International Journal of Scientific Research and Review ISSN NO: 2279-543X

Phytochemical screening by LC-MS analysis of flowers of

Allamanda neriifolia Hook
Sumathi R#1, SujapandianR*
Sujapandian R*22, Saranya G*3

#1.
Assistant Professor, Department of Biochemistry, R.A.College for women, Thiruvarur.
Tamilnadu, India

*2.
Assistant Professor, Department of Biochemistry, R.A.College for women, Thiruvarur.
Tamilnadu, India

*3
, Research Scholor. Department of Biochemistry, R.A.College for women, Thiruvarur.
Tamilnadu, India

*Corresponding Author: Sumathi R

Abstract

Medicinal plants are the nature's gift to human being to have disease-free healthy life.
This study was undertaken to investigate the bioactivity of A.neriifolia extracts and to
evaluate their chemical composition. The aim of the present study was to identify the
phytoconstituents using Liquid chromatography - Mass spectroscopy analysis. By using the
methanol extracts of A. neriifolia, molecules such as 8-O-Methylretusin, Apigenin 7- O -
(6''dihydrogalloyl) -glucosyl-8-C hamnosyl 6 C glucoside, 3’O Methylorobol,4,6 Dimethyl 3
(4'hydroxyphenyl) coumarin and 3-Hydroxy-3',4'-Dimethoxyflavone derivative were
identified by using LC-MS technique.

Key words: Allamanda neriifolia Hook, phytochemicals, LC-MS analysis, Bioactivity.

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INTRODUCTION

Indian medicinal plants have lot of potential towards curing many diseases (Santhosh
Kumar et al., 2018). Secondary metabolites are economically important in the production of drugs,
flavor and fragrances, dye and pigments, pesticides and food additives. Many of the drugs that are
derived from the secondary metabolites are simple synthetic modifications or copies of these
naturally obtained substances (Hussain et al., 2016). These phytochemicals can be used in
treatment as anticancer, antimicrobial, antioxidant, anti-inflammatory agents etc, (Wadikar and
Patki, 2016). Recent studies show that these phytochemicals are safe, broadly effective and have
less adverse effects. However in vivo studies of these phytochemicals are necessary to demonstrate
their efficacy, safety and to verify their bioavailability (Soni and Singhai, 2012). The different
phytoconstituents present in medicinal plants are flavonoid, alkaloid, phenol and tannins,
carboxylic acids, terpenes and amino acids and inorganic acids. These phytoconstituents give
specific distinctiveness and properties to plants (Parekh and Chanda et al., 2006).

Analysis of small amounts of chemicals has become easier and more cost-effective owing
to the development of hyphenated chromatographic techniques such as GC or LC-MS (Antony
Sandosh et al., 2013). Analytical techniques are generally employed for drug analysis are spectral
methods, chromatographic methods, electro analytical techniques, biological method and
microbiological methods, physical methods ,radioactive methods and other techniques like
conventional titrimetric method, gravimetric and polarometric methods.

Allamanda neriifolia Hook is a genus of flowering plants in the dogbane family,

Apocynaceae. Some species are familiar as ornamental plants cultivated for their large, colorful
flowers. Most species produce yellow flowers. The flower has five lobed sepals and a bell- or
funnel-shaped corolla of five petals, yellow in most species. Allamanda species had several
chemical compounds; including iridoid lactones such as allamandin, plumericin, and plumierides.
Due to the presence of these compounds this flower exhibit anti neoplastic, anti leukemic, anti-
inflammatory, antimicrobial, and anti-leishmanial activities (Castillo et al., 2010; Kuete et al.,
2011). antidermatophytic and antitumor activities (Tiwari et al.,2002; Sofowora,1993) . Hence, the

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present investigation was undertaken to evaluate the presence of phytochemicals present in the
flowers of A.neriifolia.

MATERIALS AND METHODS

Collection of plant material

The flowers of Allamanda neriifolia Hook was collected from Tiruchirappalli and it was
authentified by Dr.S.John Britto, Director, RAPINAT Herbarium and Centre for Molecular
Systematics, St.Joseph’s College, Tiruchirappalli (Voucher No: 002) dust were removed from
flowers and was dried at room temperature. These dried materials were macerated to powder and
stored in air tight container for further use.

Preparation of the extract

300gm of coarsely ground powder was packed into soxhlet column and extracted with
50ml of 70% methanol for 48 hours (64.5-65.5°C). The extract was filtered and concentrated
on water bath at reduced pressure (bath tem 50°C) to syrup consistency (yield: 15%). Then the
dried extract was stored in air tight container for further use.

LC- MS analysis of A.neriifolia

Liquid chromatography is a fundamental separation technique used in life sciences and

related fields of chemistry. Liquid chromatography (LC) combined with mass spectrometry (MS)
is a powerful tool for qualitative and quantitative analytics of organic molecules from various
matrices, and the use of this hyphenated technique is very common in bio analytical laboratories.
In the present study, LC/MS methods and the required sample preparation applications were
developed for detection of compounds such as flavones, terpene and sesquiterpenes lactones.

Methanolic extract was used for the analysis. For determination of secondary metabolites,
micro TOF-Q II (Bruker, Germany), UV detector at 330nm and Quadruple II for mass analysis
and TOF for mass detection was utilized. The column used was UHPLC Dionex C18 RP Acclaim
120Å, 2.1 × 150mm, 3.0μm column (Dionex, USA). Solution A: ACN (1% Acetic acid) and
Solution B: Water (1% Acetic acid) was used as mobile phase (Chitra Balasubramanian, Brindha
Pemiah, 2015).

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RESULTS AND DISCUSSION

LC-MS analysis of Allamandaneriifolia flower extract

The methanolic extract was subjected to LCMS analysis. Secondary metabolites present in
methanol extracts of A.neriifolia flower such as terpenoids, saponins, flavonoids, tannins, steroids
and alkaloids were identified with the help of this technique. The active principles with their
molecular weight, retention time and structure are presented in Table 1. The chromatogram and
the double mass spectrum of the methanolic extract of the test drug are shown in Figure 1.
The methanolic extract was subjected to LCMS analysis to understand the major molecules present
in the selected plant. In the methanol extracts of A.neriifolia flower molecules such as 8-O-
Methylretusin, apigenin 7-O-(6''-dihydrogalloyl)-glucosyl-8-C rhamnosyl-6-C-glucoside,3’-O-
Methylorobol,4,6-Dimethyl-3(4'-hydroxyphenyl)coumarinand3-Hydroxy-3',4'-
Dimethoxyflavone derivative were identified Figure 2,3,4,5 and 6.

TABLE 1: LC-MS ANALYSIS OF A.NERIIFOLIA FLOWER EXTRACT

S. No. R.T (min) Compound [M _ H] [M + H]

1. 32.9-33.3 8-O-Methylretusin 297.3 -

2. 42.7-42.9 Apigenin 7-O-(6''-dihydrogalloyl)- 893.3 -

glucosyl-8-C-rhamnosyl-6-C-
glucoside

3. 39.8-40.1 3’-O-Methylorobol 299.3 -

4. 48.6-48.8 4,6-Dimethyl-3(4'-hydroxyphenyl) 266.2 -

coumarin

5. 51.6-51.8 3-Hydroxy-3',4'-Dimethoxyflavone 297.2 -

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FIGURE 1: LC-MS ANALYSIS OF A.NERIIFOLIA FLOWER EXTRACT

FIGURE: 2 8-O-METHYLRETUSIN 297.3

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FIGURE: 3 APIGENIN 7-O-(6''-DIHYDROGALLOYL)-GLUCOSYL-8-C-

RHAMNOSYL-6-C-GLUCOSIDE

FIGURE: 4 3’-O-METHYLOROBOL

FIGURE: 5 4, 6-DIMETHYL-3(4'-HYDROXYPHENYL) COUMARIN

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FIGURE: 6 43-HYDROXY-3', 4’-DIMETHOXYFLAVONE

Liquid Chromatography - Mass Spectrometry (LC-MS) is proved to be a very useful

technique for plant metabolite profiling and allows the identification of a large variety of common
plant metabolites in a single chromatogram. The research reveals the potential of A.neriifolia
flower as a good source of bioactive compounds such as 8-O-Methylretusin, apigenin 7-O-(6''-
dihydrogalloyl)-glucosyl-8-C rhamnosyl-6-C-glucoside,3’-O-Methylorobol, 4,6-Dimethyl-3(4'-
hydroxyphenyl) coumarin and 3-Hydroxy-3',4'-Dimethoxy flavones derivatives that justify the use
of this plant for its various ailments by traditional practitioners (Chitra balasubramanian, Brindha
Pemiah, 2015). Flavonoids are the most diverse group of secondary metabolite belonging to a
subfamily of polyphenols which involve in plant growth, reproduction and seed germination. They
also involve in protection against pathogens (Prabuseenivasan et al., 2006) and predators’
.Flavonoids is of interest due to their biological effects. They are able to scavenge free-radical,
modulate enzymatic activity, inhibit cell proliferation; modify allergens, carcinogens and viruses
(Brglez Mojzer et al., 2016). Phenolic compounds as another bioactive compounds characterized
by hydroxylated aromatic rings with varying substitution patterns and functional derivatives
(Ghasemzadeh et al., 2013) and have been reported to have antioxidants, antibacterial, antiviral,
anti-cancer and anti-inflammatory properties (Mattila and Hellstrom, 2007; Sacchetti et al., 2005).

CONCLUSION

The active phyto-constituents present in the plant were the potential source for new drug
and therapeutic leads. The results of this study revealed that methanolic extract of A. neriifolia
contain pharmacologically active substances. The LC-MS analysis has brought light for the
presence of phytosterols and bioflavonoid. Therefore the flowers of A. neriifolia could be new
sources of development of new plant based therapy for management of several diseases.

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ACKNOWLEDGEMENT

The authors are thankful to Rabiammal Ahamed maideen College for Women, Tiruvarur
and Shastra University, Thanjavur.

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