Isolation Of Secondary Metabolites

Published Date: 02 Nov 2017

Isolation of secondary metabolites, which are important for the fitness of the plants as a defense and signal compounds, occurs universally in higher plants and shows very high structural diversity (Wink, 2008). Secondary metabolites are latent fungicidal, bactericidal, toxic agents to pests, repellent and herbivores (Wink, 2003 and Dewick, 1997). The aim of the isolation of this MM and MB is to summarize and investigate the mechanism of action of beneficial compounds to accelerate the discovery of new drugs derived from secondary metabolites.

4.4.1 Isolation of alkaloids by Rajukur et al, 2011

Alkaloids are defined as a natural plant compound that have a basic character and contain at least one nitrogen atom in a heterocyclic ring and having biological activities (Landanberg). Alkaloids are important chemical compounds that serve as a rich reservoir for drug discovery. Several alkaloids isolated from natural herbs exhibit antiproliferation and antimetastasis effects on various types of cancers both in vitro and in vivo (Lu et al, 2012).

The 94 mg of alkaloid was extracted with MM and 81 mg was extracted with MB and the presence was confirmed by alkaloid reagents. The Dragendorff’s reagent exhibit a red orange precipitate and Mayer’s reagent exhibit a white precipitate with alkaloid isolated from MM and MB. The TLC plate (Image 4) evidently shows the number of alkaloids presenters in MM and MB. Both the alkaloids from MM and MB exhibit a bitter tamarind odour.

The GC-MS results from the figure 2 …………………

Image 4 : TLC observation of secondary metabolites from MM and MB

Figure 2: GC-MS analysis of alkaloids isolated from MM and MB

4.4.2 Isolation of flavonids by

Flavonoids form a large class of significant naturally occurring bioactive compounds (Costa et al, 2010). The flavonoid 180 mg was isolated from MM and 134 mg was isolated from MB. The flavonoid isolated from Mm and MB showed a positive orange color in Shinoda test and a white precipitate in lead acetate test. These results and TLC results (Image 4) suggested that the MM and MB have flavanoid content.

For instance, flavonoids have been shown to have antibacterial, anti – inflammatory, anti allergic, antiviral antineoplastic activity (Alan and Miller, 1996). Many of these alleged effects have been linked to their known functions as a strong antioxidant, free radical scavenger and metal collectors (Nakayama et al, 1993).

The flavonoids isolated from MM and MB exhibit a chocolate flavor odour. The GC-MS results from figure 3………….

Figure 3: GC-MS analysis of flavanoid isolated from MM and MB

4.4.3 Isolation of phenolics and terpinoids by Rajukur et al, 2011

The plant derived phenolics and terpenoids have great remedial importance, which are a most important group of organic compounds. The presence of phenolics and terpenoids will encourage further research for possible new drugs leads (Evans, 2002)

The phenolic and terpenoids of 160 mg was isolated from MM and 110 mg was isolated from MB. The TLC results from the image 4 show evidence of a number of compounds present in phenolics and terpenoids. The presence of phenolic and terpinoids was confirmed by Liebermann-Burchard test and Libermann test, which exhibit green colour and gave positive results .The GC-MS results from figure 4…………….

Figure 4 : GC-MS analysis of phenolics and terpenoids from MM and MB

4.4.4 Isolation of quaternary alkaloids and N-oxides by Rajukur et al, 2011

The extractability of quaternary alkaloids as in N-oxides tends to pose a serious challenge to natural products Chemists, especially in the resource - limited countries. This is because of their high level of polarity and hence a significant affinity to the aqueous-acid phase usually employed in the common extraction process (Oguegbulu et al, 2012).

The 323 mg of quaternary alkaloids and N-oxides were isolated from MM and 437 mg was isolated from MB, both have a mango leaf odor. The GC-MS results from the figure 4……….

Figure 5: GC-MS analysis of quaternary alkaloids and N-oxides from MM and MB

4.5 Phytochemical screening of MM, MB, MC and MN

Phytochemical screening analysis is a process of tracing plant constituents. It provides basic information about the medicinal importance of the plant extracts, which leads to the segregation of new and novel compounds. The extracts obtained from table 8 were subjected to screening. The domino effects are shown in table 15, it revealed the presence of most important secondary metabolites like alkaloids, flavonoids, tannins, saponins, steroids, terpenoids, phenols, glycosides, anthraquinone and other constituents.

The phytochemical screening of table 8 extracts revealed the presence of alkaloids and flavonoids, but a petroleum ether extract of MN shows absence of alkaloid and also petroleum ether extract of MC and MN, aqueous extract of the MC and acetone extract of MM and MB exhibit negative results on flavonoids test. Petroleum ether and ethanol extract of MM and MB, acetone extract of MM and ethyl acetate extract of MB shows the presence of tannins. Ethanol and the chloroform extract of MM and aqueous extract of MM and MB revealed the presence of saponins. Almost all the petroleum ether extracts show the presence of terpenoids, except MC and the rest of the other solvent extracts. Petroleum ether extract of MB and MC, an ethanol extract of the MC and acetone extract of MB revealed the presence of steroids. Chloroform extract of MM and MB, an ethanol extract of the MC and aqueous extract of MC and MN shows the presence of carbohydrates and ethanol extract of the MC and aqueous extract of MC and MN revealed the presence of reducing sugar. The petroleum ether extract of MM and MC, ethyl acetate, ethanol and aqueous extract of MM, MB and MC, and chloroform extract of MM and MB shows the presence of phenol. Ethyl acetate extract of MM, MB, MC and ethanol extract of MM revealed the presence of glycosides. Almost of the extracts shows the presence of anthocyanin except ethyl acetate extract of MB, chloroform and acetone extract of MM and MB. All the extract shows the presence of anthraquinone and revealed the absence of protein and amino acids (table 15).

Alkaloids are alkaline in nature, nitrogenous substances and reveal pharmacological activities. Several alkaloids are used to cure cardiac, respiratory and cancer disorders (www.enthogen.com). Flavonoids are hydroxlated phenolic substances and distributed in higher plants. Also act as antioxidant, support entire cardiovascular system, including arterial walls and effective antimicrobial substances (Aslam et al, 2009).

Phenolic compounds are effective antifungal agents and several uncomplicated bioactive phytochemical consist of single substituted phenolic ring (Doughari and Obidah, 2008). Tannins are astringent, aromatic acidic glycosides. These occur in all parts of plants including roots, stem, bark, leaves, fruits and even hairs. Tannins promote healing of wounds. These are effective in diarrhea, colitis and peptic ulcers. Glycosides are active and complex substances containing carbon, hydrogen and oxygen. They have characteristic actions of contractile forces of cardiac muscle. Cardiac glycosides are known as cardiotonic glycosides and are characterized by their stimulative effects on the heart (Hikino and Kiso, 1988).

The saponins form strong insoluble complexes with body cholesterol and thus cause its depletion by preventing its reabsorption, due to increasing its excretion, in the same way as other cholesterol lowering drugs do. Saponins also show anti-fungal, antibacterial and anti-protozoal effects (Aslam et al, 2009). Terpenoids, which are generally deemed safe, restrain the growth of malignant cells, diminish tumor size, cholesterol level and micro-organism concentration (Gupta et al, 2011).. Anthraquinones are chemically known as 9, 10 Dione derivatives of anthracene. Anthraquinone, glycisodes are present in madder, a climbing plant and used as dye stuff (Alizarin). They possess astringent, purgative anti-inflammatory, fair anti-tumor and bactericidal belongings (Takagi et al, 1980, Fatima, 1988 and Feroz et al, 1993)

The presences of flavonoids, triterpenes and steroids in curd extract showed significant antimicrobial activity against microbes18. Steroids are potential therapeutic agents for infectious diseases. In this context, several steroid-antibiotics have been developed to mimic the antibacterial behavior of endogenous peptide antibiotic. In addition, several studies suggest that functional groups of steroid-derivative are involved in the bacterial activity. Steroidal saponins were reported for antimicrobial activities (Arif et al, 2011).

The chemical constituent and bioactive compounds occurring in several plant materials are known to have fungicidal and bactericidal property. Higher plants are contributing to produce high structure diversity of organic compounds. These accumulations in plant cell had attended of all research and academic faculty. So this knowledge is the basis of finding herbal medicine from MM, MB MC and MN.

Table 15: Phytochemical constituents of the aerial roots of Rhaphidophora aurea twined over four diverse host trees

Studies

M1

B1

C1

N1

M2

B2

C2

N2

M3

B3

C3

N3

M4

B4

C4

N4

M5

B5

M6

B6

Alkaloids

+

+

+

_

+

+

_

+

+

+

+

+

+

+

+

+

+

+

+

+

Flavonoids

+

+

_

_

+

+

+

+

+

+

+

+

+

+

_

+

-

-

+

+

Tannins

+

+

_

_

_

+

_

_

+

+

_

_

_

_

_

_

+

-

-

-

Saponins

_

_

_

_

_

_

_

+

_

_

_

+

+

_

_

-

-

-

+

Terpenoids

+

+

_

+

_

_

_

_

_

_

_

_

_

_

_

_

-

-

-

-

Steroids

_

+

+

_

_

_

_

_

_

_

+

_

_

_

_

_

-

+

-

-

Carbohydrates

_

_

_

_

_

_

_

_

_

_

+

_

_

_

+

+

-

-

+

+

Reducing sugars

_

_

_

_

_

_

_

_

_

_

+

_

_

_

+

+

-

-

-

-

Phenols

+

_

+

_

+

+

+

_

+

+

+

_

+

+

+

_

-

-

+

+

Anthraquinone

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Quinone

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

-

-

-

-

Anthocyanin

+

+

+

+

+

_

+

+

+

+

+

+

+

+

+

+

-

-

-

-

Glycosides

_

_

_

_

_

_

_

_

+

+

+

_

+

_

_

_

-

-

-

-

Protein & AA

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

-

-

+

-