Phytochemical Screening of the Extract of the Root-Bark of Morinda tinctoria (Rubiaceae) for Secondary Metabolites

 

Praveena A.*, Ramkumar G. and Sanjayan K.P.

G.S. Gill Research Institute, Guru Nanak College, Chennai-600 042, India.

Corresponding author: praveena_bioinfo@yahoo.com

 

ABSTRACT:

Morinda tinctoria Roxb. is one of the medicinally important plants belonging to the family Rubiaceae and traditionally the leaves and roots are used for treatment of various ailments. Chloroform, Ethylacetate and methanol extracts of the root-bark was subjected to standard biochemical test for the presence of several plant secondary metabolites. Alkaloids, anthroquinones, saponins, steroids, triterpenoids, quinones and cardiac glycosides were present in the root-bark. The extracts were further subjected to chromatographic separation using GC-MS. The chromatogram revealed 21 peaks from Chloroform, 17 from Ethyl acetate and 14 from Methanol extracts. The Chloroform and methanol root-bark extract consist of the potent biologically active phyto-constituents such as Heptacosane, Eicosanedioic acid and Nonadecenoic acid.

 

KEYWORDS: Morinda tinctoria, Heptacosane, Eicosanedioic acid, Nonadecenoic acid, Phytochemical screening.

 


INTRODUCTION:

In recent years, there has been renewed interest in screening higher plants for novel biologically active compounds. The genus Morinda, belonging to the family Rubiaceae, is indigenous to tropical countries and is considered an important traditional folk medicine. In particular, the species Morinda citrifolia Roxb is reported to have a broad range of therapeutic and nutritional values.1 There is great demand for its fruit juice in alternative medicine for variety of illnesses such as arthritis, diabetes, muscle aches, menstrual difficulties, heart disease, cancers, gastric ulcer, blood vessel problems, and drug addiction.2 The genus Morinda grows wild and is widely distributed in Southern India. Many species of Morinda are available in India, of which Morinda pubescens J.E. Smith (synonyms: Morinda tinctoria Roxb.) and Morinda coreia Buch. predominantly grows as a weed tree in vacant agricultural land, and especially on uncultivated lands and along the boundaries of the cultivated fields. Ancient writings reveal that Morinda has long been cultivated in different parts of the Tamil Nadu state of India. In the traditional system of medicine, leaves and roots of Morinda tinctoria Roxb. are used as astringent, deobstrent, emmengogue and to relive pain in the gout.3 Hence the objective of the present study is to identify the Phytochemical constituents of the root-bark of Morinda tinctoria with the aid of GC-MS technique.

 

MATERIALS AND METHODS:

Extract preparation:

The Root-bark of M. tinctoria was collected at Kadambathur, Thiruvallur district, Tamilnadu. They were washed well to clear off the adhering soil particles and the extracts were prepared by maceration using four different solvents in sequential order of increasing polarity viz., Chloroform, Ethyl acetate, Methanol and Water. Fifty gram of the plant material was macerated for 3 hrs first with 100ml of Chloroform and then shaken continuously in an electronic shaker for 24 hours. The macerated slurry was filtered through a Whatman No.1 filter paper and the filtrate transferred into a volumetric flask. The volume was made upto 100 ml with the solvent to form the primary stock solution of the extract.4  The pulp obtained from the filtration process was weighed accurately and then subjected to sequential extraction in ethyl acetate, followed by methanol and finally with water. Each extract obtained was made upto 100ml using the respective solvent.

 

Preliminary phyto-chemical screening:

The different chemical tests were performed for establishing profile of the extract for its chemical composition. The chemical tests for various phyto-constituents like Alkaloids5, Amino acids6, Anthraquinones7, Flavonoids7, glycosides8, Phytosterols9, Saponins10, Steroids7, Phenolic compounds11, Tannins7, Triterpenoids7, coumarins12, Quinones12, Carbohydrates13, Cardiac glycosides14 in the chloroform, ethyl acetate, methanol and water extracts of Root-bark were carried out.

 

GC-MS Analysis:

The Chloroform, Ethyl acetate, Methanol root-bark extracts of Morinda tinctoria was analysed using Agilent technologies 6890 instrument. An inlet of 0.75 mm I.D., which improves the GC resolution, was used. The carrier gas was helium (1 mL.min–1) and the injector temperature was 250°C. The analytes were separated on a HP-5MS 30 m × 0.32 mm × 0.25 mm column (Supelco, Inc., Bellefonte, USA), kept at 50°C for 2 minutes and then ramped to 240°C at 4°C/min and held at the final temperature for 10 minutes. The transfer line was kept at 250°C and the ion source was held at 230°C. Mass spectra were measured at 70 eV and collected at the rate of 1 scan/second over an m/z range of 35 to 400. Chromatographic retention indices of separated compounds were calculated relative to a C8-C22 n-alkanes mixture.

 

Constituents were identified by comparing their mass spectra to those in NIST08 database and with mass spectra of authentic standards. In many compounds, the identities were confirmed by their relative retention indices with authentic standards. Mass spectra from the literature were also compared.

 

RESULTS AND DISCUSSION:

Preliminary phyto-chemical screening:

The phytochemical analysis of the Root-bark sample of Morinda tinctoria showed presence of Alkaloids, Saponnins, Steroids, Triterpenoids and Cardiac glycosides in the water extract;  Anthraquinones, Quinones in the Chloroform extract; Alkaloid, Phytosterol, Quinone in the Ethyl acetate extract and Phytosterol, Cardiac glycosides in the methanol extract (Table 1). It has been reported that many medicinal plants are rich in varieties of secondary metabolites such as alkaloids, flavonoids, tannins and terpenoids.15,16 Secondary plant metabolites show a wide range of biological activities on physiological systems with valuable therapeutic index.17 The anti-inflammatory effects of phytochemicals such as saponins, terpenoids, flavonoids, tannins, steroids and alkaloids have been reported by many researchers.18, 19, 20, 21, 22 Rupasinghe et.al. (2003)  reported that saponins possess hypocholesterolemic and antidiabetic properties.23 Steroids and triterpenoids has analgesic properties.24,25 Gopieshkhanna and Kannabiran (2007) observed the presence of carbohydrates, saponins, phytosterols, phenols, flavonoids and tannins in the plant extract having mosquito larvicidal activity.26

 

GC-MS Analysis:

Gas chromatography–mass spectrometry (GC–MS) is a useful tool for analysis of a wide range of relatively volatile compounds, and the technique has been widely applied in medical, biological, and food research. The GC-MS chromatogram of Root-bark extracts showed 21 peaks from Chloroform, 17 from Ethyl acetate and 14 from Methanol out of which compounds identified using NIST08 library are 11 from Chloroform, 10 from Ethyl acetate and 5 from Methanol (Table 2, Fig 1, 2, 3). The Indian Neem, Azadirachta indica (A.Juss), is attributed to possess different biological and pharmacological activities include antiplasmodial, antitrypanosomal, antioxidant, anticancer, antibacterial, antiviral, larvicidal and fungicidal activities.27 The alkanes (pentacosane, hexacosane, heptacosane (Table 3), nonacosane, triacontane and hentriacontane), olefin (squalene), esters (hexadecanoic acid, ethyl ester and 9,12.15-octadecatrienoic acid, ethyl ester, (Z,Z,Z)-) and  amide (erucyl amide) identified in the Petroleum ether extract of Neem leaves showed insecticidal activities against 5th instar of Oxya chinensis (Thunberg). Annona squamosa L. is reported to have significant anticancer, cytotoxic, antiparasitic, insecticidal or immunosuppressive activity and investigations into its chemical components showed the presence of Nonadecanoic acid (Table 3).28 Mass spectrometry analysis of roots of sea-lavender which have antifungal, antibacterial and insecticidal activity showed fatty acid methyl esters, hexadecanedioic, eicosanedioic (Table 3), and docosanedioic acids.29

 


 

Table 1: Preliminary phytochemical screening of Root-bark extracts of Morinda tinctoria

Tests

Water extract

Chloroform extract

Ethyl acetate extract

Methanol extract

Alkaloids

+

-

+

-

Protein

-

-

-

-

Anthraquinones

-

+

-

-

Flavonoids

-

-

-

-

Glycosides

-

-

-

-

Phytosterols

-

-

+

+

Saponnins

+

-

-

-

Steroids

+

-

-

-

Phenolic compounds

-

-

-

-

Tannins

-

-

-

-

Triterpenoids

+

+

-

-

Carbohydrates

-

-

-

-

Coumarins

-

-

-

-

Quinones

-

+

+

-

Cardiac glycosides

+

-

-

+

 

Table 2: List of compounds identified by GC-MS from the root-bark extract of Morinda tinctoria.

Chloroform extract

Ethyl acetate extract

Methanol extract

Name of the compound

RT value

Name of the compound

RT value

Name of the compound

RT value

9-Octadecen-1-ol (E)

25.48

Z-11-Pentadecenal

27.67, 35.41

Eicosanedioic acid

43.65, 45.68

9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)

33.29

3,4-Bis [[trimethylsilyl]oxy]benzaldehyde N-[tert-butyl] thiosemicarbazone

36.99

Urea, N,N-dimethyl-N’ [4-[1-methyl ethyl] phenyl]

20.89

Eicosen-1-ol,cis 9

29.58

Methyl vanilpyruvate bis (trimethylsilyl) ether

39.4, 44.09

18-Nonadecenoic acid

51.45

Ethanone, 1- [2,4,5-triethyl phenyl]

19.02

9H-purine, 9(trimethylsilyl)-2,6-bis [(trimethlsilyl)oxy]-

31.52

1,3 dimethyl adamantan 5-ol-7-carboxylic acid, bis[O-trimethylsilyl]

31.52

E-15 Heptadecenoic acid

28.23

Cyclodecanamine, N-methyl

40.42, 41.98

Z-11-Pentadecenal

48.22

7, 10-Octadecadienoic acid, methyl ester

31.59

5-Formylsalicylic acid

41.21

-

-

26,27-di(nor)-cholest-5,7,23-trien-22-ol, 3-methoxymethoxy

32.33

1,2-oxathiane, 6-dodecyl-2,2-dioxide

43.64

-

-

Benzoic acid, 4-(aminocarbonyl)

39.2

N-[6-cyclododecyl aminohexyl]aziridine

45.67, 48.21

-

-

Heptacosane

41.69

Urea, N,N-dimethyl-N’ [4-[1-methyl ethyl] phenyl]-

20.75

-

-

Cyclooctacosane

43.74

N-[3-[N-Aziridyl] propyl]cyclohexylamine

38.82

-

-

Heptanoic acid, 7(5-acetylamino methylthien-2-yl)

46.3

-

-

-

-

 

Fig 1: GC-MS Chromatogram of Chloroform extract of the root-bark of Morinda tinctoria

 

 

Table 3: Biological activities of the compounds identified from the Root-bark extracts of Morinda tinctoria.

Name of the Compound

Biological activity

Heptacosane

Insecticidal activity (Sunday et.al., 2008)

Nonadecanoic acid

Anticancer, cytotoxic, antiparasitic, insecticidal, immunosuppressive activity (Xiang et.al., 2010)

Eicosanedioic acid

Antifungal, antibacterial and insecticidal activity (Korulkina et.al., 2004)

Heptadecenoic acid

Antioxidant (Dr.Dukes phytochemical and ethnobotanical databases)

 

Fig 2: GC-MS Chromatogram of Ethyl acetate extract of the root-bark of Morinda tinctoria

 

Fig 3: GC-MS Chromatogram of Methanol extract of the root-bark of Morinda tinctoria

 

 


CONCLUSION:

In the present study chemical constituents have been identified from Chloroform, Ethyl acetate and methanol extract of the root-bark of Morinda tinctoria by Gas Chromatogram- Mass spectrometry (GC-MS) analysis. The presence of various bioactive compounds such as Heptacosane in Chloroform extract, Eicosanedioic acid and Nonadecenoic acid in the Methanol extract showed that root-bark of Morinda tinctoria might have potent biological activities on physiological systems such as antifungal, antibacterial and insecticidal activity.

 

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Received on 04.10.2011          Modified on 20.10.2011

Accepted on 28.10.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 83-87