Research Article

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

Ravendra Kumar, Gaurav Verma, Om Prakash and A.K. Pant
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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.

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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.


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.

Table 1: In vitro antiplasmodial activity of pericarp extracts of T. connaroides against P. falciparum isolates
Image for - 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
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, 1995).

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.


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.

Table 2: Chemical composition of diethyl ether extracts (%) of Trichilia connaroides
Image for - 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

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., 2007).

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.


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