Int J Pharm Pharm Sci, Vol 6, Issue 9, 123-127Original Article

GC-MS ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS IN LEAF EXTRACTS OF NEOLAMARCKIA CADAMBA (RUBIACEAE) FROM MALAYSIA

MOHAMED ZAKY ZAYED1, FASIHUDDIN BADRUDDIN AHMAD2, WEI-SENG HO1*, SHEK-LING PANG3

1Forest Genomics and Informatics Laboratory (fGiL), Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak, 2Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak, 3Applied Forest Science and Industry Development (AFSID), Sarawak Forestry Corporation, 93250 Kuching, Sarawak.
Email: wsho@frst.unimas.my

Received: 07 Jul 2014 Revised and Accepted: 14 Aug 2014

ABSTRACT

Neolamarckia cadamba is one of the medicinal plants used in the treatment of various diseases traditionally. This study was conducted to identify the phytochemical constituents of N. cadamba leaf extracts using gas chromatography mass spectrometry (GC-MS). Solvents with increasing polarities viz. hexane, petroleum ether, chloroform, ethyl acetate and methanol were used in this study. The solvent extracts were analyzed using GC-MS and the mass spectra of the compounds found in the respective extract were matched with the National Institute of Standards and Technology (NIST) library. A total of 26 compounds were identified and the major chemical constituents were n-hexadecanoic acid (44.88%), hexadecanoic acid ethyl ester (17.96%) and octadecanoic acid ethyl ester (11.71%). Some of the identified compounds have been reported to possess various biological activities such as antioxidant, antimicrobial, anesthetic, antiseptic, antidiabetic, hypocholesterolemic and etc. The results thus concluded that N. cadamba leaves possess various potent bioactive compounds and is recommended as a plant of phytopharmaceutical importance.

Keywords: Neolamarckia cadamba, Gas chromatography mass spectrometry (GC-MS), Phytochemical, Secondary metabolite, Antioxidant

INTRODUCTION

Higher plants as sources of bioactive compounds continue to play a dominant role in the maintenance of human health. Plants are rich sources of secondary metabolites with interesting biological activities [1-2]. It is also the best sources for obtaining natural antioxidants for various medicinal uses such as aging and disease related to radical mechanism such as cancer [3]. Neolamarckia cadamba is one of the medicinal plants traditionally used by the Indian. It has been mentioned in many Indian medical literatures for the treatment of fever, anaemia, diabetes, uterine and liver complaints, menorrhagia, blood and skin diseases, diarrhoea, colitis, stomatitis, dysentery and in improvement of semen quality [4-8]. Various parts of this plant have been traditionally used to treat various diseases [9-10]. Bioactivity studies on this plant revealed its antimicrobial, antioxidant and wound healing properties, antimalarial, antihepatotoxic, hepatoprotective, analgesic, anti-inflammatory, antipyretic, anthelmintic, diuretic, laxative and antidiabetic activities [10-11]. In addition, the leaves and barks extracts of this plant showed antifungal activity against Aspagillus fumigates and Candila albicans [12]. The tribes of Ganjam district of Orissa drink the root paste duly suspended in water for antimicrobial and anthelmintic activities.

Various phytochemical compounds have been identified from N. cadamba using phytochemistry approaches to date. The leaf extracts of N. cadamba revealed the presence of various secondary metabolites and these include glycosides, alkaloids, tannins, phenolic, steroids, and flavonoids [13-14]. The bark contains alkaloids like cadambine and its derivatives, saponins, glycosides, triterpenoids, cadambagic acid, quinovic acid and β-sitosterol [7, 10]. Those alkaloids, steroids and flavonoids have potent antiepileptic effect in various seizure models [15]. In addition to this, saponins have also been able to modulate the neurotransmitter levels in the brain and to possess potent anti-convulsant activity [16]. The qualitative chemical tests revealed the presence of saponins, proteins, terpenes, carbohydrates and alkaloids in the bark powder of N. cadamba [12]. Phytochemical evaluation of methanolic extract of N. cadamba showed the presence of flavonoids, alkaloids, carbohydrate, proteins and glycoside compounds [17]. The flowers of N. cadamba yield essential oil and the main constituents of the essential oils were linalool, geraniol, geranylacetate, linalyl acetate, α-selinene, 2-nonanol, β-phellandrene, α-bergamottin, p-cymol,curcumene, terpinolene, camphene and myrcene [18]. The seeds of N. cadamba are composed of water-soluble polysaccharides D-xylose, D-mannose and D-glucose in the molar ratio 1:3:5 [19]. From literature survey it was found that almost every part of the N. cadamba is used in the treatment of various diseases traditionally. Unfortunately, thus far there is no phytochemical study on N. cadambafrom Malaysia. In fact, it has been selected as one of the plantation tree species in forest rehabilitation projects in Malaysia due to its short rotation period which can give early commercial returns within 8-10 years [20-23]. Therefore ,the present study was carried out to determine the phytochemical constituents of N. cadamba leaves by using gas chromatography mass spectrometry (GC-MS). It is hoped that this study will provide another useful resource for future extraction of phytochemicals from this species which can be used as dietary supplements. To date, all published scientific findings are in agreement with the traditional use of the plant.

MATERIALS AND METHODS

Plant materials

N. cadamba seeds were obtained from the Seed Bank of Sarawak Forestry Corporation, Sarawak. The seeds were planted in trays of 50 holes and contained sand and compost (3:1) for one month and then planted in seed beds for the next 6 months. The leaves samples were dried in the shade in the open air condition for 6 – 12 days prior to extraction.

Sample extraction and column chromatography (CC)

About 300 g of dried leaves were ground into fine powder by using electric blender. The ground sample was percolated with methanol at room temperature for three days and filtered. The residue was extracted two more time to ensure complete extraction process. The residue was discarded and the filtrates were combined and evaporated to dryness using rotary evaporator. The crude dried methanol extract was partitioned with hexane, petroleum ether, chloroform, ethyl acetate and methanol. The entire steps were performed in three times in order to increase the effectiveness of the extraction process. All the hexane, petroleum ether, chloroform, ethyl acetate and methanol partitions were evaporated to dryness using rotary evaporator and the weight of each fraction was determined. The selected partitions were separated by column chromatography (60 cm length and diameter of 3.2 cm) using silica gel 280-400 mesh as stationary phase. Initially, the column was rinsed with suitable solvent systems. The column was filled with the slurry of silica gel, which was prepared by adding 220 g of the silica gel into 350 ml suitable solvent systems. Glass rod was used to knock off the silica gel slowly into the column to ensure the silica gel was compacted and no air bubbles were present in the column. Then, the silica gel was rinsed with hexane. Combinations of solvents with increasing polarity were used. About 3 g of partitions were added into the column and then a number of fractions (25 ml each) were collected.

Table 1: Weight, percentage yield and colour of N. Cadamba leaf extracts with different solvents

Solvent Extracts Colour Weight (g) Percentage (%)
Hexane Yellowish 0.97 1.07
Petroleum ether Yellowish brown 2.23 2.48
Chloroform Blackish green 1.50 1.67
Ethyl acetate Reddish 3.28 3.64
Methanol Black bluish 4.62 5.13

Table 2: Phytochemicals screening of solvent extracts of N. cadamba leaves by GC-MS

No. RT Name of the compounds Molecular Formula MW Peak Area (%)
Hexane extract
1 26.27 Hexadecanoic acid, methyl ester C17H34O2 270 2.84
2 27.25 Hexadecanoic acid, ethyl ester C18H36O2 284 17.96
3 28.68 Heptadecanoic acid, ethyl ester C19H38O2 298 0.41
4 29.17 Stearic acid, methyl ester C19H38O2 298 3.09
5 30.05 Octadecanoic acid, ethyl ester C20H40O2 312 11.71
6 34.27 Docosanoic acid, methyl ester C23H46O2 354 0.74
7 34.50 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 1.44
8 35.43 Tricosanoic acid, methyl ester C24H48O2 368 0.16
9 37.61 Pentacosanoic acid, methyl ester C26H52O2 396 0.15
10 38.16 Tetratetracontane C44H90 618 0.43
Petroleum ether extract
1 34.53 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 1.15
2 34.95 Heneicosane C21H44 296 0.06
3 37.15 Tetratetracontane C44H90 618 0.13
Chloroform extract
1 26.34 Hexadecanoic acid, methyl ester C17H34O2 270 0.49
2 28.87 Octadecenoic acid, methyl ester C19H36O2 296 0.82
3 29.22 Stearic acid, methyl ester C19H38O2 298 0.14
4 33.79 Eicosane C20H42 282 0.09
5 34.53 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 1.27
6 37.14 Tetratetracontane C44H90 618 0.44
Ethyl acetate extract
1 7.93 Benzaldehyde C7H6O 106 6.08
2 9.82 Benzyl Alcohol C7H8O 108 9.07
3 22.07 Pentanoic acid, 4-oxo-, phenylmethyl ester C12H14O3 206 0.82
4 22.49 Benzyl ether C14H14O 198 0.77
5 24.13 Tetradecanoic acid C14H28O2 228 0.52
6 27.48 n-Hexadecanoic acid C16H32O2 256 6.35
7 30.04 Octadecanoic acid, ethyl ester C20H40O2 312 0.31
8 34.51 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 1.32
9 39.34 Progesterone C21H30O2 314 1.87
10 40.13 Tetratetracontane C44H90 618 0.23
Methanol extract
1 20.88 Dodecanoic acid C12H24O2 200 0.32
2 24.24 Myristic acid C14H28O2 228 3.83
3 25.14 2-cyclohexen-1-one, 4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl) C13H18O3 222 0.55
4 25.74 Pentadecanoic acid C15H30O2 242 0.26
5 27.73 n-Hexadecanoic acid C16H32O2 256 44.88
6 30.44 Hexadecanamide C16H33NO 255 0.56
7 33.03 Octadecanamide C18H37NO 283 0.10
8 33.76 Heneicosane C21H44 296 0.10
9 34.50 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 2.15
10 37.11 Tetratetracontane C44H90 618 0.20

Gas Chromatograph-Mass Spectroscopy (GC-MS)

GC-MS (Shimadzu QP 5000) was performed by using non-polar DB-5 cross linked column (30 m long x 0.25 mm ID x 0.25 μm film thickness composed of 5% phenyl methyl polysiloxane). The initial temperature was programmed at 50oC and held for two minutes, and then it was increased to 300oC with the rate of 6.5oC/min. The final temperature was held for ten minutes. The temperature of the injector and detector were set up to 280oC and 300oC, respectively. Helium gas was used as a carrier gas. 1 μl of the fractions was diluted in 200 μl dichloromethane and then injected into the GC-MS [24-25]. Interpretation of mass-spectrum was conducted using the database of National Institute Standard and Technology (NIST). The spectrum of the unknown components was compared with the spectrum of known components stored in the NIST library. The name, molecular mass and structure of the components of the test materials were ascertained.

RESULTS AND DISCUSSION

A total of 300 g of powdered dried leaves was percolated with methanol for three days and then filtered. The percolating procedure was repeated twice in order to complete the extraction process and increase the effectiveness of the extraction. A total of 180.34 g crude extract was obtained from the leaves of N. cadamba. 90 g of the crude extract were used for solvent partition using solvent with different polarities. Solvents with increasing polarities used in this study were hexane, petroleum ether, chloroform, ethyl acetate and methanol. The solvent partition was repeated until the solvent in the thimble becomes clear indicating the completion of extraction process. The weights and the percentage of yields of the solvent partitions are shown in Table 1. Methanol partition gave the highest yield compared to other partitions. This result showed the presence of polar compounds in the leaves of N. cadamba. Each partition was subjected to GC-MS analysis. The chemical components present in the leaf extracts of N. cadamba were identified by GC-MS analysis. The active principals with their retention time (RT), molecular formula, molecular mass (MW) and concentration (%) in the solvent extracts of N. cadamba leaves are presented in Table 2. Ten important compounds detected in the hexane extracts were hexadecanoic acid methyl ester, hexadecanoic acid ethyl ester, heptadecanoic acid ethyl ester, stearic acid methyl ester, octadecanoic acid ethyl ester, docosanoic acid methyl ester, 1,2-benzenedicarboxylic acid diisooctyl ester, tricosanoic acid methyl ester, pentacosanoic acid methyl ester and tetratetracontane (Fig.1). Meanwhile, three important compounds were detected in the petroleum ether extract of N. cadamba leaves. The compounds were 1, 2-benzenedicarboxylic acid, diisooctyl ester, heneicosane and tetratetracontane (Fig.2). Six compounds identified in the chloroform extract as hexadecanoic acid, methyl ester, octadecenoic acid, methyl ester, stearic acid, methyl ester, eicosane, 1,2-benzenedicarboxylic acid, diisooctyl esterand tetratetracontane (Fig.3). Inethyl acetate extract, ten compounds were detected and these include benzaldehyde, benzyl Alcohol, pentanoic acid 4-oxo- phenylmethyl ester, benzyl ether, tetradecanoic acid, n-hexadecanoic acid, octadecanoic acid ethyl ester, 1,2-benzenedicarboxylic acid diisooctyl ester, progesterone and tetratetracontane (Fig.4). Ten important compounds were also successfully identified in the methanol extract of N. cadamba leaves. The compounds were dodecanoic acid, myristic acid, 2-cyclohexen-1-one, 4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl), pentadecanoic acid, n-hexadecanoic acid, hexadecanamide, octadecanamide, heneicosane, 1,2-benzenedicarboxylic acid diisooctyl ester and tetratetracontane (Fig. 5).

Fig. 1: Gas chromatogram of the hexane extract of the leaves of N. cadamba. Hexadecanoic acid, methyl ester (1), hexadecanoic acid, ethyl ester (2), heptadecanoic acid, ethyl ester (3), stearic acid, methyl ester (4), octadecanoic acid, ethyl ester (10), docosanoic acid, methyl ester (14), 1,2-benzenedicarboxylic acid, diisooctyl ester (15), tricosanoic acid, methyl ester (17), pentacosanoic acid, methyl ester (22), and tetratetracontane (23)


Fig. 2: Gas chromatogram of the petroleum ether extract of the leaves of N. cadamba. 1,2-benzenedicarboxylic acid, diisooctyl ester (18), heneicosane (19), and tetratetracontane (27)


Fig. 3: Gas chromatogram of the chloroform extract of the leaves of N. cadamba. Hexadecanoic acid, methyl ester (8), octadecenoic acid, methyl ester (10), stearicacid, methyl ester (12), eicosane (23), 1,2-benzenedicarboxylic acid, diisooctyl ester (24), and tetratetracontane (40)


Fig. 4: Gas chromatogram of the ethyl acetate extract of the leaves of N. cadamba. Benzaldehyde (1), benzyl alcohol (2), pentanoic acid, 4-oxo-, phenylmethyl ester(6), benzyl ether (8), tetradecanoic acid (10), n-hexadecanoic acid (16),octadecanoic acid, ethyl ester (21), 1,2-benzenedicarboxylic acid, diisooctyl ester (28), progesterone (42), and tetratetracontane (45)


Fig. 5: Gas chromatogram of the methanol extract of the leaves of N. cadamba. Dodecanoic acid (2), myristic acid (4), 2-cyclohexen-1-one, 4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl) (10), pentadecanoic acid (11), n-hexadecanoic acid(15), hexadecanamide (30), octadecanamide (34), heneicosane (35), 1,2-benzenedicarboxylic acid, diisooctyl ester (36), and tetratetracontane (40)


Table 3: Summary of phytochemical compounds identified from the N. cadamba leaf extracts and their general biological activities (Modified from Dr. Duke’s: Phytochemical and Ethnobotanical Databases [9])

S. No. Compounds Secondary metabolites Biological activity
1 Progesterone Steroid Neuroactive, analgesic, anesthetic, antioxidant activity, anticancer
2 Hexadecanoic acid, ethyl ester (palmitic acid ethyl ester) Fatty acid ester Antioxidant, hypocholesterolemic, nematicide, pesticide, antiandrogenic, flavor, hemolytic, 5-alpha reductase inhibitor
3 Hexadecanoic acid methyl ester Fatty acid ester Antioxidant, nematicide,

pesticide, flavour, antiandrogenic
4 Stearic acid methyl ester Fatty acid ester 5-Alpha reductase inhibitor, cosmetic, flavour, hypocholesterolemic, perfumery, suppository
5 n-hexadecanoic acid Fatty acid Antioxidant, hypocholesterolemic, nematicide, pesticide, antiandrogenic, flavour, hemolytic, 5-alpha reductase inhibitor
6 Tetradecanoic acid Fatty acid Antioxidant, hypercholesterolemic, cancer-preventive, cosmetic
7 Heptadecanoic acid Fatty acid Antioxidant
8 Dodecanoic acid Fatty acid Flavour
9 Myristic acid Fatty acid Antioxidant, hypercholesterolemic,cancer-preventive, cosmetic, nematicid.
10 Pentadecanoic acid Fatty acid Antioxidant
11 Benzaldehyde Aromatic compounds Allergenic, anesthetic, antibacterial, anticancer, antimutagenic, antipeptic, antiseptic, antispasmodic,

antitumor, candidicide, flavour, insecticide, nematicide, pesticide, sedative, termiticide, tyrosinase inhibitor
12 Benzyl alcohol Aromatic compounds Allergenic, anesthetic, antiodontalgic, antipruritic, antiseptic, flavour, fungicide, pesticide, sedative
13 1,2-Benzenedicarboxylic acid, diisooctyl ester Plasticizer compound Antimicrobial, antifouling

In accordance with the previous findings, most of the identified compounds from this study have also been reported elsewhere in other species. For instance, n-hexadenoic acid was identified as the major compound (44.88%) of methanol extract of N. cadamba leaves and this compound was also reported as a major compound in the methanolic leaf extract of Trichilia connaroides [26]. 17 compounds were identified in the leaves of Cleistanthus collinus and n-hexadecanoic acid was identified as the major compound [27]. Eicosane, 1,2-benzenedicarboxylic acid, diisooctyl ester, n-hexadecanoic acid and ethyl ester were present in Cassia italica leaf methanol extract [28]. Heptadecanoic acid, ethyl ester, tricosanoic acid and stearic acid have been reported in the hexane extract of the leaves of Desmodium elegans [29]. The aromatic metabolites such as benzoic acid (BA) or benzyl alcohol constitute the backbone of numerous compounds in plants [30-31]. Benzaldehyde and benzyl alcohol were present in the dichloromethane extract of the leaves of Drypetes gossweileri and pentanoic acid-4-oxo- phenylmethyl ester has been reported in the ethyle acetate extract of the leaves of the same plant [32]. Palmitic acid, stearic acid, myristic acid and hexadecanamide were present in the methanolic extract of Gigantochloa apus leaf [33]. Octadecanamide was isolated from the aqueous extract of Bacopa monnieri [34].

The phytochemical screening has identified 26 compounds from all the solvent extracts from the N. cadamba leaves and some of the identified compounds have been reported to possess various biological activities such as anti-microbial, anti-cancer, antimutagenic, antipeptic, antiseptic, antispasmodic, anti-adrenogenic and hypocholesterolemic activities as summarized in Table 3. Palmitic acid is an intermediate in the biosynthesis of sexual pheromones of some insects [35]. It is used in the preparation of the ingredients of some drugs to decrease the hydrophobicity of virginiamycin, a drug used against Mycobacterium avium [36-37] and it is well known as insecticide and anti-microbial agents [38]. Myristic acid is one of saturated fatty acids in animal and vegetable fats that are commonly used in soaps, cosmetics, flavourings and perfumes. It has hypercholesterolemic activity as it increases low density lipoprotein cholesterol production [39].

CONCLUSION

The present phytochemical study of N. cadamba leaf extracts was studied for the first time using five different solvent extracts with increasing polarities. In fact, this is the first available information about the phytoconstituents of N. cadamba from Malaysia. N. cadamba possess various potent bioactive compounds and is recommended as a plant of phytopharmaceutical importance. Its leaves can be used as antimicrobial, antiparasitic insecticide, nematicide, pesticide, antiandrogenic, hypocholesterolemic, antioxidant, cancer preventive, anticoronary, antiarthritic, hepatoprotective, neuroactive, analgestic and anesthetic, flavour and allergenic. The presence of various bioactive compounds has justified the use of N. cadamba leaf extracts for various ailments by traditional practitioners and therefore, further studies on isolation and identification of individual constituent are very much needed. It is also timely to explore its pharmacological values at the molecular level with the help of various biotechnological techniques in future.

CONFLICT OF INTERESTS

Declared None

ACKNOWLEDGMENT

This work is part of the joint Industry-University Partnership Programme, a research programme funded by the Sarawak Forestry Corporation (SFC), Sarawak Timber Association (STA) and Universiti Malaysia Sarawak under grant no. 02(DPI09) 832/ 2012(1), RACE/a(2)/884/2012(02) and GL(F07)/06/ 2013/STA-UNIMAS(06).

REFERENCES

  1. Chaman L, Verma LR. Use of certain bioproducts for insect pest control. Indian J Traditional Knowledge 2006;5(1):79-82.
  2. Yousuf S, Bachheti RK, Joshi A, Mathur A. Evaluation of antioxidant potential and phytochemicals of Morina longifolia. Int J Pharm Pharm Sci 2014;6(6):208-12.
  3. Alekhya V, Deepan T, Shaktiprasanna S, Dhanaraju MD. Preliminary phytochemical screening and evaluation of in vitro antioxidant activity of Anthocephalous cadamba by using solvent extracts. Eur J Biol Sci 2013;5(1):34-7.
  4. Slkar IV, Kakkar KK, Chakre OJ. Glossary of Indian medicinal plants with active principles. CSIR: New Delhi;1992.
  5. Kirtikar KR, Basu BD. Indian medicinal plants. Allahabad: Lalit Mohan Basu publishers; 1999.
  6. Prajapati N, Purohit D, Sharma SS, Kumar AK. A handbook of medicinal plants: a complete source book. Agrobios (India) publisher; 2007.
  7. Kumar V, Mahdi F, Chander R, Singh R, Mahdi AA, Khanna AA, et al. Hypolipidemic and antioxidant activity of Anthocephalus indicus (Kadam) root extract. Indian J Biochemistry Biophysics 2010;47:104-9.
  8. Dubey A, Nayak S, Goupale DC. A review on phytochemical, pharmacological and toxicological studies on Neolamarckia cadamba. Der Pharmacia Letter 2011;3(1):45-54.
  9. Duke JA. Phytochemical and Ethnobotanical Databases. (http: //www. ars-grin. gov/duke/chem-activities. html); 2007.
  10. Umachigi SP, Kumar GS, Jayaveera KN, Kumar KDV, Kumar ACK, Dhanpal R. Antimicrobial, wound healing and antioxidant activities of Anthocephalus cadamba. Afr J Tradit Complementary Altern Med 2007;4(4):481-7.
  11. Acharyya S, Dash DK, Mondal S, Dash SK. Studies on glucose lowering efficacy the Anthocephalus cadamba (Roxb.) Miq roots. Int J Pharm Bio Sci 2010;2(1):1-9.
  12. Patel DA, Patel YK, Shah PB. Pharmacognostical study of Neolamarckia cadamba (Roxb.) Bosser bark. Int Res J Pharm 2012;3(6):120-21.
  13. Usman MRM, Purushottam SR, Abullais MD, Usman MD. Evaluation of antipyretic activity of Anthocephalus cadamba Roxb. leaves extracts. Res J Pharm Biol Chem Sci 2012;3(1):825-34.
  14. Madhu C, Sharma U, Kumar N, Singh B, Satwinderjeet K. Antioxidant activity and identification of bioactive compounds from leaves of Anthocephalus cadambaby ultra-performance liquid chromatography/electrospray ionization quadrupole time of flight mass spectrometry. Asian Pacific J Tropical Medicine 2012;5(12):977-85.
  15. Hegde K, Thakker SP, Joshi AP, Shastry CS, Chandrashekhar KS. Anticonvulsant activity of Carissa carandas Linn. root extract in experimental mice. Tropical J Pharm Res 2009;8:117–25.
  16. Pal D, Sannigrahi S, Mazumder UK. Analgesic and anticonvulsant effects of saponins isolated from the leaves of Clerodendrum infortunatum Linn in mice. Indian J Experimental Biology 2007;47(9):743-7.
  17. Himanshu GSK, Nandanwar JR, Vinod KS. Phytochemical screening on the stem bark of Anthocephalus cadamba (Roxb.) Miq. Int J Pharm Sci Res 2010;1(7):108-15.
  18. The Wealth of India. A dictionary of Indian raw materials and industrial products. New Delhi: NISCAIR press publishers; 2006.
  19. Chandra O, Gupta D. A complex polysaccharide from the seeds of Anthocephalus indicus. Carbohydrate Res 1980;83:85-92.
  20. Lai PS, Ho WS, Pang SL. Development, characterization and cross-species transferability of expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from kelampayan tree transcriptome. Biotechnol 2013;12(6):225-35.
  21. Tchin BL, Ho WS, Pang SL, Ismail J. Association genetics of the cinnamyl alcohol dehydrogenase (CAD) and cinnamate 4-hydroxylase (C4H) genes with basic wood density in Neolamarckia cadamba. Biotechnology 2012;11(6):307-17.
  22. Tiong SY, Ho WS, Pang SL, Ismail J. Nucleotide diversity and association genetics of xyloglucan endotransglycosylase/ hydrolase (XTH) and cellulose synthase (CesA) genes in Neolamarckia cadamba. J Biol Sci 2014;14(4):267-375.
  23. Ho WS, Pang SL, Julaihi A. Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)”. Physiol Mol Biol Plants 2014;20(3):393-7.
  24. Houghton PJ, Raman A. Laboratory handbook for the fractionation of natural extracts. 1st ed. London: Chapman and Hall; 1998.
  25. Pavia DL, Lampman GM, Kritz GS, Engel RG. Introduction to organic laboratory techniques: A microscale approach. 4th Ed. St. Paul: Cengage Learning, Brooks/Cole Publishing Co; 2006.
  26. Senthilkumar N, Murugesan S, Vijayalakshmi KB. C-MS-MS analysis of Trichilia connaroides (Wight & Arn.) Bentv (Meliaceae): A tree of ethnobotanical records. Asian J Plant Sci Res 2012;2(2):193-7.
  27. Parasuraman S, Raveendran R, Madhavrao C. GC-MS analysis of leaf extracts of Cleistanthus collinus Roxb. (Euphorbiaceae). Int J Pharm Sci 2009;1(2):284-6.
  28. Sermakkani M, Thangapandian V. GC-MS analysis of Cassia italica leaf methanol extract. Asian J Pharm Clinical Res 2012;5(2):90-4.
  29. Khan A, Usman R, Wang M, Rauf A, Muhammad N, Aman A, Tahir TH. In vitro biological screening of the stem of Desmodium elegans. Asian Pacific J Tropical Biomedicine 2013;3(9):711-5.
  30. Bjorklund J, Leete E. Biosynthesis of the benzoyl moiety of cocaine from cinnamic acid via (R)-(+)-3-hydroxy-3-phenylpropanoic acid. Phytochemistry 1992;3:357–60.
  31. Walker K, Croteau R. Taxol biosynthesis: molecular cloning of a benzoyl-CoA: taxane 2a-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 2000;97:13591–6.
  32. Lucie AT, Dogo S, Lakouetene DB, Florent BO, Talla GM, Anna T, et al. Chemical characterization and insecticidal activity of ethyl acetate and dichloromethane extracts of Drypetes gossweileri against Sitophilus zeamais, Tribolium castaneum and Rhyzopertha dominica. J Life Sci 2013;7(10):1030-40.
  33. Mulyono N, Bibiana WL, Ocktreya L, Rahayu S. Antidiarrheal activity of apus bamboo (Gigantochloa apus) leaf extract and its bioactive compounds. American J Microbiol 2013;4(1):1-8.
  34. Subashri B, Pillai YJ. A comparative study of antioxidant activity of Baccopa monnieri (L.) Pennell using various solvent extracts and its GC-MS analysis. Int J Pharm Pharm Sci 2014;6(2): 494-8.
  35. Trabalon M, Niogret J, Legrand-Frossi C. Effect of 20-hydroxyecdysone on cannibalism, sexual behavior, and contact sex pheromone in the solitary female spider, Tegenaria atrica. General Comparative Endocrinology 2005;144:60–6.
  36. Rastogi N, Moreau B, Capmau ML, Goh KS, David HL. Antibacterial action of amphiphatic derivatives of isoniazid against the Mycobacterium avium complex. Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene 1988;268:456–62.
  37. Mingarro I, Lukovic D, Vilar M, Perez-Gil J. Synthetic pulmonary surfactant preparations: new developments and future trends. Current Medicinal Chemistry 2008;15(4):393-403.
  38. Praveen KP, Kumaravel S, Lalitha C. Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo. African J Biochemistry Res 2010;4(7):191-5.
  39. Huges TA, Heimberg M, Wang X. Comparative lipoprotein metabolism of myrisate palmitate and stearate in normalipidemic man metabolism. Int J Res Pharm Sci 1996;5(9):1108-18.