Head Space GC/MS Analysis of Volatile Constituents of Trichilea connaroides Wight and Arn. Extracts and their in vitro Anti- Plasmodium Activity Against Plasmodium falciparum Isolates
Head space GC-MS analysis ethyl acetate extracts of leaves, bark, root and pericarps of Trichilia connnaroides Wight and Arn. Revealed the presence of over 45 compounds of which 22.06, 97.24, 46.42 and 58.27% of the total volatiles from extracts of leaves bark, root and pericarps were identified, respectively. The volatile constituents of bark extract were rich in sesquiterpenoides. Copaene (24.71%), azulene (17.47%), α-cubebene (14.98%), β-cadinene (12.58%), α-bergamotene (4.96%) and ylangene (5.50%) were the major constituents of the total volatiles of the extract. The identified constituents in other extract were 22.06% in leaves extract, 46.42% in root extract and 58.26% in pericarp extract, respectively. In vitro antiplasmodium activity of five extract were tested against K 1 strain of Plasmodium falciparum isolates. All the extract showed antiplasmodial activity. Relatively high activity was observed in dichloromethane extract.
to cite this article:
Ravendra Kumar, Gaurav Verma, Om Prakash and A.K. Pant, 2011. Head Space GC/MS Analysis of Volatile Constituents of Trichilea connaroides Wight and Arn. Extracts and their in vitro Anti- Plasmodium Activity Against Plasmodium falciparum Isolates. Research Journal of Phytochemistry, 5: 41-47.
Received: September 14, 2010;
Accepted: December 18, 2010;
Published: February 26, 2011
Preparations from leaves, seeds, stem, bark and roots of many plants belonging
to the family Meliaceae have been widely used in traditional medicine. Antiviral,
antihelmintic, antitumoral, anti-inflammatory and antirheumatic activities of
the plant family Meliaceae have been described Bhakuni et
al. (1969), Fujiwara et al. (1982), Patel
(1986), Andrei et al. (1990), Bray
et al. (1990) and Coulombie et al. (1992).
The family Meliaceae is known to be a rich source of limonoids, which possess
interesting biological activities against insects such as antifeeding, deterrent
and inhibitors of ecdyasis (Champagne et al., 1992).
Malaria is one of the most prevalent diseases in the world. It affects more
than 500 million each year, mostly from sub-Saharan Africa and Asia causes about
2.3 million deaths a year (World Health Organization, 1996).
The problems of the resistance of the vector mosquitoes to insecticides and
to the parasites to most of the commercially available antimalarials are a serious
problem (Wernsdorfer and Trigg, 1988). Members of the
Meliaceae have been used for generations in Africa, India and tropical America
to treat malaria. In tropical America Cedrela odorata, Carapa quianensis
and Swietenia mahagoni have been used while in Africa and India the
Neem tree or Azadirachta indica is used (Mac-Kinnon
et al., 1997). Some other plant species belonging to the Meliaceae
family viz. Khaya grantifoliola, Entendrophragma utile and Morinda
lucida are also widely used as antimalarials or antipyretics in traditional
medicine (Bray et al., 1990; Bickii
et al., 2000; Obih et al., 1985; Weenen
et al., 1990). These plants are sources of poly oxygenated terpenoids
called limonoids, biosynthetically related to quassinoids whose antiplasmodial
and cytotoxic activities have been demonstrated (Bray et
al., 1990; Connolly, 1983).
Trichilia connaroides (Wight and Arn.) Bentv. Syn. Heynea trijuga
Roxb. is a tall tree widely distributed in the South and East of Asia, such
as India, Indonesia and South China (Chen et al.,
2007). It has been reported that of T. Connaroides posses analgesic
and anti-inflammatory activity (Purnima et al., 2006).
Larvicidal activity against Peridroma saucia and Spodoptera litura
have also been reported (Xio et al., 1994).
We also have earlier reported hypotensive activity of T. connaroides
extracts in rats (Agarwal et al., 2006) and
growth regulatory activity of T. connaroides leaf extracts against the
Bihar hairy caterpillar Spilosoma oblique (Lepidoptera: Arctiidae) (Tandon
et al., 2009).
The present study is on investigation of head-space GC/MS analysis of volatiles from plant parts and in vitro antimalarial activity of different extracts of T. connaroides Wight. and Arn. fruit pericarps against P. falciparum isolates.
MATERIALS AND METHODS
Plant collection: The plant Trichilia connnaroides Wight and
Arn. was collected from Ranibagh, District Nainital, Uttarakhand, India in the
month of October, 2008. Identification of this plant was confirmed by Prof Y.P.S.
Pangty, Professor of botany and plant taxonomist, Kumaon University, Nainital.
The herbaria was deposited and maintained in Department of Chemistry, G.B. Pant
University of Ag. and Tech., Pantnagar.
Preparation of extracts: Fresh leaves, bark, root and seeds of Trichilia connaroides were collected, seed coats (pericarps) removed separately, shade dried, powdered and extracted with diethyl ether and the solvent was removed under vacuum. Seed coats (paricarps) were also subjected to extraction using cold extraction process in a percolator first in petroleum ether for three days. The process was repeated for three times. Same process was followed with diethyl ether,dichloromethane, chloroform and methanol. The solvents were evaporated using thin film vacuum ratatory evaporator. The extracts were kept in refrigerator for further use. The yields are presented in Table 1.
Gc-ms analysis (head space analysis): GC-MS analysis of the diethyl
ether extracts of leaves, bark, root and pericarps were performed on a Agilent
19091s-433 with MS detector, using a HP-SMS capillary column (30 m x 0.25 mm
id) with a temperature program from 50 to 280°C at 1.5°C min-1
and finally held at 280°C.
|| In vitro antiplasmodial activity of pericarp extracts
of T. connaroides against P. falciparum isolates
|Data shown are values from two replicate experiments
Helium was the carrier gas (flow rate 1.1 mL min-1), ion source
temperature was 230°C and the ion inlet temperature 220°C. The MS were
recorded under EI conditions (70 ev) with a split less mode. Sample components
were identified by comparing their mass spectra with those in the NIST/Wiley
Library and by comparison with literature data and GC retention indices (Adams,
One gram of sample is taken in 20 mL headspace vial. Headspace septum was obesotead and these vapours were injected in GC equipped with MSD.
||Zone temp: Vial temperature: 120°C
||Loop temperature: 130°C
||Transfer line temp: 150°C
||Event time: Vial equilibration time: 15.0 min
||Pressurizing time: 0.20 min
||Loop fill time: 0.05 min
||Loop equilibration time: 0.05 min
||Injection time: 10.0 min
Drug sensitivity assays: Plant extracts were dissolved in dimethyl sulfoxide (DMSO) to obtain desired concentrations and were screened for antiplasmodial activity against K1 strain of Plasmodium falciparum isolates.
In vitro antiplasmodium activity: In vitro drug sensitivity
of extract was carried out at National Institute of Malaria Research, Delhi,
India as per procedure described by Trager and Jensen (1976).
Chloroquine sensitive Plasmodium falciparum FSG strain derived from an
Indian patients of Uttar Pradesh was used for the study using in vitro
candle-jar method as described by Trager and Jensen (1976).
Culture was maintained in A+ erythrocytes using RPMI 1640 medium
supplemented with AB Rh +ve human serum (10%), sodium bicarbonate (0.2%), HEPES
buffer (25 mM) and gentamycin (50 μg mL-1). The culture was
treated with selected concentrations (50, 10, 5, 2.5, 1.25 μg well-1)
of T. connaroides extracts. After 72 h of incubation, blood smears were
prepared and stained with Giemsa strain. Percentage maturation of schizonts
against control was recorded. Chloroquine was used as a standard drug. The inhibitory
concentration values which kills 50% of the parasites (IC50) were
considered for anti-plasmodial activity.
RESULTS AND DISCUSSION
Chemical composition of diethyl ether extract of leaves, bark, root and
pericarps: The results of Head Space analysis of diethyl ether extracts
of leaves, bark, root and seed pericarp are recorded in Table
2. Bark volatiles are rich in sesquiterpenoides. The major components identified
were azulene (17.47%), cubebene (14.98%), ylengene (5.5%) and copaene (24.71%)
of the total volatiles. The total volatile volatiles identified in other extracts
were 22.06% in leaves extract, 46.42% in root extract and 58.27% in pericarp
extract respectively. The major volatile compounds identified in leaf extract
were azulene (8.62%), caryophyllene (1.76%), copaene (1.10%) and β-bourbonene
(1.02%) of the total volatiles. The major volatiles identified in root extract
were azulene(10.62%), 2-methyl-2-bornene(2.36%), α-bergamotene (5.03%),
β-cedrene(3.65%) and β-chamigrene(10.2%)of the total volatile of root
extract. Caryophyllene (14.13%), cis- calamenene (6.34%), copaene (4.92%), cadiene-1,4-diene
(3.10%), α-caryophyllene (3.01%) were the major components present in the
total volatiles of pericrap extract. The detailed compositions of the volatiles
are recorded in Table 2.
||Chemical composition of diethyl ether extracts (%) of Trichilia
In vitro antiplasmodium activity different extracts of pericarps: Five crude organic extracts obtained from T. connaroides pericarps were tested in vitro against P. falciparum. Two extracts (dicloromethane and chloroform) of seed pericarp of T. connaroides were effective against Plasmodium falciparum K1 strain. Other extracts viz., petroleum ether, diethylether and methanol showed weak antiplasmodial activity against P. falciparum K1 strain as comparison to the standard drug chloroquine (IC50 = 0.051μg mL-1). IC50 values of extracts were between 6 and 22 μg mL-1 Table 1.
Earlier, Rochanakij et al. (1985) identified
nimbolide as the active antimalarial principle of the Neem tree. Gedunin and
its dihydro derivative were also found to be active in vitro against P. falciparum
(Mac-Kinnon et al., 1997). Limonoids, which exhibit
in vitro antimalarial activities, have been reported from Cedrela odorata
(Bray et al., 1990), Khaya senegalensis (Khalid
et al., 1998) and Khaya grantifoliola (Bickii
et al., 2000). Two limonoids, trichirubine A and B have been isolated
from T. rubescens with significant antimalarial activity (Krief
et al., 2004). Their antimalarial activities may be related to the
presence of reactive groups on ring A like the carbonyl group at C-3 and unsaturation
in C-1/C-2 positions. The limonoid derivatives 7-deacetylgedunin and 7-deacetyl-7-oxogedunin
isolated from the roots of Pseudocedrela kotschyi have been reported
to display moderate antimalarial activity (Hay et al.,
Trichilia connaroides is a rich source of triterpenoids, limonoids and
a steroid have been characterized from the leaves, flowers, roots, or pericarps
of Trichilia connaroides (Puroshothaman et al.,
1983, 1987; Venkatanarsimhan
et al., 1990; Inada et al., 1994;
Zhang et al., 2003; Wang
et al., 2008; Geng et al., 2009).
Therefore the antiplasmodial activity of T. connaroides pericarp extracts
could be due to limonoids.
The volatiles from leaves, bark, roots and fruit preicarp of Trichilia connaroides are being reported for the first time. The extracts of the plant parts posses antiplasmodial activity. The results are of pharmaceutical interest for further drug discovery programmes.
Thanks are due to University Grant Commission, New Delhi for providing research fellowship to Ravendra kumar.
Adams, R.P., 1995.
Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured Publishing Co., Carol Stream, IL., USA., pp: 69-351
Agarwal, G., S.K. Hore, O. Prakash and A.K. Pant, 2006.
Hypotensive activity of T. connaroides
extracts in rats. J. Vet. Pharmcol. Toxicol., 5: 72-73.
Andrei, G.M., F.C. Coulombie, M.C. Courreges, R.A. de Torres and C.E. Coto, 1990.
Melicine, an antiviral compound from Melia azedarach
L., inhibits interferon production. J. Interferon Res., 10: 469-475.
Bickii, J.N., J. Njifutie, J.A. Foyere, L.K. Basco and P.J. Ringwald, 2000. In vitro
antimalarial activity of lomonoids from Khaya grantifolia
C.D.C (Meliaceae). Ethnopharmacology, 69: 27-33.
Bhakuni, D.S., M.L. Dhar, M.M. Dhar, B.W. Dhawan and B.N. Mehratra, 1969.
Screening of plants for biological activity. Part II. Indian J. Exp. Biol., 7: 250-262.
Bray, D.H., D.C. Warhurst, J.D. Connolly, M.J. O`Neill and J.D. Phillipson, 1990.
Plants as sources of antimalarial drugs. Part 7. Activity of some species of meliaceae plants and their constituent limonoids. Phytotherapy Res., 4: 29-35.CrossRef | Direct Link |
Champagne, D.E., O. Koul, M.B. Isman, G.G.E. Scudder and G.H.N. Towers, 1992.
Biological activity of limonoids from the Rutales. Phytochemistrym, 31: 377-394.CrossRef |
Chen, Y.Y., X.N. Wang, C.Q. Fan, S. Yin and J.M. Yue, 2007.
Swiemahogins A and B, two novel limonoids from Swietenia mahogany
. Tetrahedron Lett., 48: 7480-7484.CrossRef |
Connolly, D.L., 1983.
Chemistry of Limonoids of the Meliaceae and Cneoraceae. In: Chemistry and Chemical Taxonomy of the Rutales, Waterman, P.G. and M.F. Grundon (Eds.). Academic Press, London
Coulombie, F.C., G.M. Andrei, R.P. Laguens, R.A. de Torres and C.E. Coto, 1992.
Partially purified leaf extracts of Melia azedarach
L. inhibit Tacaribe virus growth in neonatal mice. Phytotherapy Res., 6: 15-19.CrossRef | Direct Link |
Fujiwara, T., T. Takeda, Y. Ogihara, M. Shimizu, T. Nomura and T. Tomita, 1982.
Studies of the structure of polysaccharides from the bark of Melia azadirachta
. Chem. Pharm. Bull., 30: 4025-4030.PubMed |
Geng, Z.L., X. Fang, Y.T. Di, Q. Zhang, Y. Zeng, Y.M. Shen and X.J. Hao, 2009.
Trichilin B, a novel limonoid with highly rearranged ring system from Trichilia connaroides
. Tedrtraheon Lett., 50: 2132-2134.CrossRef |
Hay, A.E., J.R. Ioset, K.M. Ahua and D. Diallo, R. Brun and K.J. Hostettmann, 2007.
Limonoid orthoacetates and antiprotozoal compounds from the roots of Pseudocedrela kotschyi
. J. Nat. Prod., 70: 9-13.CrossRef | PubMed |
Inada, A., M. Konishi, H. Murata and T. Nakanishi, 1994.
Structure of new limilnoid and a new triterpenoid derivative from pericarp of Trichillia connaroides
. J. Nat. Prod., 57: 1446-1449.CrossRef |
Khalid, S.A., G.M. Friedrichsen, A. Kharazmi, G.T. Theander, C.E. Olsen and C.S. Brogger, 1998.
Liminoids from Khaya senegalensis
. Phytochemistry, 49: 1769-1772.CrossRef |
Krief, S., M.T. Martin, P. Grellier, J. Kasenene and T. Sevenet, 2004.
Novel antimalerial compounds isolated in a survey of self medicative behaviour of wild chimpanzees in Uganda. Antimicrob. Agents Chemother., 48: 3196-3199.CrossRef | Direct Link |
MacKinnon, S., T. Durst, J.T. Arnason, C. Angerhofer and J. Pezutto et al
Antimalarial activity of tropical Meliaceae extracts and Gedunin derivatives. J. Nat. Prod., 60: 336-341.PubMed |
Obih, P.O., J.M. Makinde and J. Laoye, 1985.
Investigation of various extracts of Morinda lucida
for antimalarial actionsm on Plasmodium berghei
in mice. Afr. J. Med. Sci., 14: 45-49.
Patel, N.G., 1986.
Indian Traditional Medicine. In: Folk Medicine: The Art and Science, Steiner, R.P. (Ed.). American Chemical Socity, Washinggton DC., pp: 57
Purnima, A., G.S. Prasanna and V. Mathuram, 2006.
Analgesic and antiinflammatory activity of the chloroform extract of Trichilia connaroides
(W. and A.) Bentilizen. Indian J. Pharmaceutical Sci., 68: 231-233.CrossRef | Direct Link |
Puroshothaman, K.K., A. Sarada and V. Mathuram, 1983.
Structure of Heynic acid: A new triterpene acid from Heynea trijuga
roxb. Ind. J. Chem., 22B: 820-821.
Puroshothaman, K.K., M. Sarada, Venkatanarsimhan, A. Sarada, J.D. Connolly and D.S. Rycroft, 1987.
Trigugins A and B, tetranorterpenoids with a novel rearranged carbon skeleton from Heynea trigjuga
(Meliaceae). Can. J. Chem., 65: 35-37.
Rochanakij, S., Y. Thebtaranonth, C.H. Yenjal and Y. Yuthavong, 1985.
Nimbolide, a constituent of Azadirachta indica
inhibits plasmodium falciparum in culture Southeast Asian. J. Trop. Med. Public Health, 16: 66-72.PubMed |
Tandon, S., A.K. Mittal and A.K. Pant, 2009.
Growth regulatory activity of T. Connaroides
(syn. Heynea trijuga
) leaf extracts against the Bihar hairy caterpillar Spilosoma oblique
(Lepidoptera: Arctiidae). Int. J. Trop. Insect Sci., 29: 180-184.CrossRef | Direct Link |
Trager, W. and J.B. Jensen, 1976.
Human malaria parasites in continuous culture. Sciences, 193: 673-675.CrossRef | PubMed | Direct Link |
Venkatanarsimhan, M., A.B. Kundu and A. Patra, 1990.
Isolation and characterization of trijugin-B acetate from Heynea trijuga
Roxb. Indian J. Chem., 29B: 970-970.
Wang, X.N., C.Q. Fan, S. Yin, L.S. Gan and J.M. Yue, 2008.
Structural elucidation of limonoids and steroids from Trichilia connaroides
. Phytochemistry, 69: 1319-1327.CrossRef |
Weenen, H., M.H. Nkunya, D.H. Bray, L.B. Mwasumbi, L.S. Kinabo and V.A. Kilimali, 1990.
Antimalarial activity of Tanzanian medicinal plants. Planta Medica, 56: 368-370.PubMed |
Wernsdorfer, W.H. and P.I. Trigg, 1988.
Recent Progress of Malaria Research: Chemotherapy. In: Malaria Principle and Practice of Malariology, Wernsdorfer, W.H. and S.I. McGregor (Eds.). Churchill Livingstone, London, pp: 1569-1674
World Health Organization, 1996.
Malaria distribution. A Weekly Epidemiol. Record, 71: 17-24.
Xio, Y.S., M.B. Isman, P. Gunning, S. MacKinnon and J.T. Arnason et al
Biological activity of extracts of Trichilia species and the limonoid hirtin against lepidopteran larvae. Biochem. Syst. Ecol., 22: 129-136.CrossRef |
Zhang, H.P., S.H. Wu, Y.M. Shen, Y.B. Ma, D.G. Wu, S.H. Qi and X.D. Luo, 2003.
Apentanortriterpenoid with a novel carbon skeleton and new pregnane from atrichilia connaroides. Can. J. Chem., 81: 253-257.CrossRef |