hypocholestero-lemic, 5-Alpha reductase inhibitor, Antihistaminic, Insecti-fuge, Antieczemic, Antiacne
8 9,12,15-Octadecatrienoic acid (Z,Z,Z)
α-linolenic acid Analgesic, Anesthetic, Allergenic, Antibacterial, Anticon-vulsant, inflammatory, Antioxidant, Antipyretic, Anti-salmonella, Antiseptic, Antistaphylococci
9 1,2-benzenedicarboxylic acid,diisooctyl ester
Plasticizer com-pound
Antimicrobial, Antifouling
10 Squalene Triterpene Antibacterial, Immunostimulant, Chemo preventive, Anti-oxidant, Antitumor, Cancer preventive, Lipoxygenase-inhibitor, Pesticide
11 Retinoic acid, methyl ester
Fatty acid ester Analgesic, Antibacterial, Antiinflammatory, Sedative, Fun-gicide
12 Androstan-17-one,3-ethyl-3-hydroxy-,(5α)
Steroid Neuroactive, analgesic, anesthetic
13 β-sitosterol Steroid Anti-hypercholesterolemia, reduces blood levels of
choles-terol, antioxidant activity, anticancer
Discussion
Previous studies determined the chemical profile of chloroform extracts of A. paniculata leaves using GC-MS (Roy et al. 2010). They observed the presence of phenols, aromatic car-boxylic acids and esters in the chloroform ex-tracts of A. paniculata. The therapeutically im-portant active principle andrographolide (Kal-megh) was observed in the aerial parts of A.
pa-niculata (Chem and Liang 1982; Siripong et al.
1992; Rastogi and Mehrotra 1998; Rastogi and Mehrotra 1999; Jain et al. 2000; Wu et al. 2008; Chandrasekaran et al. 2009). Other therapeutic
compounds include
14-deoxy-11-oxoandrographolide, 14-deoxy-11,12-didehydroandrorapholide/andrographolide D, 14-deoxy-andrographolide, non-bitter com-pound neoandrographolide,
homoandrographo-lide, andrographosterol, andrographane,
andro-graphosterin, andrograpanin, α-sitosterol, stig-masterol, apigenin-7,4’-di-O-methyl ester,
5-hydroxy 7,8,2’,3’-tetramethoxy flavones, mo-nohydroxy trimethyl flavones, andrographnin, dihydroxy-di-methoxy flavones, panico-lin,andrographoneo, andrographoside, andropa-niculosin A; andropani-culoside A, andrograpa-nin, isoandrographolide and skullcaflavone. Shen et al. 2006 reported six entlabdane diter-penoids i.e. 3-O-beta-D-glucopyranosyl-14, 19-dideoxyandrographolide, 14-deoxy-17-hydroxyandrographolide, 19-O-[beta-D-apiofuranosyl(1-2)-beta-D-glucopyranoyl]-3, 14-dideoxyandrographolide, 3-O-beta-D-glucopyranosyl andrographolide, 12S-hydroxyandrographolide and andragraphatoside from the aerial parts of the plant. These
com-Int. J. Med. Arom. Plants GC-MS studies on A. paniculata pounds showed inhibitory activity against
sev-eral bacterial and fungal strains. Four xanthones 1,8-di-hydroxy-3,7-dimethoxy-xanthone, 4,8-di hydroxy-2,7-dimethoxy-xanthone and 3,7,8-trimethoxy-1-hydroxyxanthone are reported from the roots. In the present study, we ob-served 13 different compounds from the etha-nolic extracts of A. paniculata leaves. Similar to our studies Ghannadi and Dezfuly (2011) also identified seventeen compounds from Myrtus
communis and Chibani et al. 2011 charaterized
eighteen compounds from Ferula communis using GS-MS analysisThe results of the present study confirm the traditional applications of the medicinal plant A. paniculata. The mass spectra are fingerprint of the compounds which can be used as a pharmacognostical tool for the identi-fication of adulterants. Rawat and Vashistha (2011) produced an easy and low cost method for the propagation of this medicinally impor-tant plant for the sustainable utilization and management.
Conclusion
The results of the present study suggest that
A. paniculata leaves can be used as nematicide,
fungicide, pesticide, lubricant. The leaves of A.
paniculata possess antibacterial,
staphylococci, allergenic, anticonvulsant, inflammatory, antisalmonella, antiseptic, fouling, antioxidant, antipyretic, antiacne, anti-coronary, antiarthritic, antiandrogenic, analges-ic, 5-antihistaminanalges-ic, anesthetanalges-ic, antisecretory, antispermigenic, antitonsilitic, antitubercular, antieczemic, cancer preventive, choleretic, con-traceptive, chemo preventive, hepatoprotective, hypocholesterolemic, immunostimulant, seda-tive properties. In addition they reduce blood levels of cholesterol and act as 5-α reductase
inhibitor, etc. The present study enhances the traditional usage of A. paniculata which possess several known and unknown bioactive com-pounds. By isolating and identifying these bio-active compounds, new drugs can be formulated to treat various diseases.
Acknowledgement: The authors are grateful to the Indian Institute of Crop Processing Technol-ogy (IICPT), Thanjavur, for providing the la-boratory facilities.
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