PHYTOCHEMICAL ANALYSIS OF CRUDE RHIZOME EXTRACTS FROM K. galanga

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PHYTOCHEMICAL ANALYSIS OF CRUDE RHIZOME EXTRACTS FROM K. galanga

4.1 INTRODUCTION

Developing countries mostly depend on traditional and medicinal plants focusing towards healthcare applications (Allameh et al.,2002). The traditional medicines involve the use of different medicinal plant extracts or the bioactive constitutions that are helpful todiagnose, prevent, treat, control or curediseases (Davis and Robson, 1999). The study on ethnomedicine intensely represents one of the finest avenues in searching novel economic plants for medicine (Esonu et al., 2006). Qualitative phytochemical analysis of Momordica charantia and Nerium oleander leaf extracts has already confirmed the presence of various phytochemicals like carbohydrates, cholesterol, protein, amino acid, alkaloids, flavinoids, tannins, saponins, cardiac glycosides, terpenoids, coumerins, anthocyanins, steroids, fatty acids, phlabotanins,phenols and starch in their aqueous, ethanol, methanol, ethyl acetate and chloroform plant extracts (Haller, 1990). Phytonutrients as remedies for human diseases in curing various ailments possess essential antioxidant and anticancer properties (Koul et al., 1989). The reports provided origin for the present investigation to carry out research on phytochemical studies for rhizome crude solvent extracts in K. galanga.

The world depends upon the herbal medicine for the largest source of the plant biodiversity. Nearly 70 to 80 percent of world populations are being used since the ages as the traditional healthcare systems (Lale, 2003). The medicinal plants contain bioactive phytochemical constituents that produce definite physiological effects on human body (Kaladhar et al., 2010). Natural compounds in medicinal plants containing phytochemicals protect the human body from various diseases (Apparao et al., 2011). The phytochemicals are having medicinal properties that are having nutritive and non-nutritive plant chemicals. Phytochemicals are divided into two groups according to their functions in plant metabolism. They are primary and secondary metabolites (Kaladhar etal., 2013). Primary metabolites consist of carbohydrates, amino acids, proteins, chlorophyll while secondary metabolites consist of alkaloids, saponins, flavonoids etc. (Prabha et al., 2013).

Plants always contains common source of medicaments in traditional preparations or as pure bioactive principles. The identification of plants or plant extracts could be used as drugs, or that could replace some pharmaceutical preparations are being purchased and imported from different countries(Farnsworth et al., 1985). The research in drug finding from medicinal plants involves a versatile approach combining the phytochemical, botanical, biological, and molecular techniques (Barboza et al., 2009). The medicinal plant drug discovery continues to provide innovative and significant lead against several pharmacological target include Inflammation, Cancer, HIV/AIDS, Alzheimer's, Malaria, and Arteriosclerosis (Moridi et al., 2011). Several natural products of plant origin recently introduced to the United States market include artether, galantamine, nitisinone, and tiotropium (De et al., 2008).

The drug discovery from medicinal plants is to provide a source of new drug leads against dreadful diseases. Many challenges are encountered with the procurement of plant parts, the selection and implementation of the scale-up of active compounds and bioassays (Balunas and Kinghorn, 2005). The Ethanopharmacologists, botanists, microbiologists, biochemists and natural-product chemists are combining the phytochemicals that could be used for treatment of various infectious diseases. Nearly 30% to 50% of existing pharmaceuticals are resulting from plants which are used as antimicrobials (Palombo, 2011). The traditional healers have used medicinal plantsfrom ancient times in cure of infectious conditions. The Plants that are prosperous in an extensive variety of secondary metabolites like saponins, phenols, tannins, terpenoids, alkaloids, and flavonoids found in vitro show antimicrobial properties (Cowan, 1999).

A valuable association between the traditional and western medical practitioners is due to the use of traditional and herbal medicines (Cheng, 2000). Various non-scientific reports provide enhanced interest from western countries in herbal remedies that provide urgent need to develop new effective drugs from traditionally used medicinal plants (Offiah et al., 2011). Recently phytomedicinal components predictable the attention of the pharmaceutical and scientific communities to produce novel medicines towards diseases is to be investigated. It involves the isolation, differentiation and identification of the secondary metabolites that are produced by various medicinal plants and used as the effective principles in medical preparations (Taylor et al., 2001).

Higher plants are having solar-powered biochemical factories (both primary and secondary metabolites) which manufacture their requirements to survive from microbial species. Many species of higher plants synthesize and accumulate organic substances in different quantities to be economically useful as chemical feed stocks or as raw materials for various scientific and commercial applications (Brown et al., 2008). The natural substances by a huge number of industries and natural plant products are applied directly or indirectly upon humans and surrounding species. Phytochemicals are used to a large amount by the pharmaceutical, food, cosmetics and agrochemical industries (Franz et al., 2011).The economically important plants provide as unique sources of flavors and fragrances, industrial oils (both volatile and fixed), natural rubber, surfactants, resins (e.g., rosin and tall oil), gums, saponins, pharmaceuticals, waxes, dyes, pesticides and many specialty products (Balandrin et al.,1985).

4.2 MATERIALS AND METHODS

4.2.1 Collection and Extraction of Plant Materials

The collection and extraction of the solvents (ethyl acetate, methanol, ethanol, chloroform and water) rhizome extracts of K. galangaseparately was done as mentioned in Chapter 3 (page number 30 (3.2.1 & 3.2.2)).

DSCN1417

  1. Rhizomes

DSCN1440

  1. Plant rhizome powder

Figure 4.1: Rhizomes and powder of K.galanga

4.2.2 Preliminary Phytochemical Screening

Various extracts were used for preliminary screening for phytochemicals such as carbohydrates (Molisch’s test), cholesterol (Liberman Burchard test), protein (Biuret test), amino acid (Ninhydrin test), alkaloid (Mayer’s and Dragandreff’s test), saponins, tannins, flavinoids, cariac glycosides, terpenoids and phlobatanins.

4.2.2.1 Test for Amino Acids

To 2 ml of the extract, 2 ml of Ninhydrin reagent is to be added and keep the solution in hot water bath for 15 minutes. The formation of purple color indicates the presence of amino acids in the sample.

4.2.2.2 Test for Proteins

To 2 ml of the extract, 2 ml of Biuret reagent is to be added. An appearance of violet color ring indicates the presence of protein (peptide linkages of a molecule).

4.2.2.3 Test for Carbohydrates

To 2 ml of extract add 2drops of molisch’s reagent and mix the solution. Nearly 2 ml of Conc. H2SO4 is to be added drop by drop from the sides of the test tube. A reddish violet color ring appearance at the junction of two layers immediately indicates the presence of carbohydrates.

4.2.2.4 Test for Alkaloids

To 2 ml of extract, add 1% HCl, few drops of Mayer’s reagent (potassium mercuric iodide) and 6 drops of Dragendroff’s reagent (potassium bismuth iodide). An organic precipitate indicates the presence of alkaloids in the extract.

4.2.2.5 Test for Steroids

To 2 ml of acetic anhydride, 0.5 ml of extract and 2 ml of H2SO4 is to be added. The color changed from green or violet to blue indicates the presence of steroids.

4.2.2.6 Test for Cholesterol

To 2 ml of chloroform taken in a dry test tubeand add 2 ml of the extract. About 10 drops of acetic anhydride and 2 to 3 drops of Conc. H2SO4 are to be added to the solution. A change from red rose color solution to blue green color solution indicates the presence of cholesterol.

4.2.2.7 Test for Cardiac Glycosides

To 5 ml of extract add 2 ml of glacial acetic acid contain one drop of ferric chloride solution is to be added. The solution was underlayed with 1ml of Conc.H2SO4. A brown colored ring of the edge indicates a deoxysugar is a feature of cardenolides.A violet ring may appear under the brown ring indicates an acetic acid layer and a greenish ring might form just progressively throughout thin layer.

4.2.2.8 Test for Flavonoids

To 5 ml of dilute ammonia solution, afew drops of Conc.H2SO4 are to be added. A yellow colored solution confirms the presence of flavonoids and will disappears on long standing.

4.2.2.9 Test for Saponins

To the 5 ml of plant extract, 20 ml of distilled water is to be added and is disconcerted in a graduated container for 15 minutes. Development of 1cm layer of spume in the container shows the presence of saponins.

4.2.2.10 Test for Tannins

To 5 ml of extract, few drops of 1% of lead acetate are to be added. A yellow colored precipitate formed in the test tube shows the presence of tannins.

4.2.2.11 Test for Terpenoids

To 2 ml of extract add 2 ml of chloroform and 3 ml of Conc.H2SO4. Formation of a monolayer of reddish brown coloration of an interface shows a positive result for the terpenoids.

4.2.2.12 Test for Phlobatinins

To 2 ml of extract add 1%aqueous HCl and boiled for few minutes. A red precipitate formed and deposited in the test tube is an evidence for the presence of phlobatinins.

4.2.2.13 Test for Fatty Acids

To 0.5 ml of extract, 5 ml of ether is to be added. The solution was allowed for evaporation on a filter paper and dried for few minutes. The emergence of transparence on filter paper indicates the occurrence of fatty acids.

4.2.2.14 Test for Anthocyanins

To 2 ml of extract, 2 ml of 2N NH4Cl and ammonia are to be added. The appearance of pink-red color turning to blue or violet color indicates the presence of anthocyanins.

4.2.2.15 Test for Coumarins

To 3 ml of 10% NaOH, 2 ml ofplant extract is to be added. The formation of yellow color solution indicates the presence of coumarins.

4.2.2.16 Test for Leucoanthocyanins To 5 ml of extract, 5 ml of isoamyl alcohol is to be added. The red colorappears in upper side of the solution of test tube indicates the presence of leucoanthocyanins.

4.2.2.17 Test for Phenols

About 2 ml of extract, 3ml of ethanol and a pinch of FeCl3 is to be added. The formation of greenish yellow color solution indicates the presence of phenols.

4.2.2.18 Test for Quinones

To 2 ml of extract, 3 ml of Conc. HCl is to be added. Formation of green color solution indicates the presence of quinones.

4.2.2.19 Test for Emodins

To 2 ml of NH4OH, 3 ml of benzene and 5 ml of plant extract is to be added. The formation of red color indicates the occurrence of emodins.

4.3 RESULTS AND DISCUSSION

The useful medicinal properties from plant materials usually resultsdue to the combination of secondary metabolites present in the plant (Rates, 2001). The medicinal procedures of plants are exclusive to particular plant groups or species and are constant with the perception as the combination of secondary products in a specific plant are often taxonomically distinctive (Donald, 2000).

The development in medicinal plant research has undergone a unique growth during the last two decades (Tilman et al., 2002). An extensive trend towards the use of natural plant remedies has produced enormous need for information about the properties and uses of medicinal plant. The available information regarding medicinal plants can show antitumor, antianalgesic and insecticide properties (Baker et al., 2007). Moreover a medicine, the plants provides thousands of new compounds for instance foods, beverages, fragrances, flavorings, dyes, fibers and building materials etc (Mungole and Chaturvedi, 2011).

The present study carried out on the plant rhizomesis to reveal the occurrence of medicinally vigorous metabolites. The phyto-constituentsevaluation of K. galanga rhizome extracts has been shown in Table 4.1.

The aqueous extract of mature rhizomes of K. galanga contains metabolites like carbohydrates, amino acids, alkaloids, cardiac glycosides, tannins, phlobatinins, fatty acids and coumarins.

A comparative study with the tested extracts, ethanolic extract from mature rhizomes of K. galanga is found to be containing more number of phytocompounds like carbohydrates, cholesterol, amino acids, steroids, alkaloids, cardiac glycosides, saponins, tannins, terpenoids, phlobatanins, fatty acids, coumarins and phenols.

The methanolic rhizomes of K. galanga extract has shown various metabolites like carbohydrates, steroids, amino acids, steroids, alkaloids, cardiac glycosides, tannins, terpenoids, phlobatanins, fatty acids, coumarins and phenols based on the phytochemical tests.

The ethyl acetate from rhizomes of K. galanga had shown very less number metabolites in their extract like amino acids, cardiac glycosides, tannins and fatty acids.

The choloroform extract from mature rhizomes of K. galanga is found to be containing carbohydrates, cholesterol, amino acids, alkaloids, cardiac glycosides, tannins, saponins and fatty acids. As there is less phytochemical constituents in chloroform extract of K. galanga, the further analysis on biological activities was not been considered in the present work.

Based on the previous literature, the phenolic phytocompounds have a great importance in relation to potential for beneficial effects on health (Devasagayam et al., 2004). Over the last few decades, several experimental studies have revealed importance of phenolic compounds with biological and pharmacological properties/activities like cytotoxic, anti-inflammatory, antioxidant, and antiviral activities. Phenolic compounds from plants or synthetics are also used in treatment of kidney and stomach problems (Boudreau and Beland, 2006). Pancharoen et al., 2000 has shown the anti-inflammatory properties isolated from K. galanga.

Saponins present in bioactive compounds in foods may have protective strategy in lowering the risk of cardio-vascular diseases and human cancers (Kris-Etherton et al., 2002).

Tannins are also present in all the extracts of the plants and may show decrease in the bacterial proliferation by blocking the key enzymes at microbial metabolism (Mungole et al., 2010). Tannins play an important role as potent antioxidant (Rahimi et al., 2005). Herbs have tannins as their main constituents and are used for treating intestinal disorders such as dysentery and diarrhea (Palombo, 2006).

Flavonoids are well known for their anti-inflammatory, antiviral, antioxidant and cytotoxic activity. Flavonoids in the plant extracts may also be used in the treatment of rheumatic fever,diabetes and hypertension etc (Duan and Wang, 2002).

The class of cardiac glycosides are utilized in the cure of congestive heart failure may have anti-arrhythmic agents. These agents play an emerging role with adiverged category of compounds in the prevention and treatment of proliferative diseases such as cancer (Newman et al., 2008).

Various herbsare using as food and also as medicinal purposes from centuries to till date. The herbs that possess antiplatelet, antitumor, hypolipidemic or immune-stimulating properties are useful adjuncts in reducing the risk of cardiovascular disease and cancer. In different herbs, a large variety of active phytochemicals, including the terpenoids, lignans,flavonoids, polyphenolics, sulfides, coumarins, plant sterols, saponins, carotenoids, curcumins, and phthalides, have been identified in the previous literature (Craig, 1999; Craig, 2002).

Biochemicals and phytochemicals present in plants may show good biological activities. The phytochemical screening and qualitative estimation of the plants in the present study showed that the rhizomes were rich in phyto-components like aminoacids, cardiac glycosides, and fatty acids in all the extracts. Methanolic and ethanolic extracts has shown some common metabolites like carbohydrates, aminoacids, cholesterol, steroids, alkaloids, cardiac glycosides, tannins, terpenoids, phlobatanins, fatty acids and phenols.

Table 4.1: Phytochemical screening of Kaempferia galanga rhizome extracts

Name of the compound

Aqueous

extract

Chloroform

extract

Ethanol

extract

Ethyl acetate

extract

Methanol

extract

Amino acids

++

++

+++

++

+++

Protein

-

-

-

-

-

Carbohydrates

+++

+

+++

-

+++

Alkaloids

+

+

++

-

++

Steroids

-

+

++

-

++

Cholesterol

-

+

+++

+

+++

Cardiac glycosides

+++

+

+++

++

++

Flavonoids

-

-

++

-

+

Saponins

-

+

++

-

-

Tannins

+

+

++

+

++

Terpenoids

-

-

++

-

++

Phlobatinins

+

-

++

-

+

Fatty acids

-

++

+++

+

++

Anthocyanins

-

-

-

-

-

Leucoanthocyanins

-

-

-

-

-

Coumarins

+

-

++

-

+

Phenols

+++

-

+++

-

+++

Quinones

-

-

-

-

-

Emodins

-

-

-

-

-

Note: +++ isstronglypositive (Color with more intensity); ++ ismoderately positive (Color intensity medium); + is weakly positive (Colorintensity low); -is Negative

The presence of cardiac glycosides in medicinal plants is used in the ancient Indian medicinal systems (Shellon, 1996; Sofowora, 1993). The plant studies can be seen as potential source of useful drugs. Further studies onK. galanga rhizome extracts have been conducted to identify benefits and uses in the field of medicine. The medicinal plants have phytochemical compounds that are helpful as medicine in the control of diseases and disorders. In the present studies, K. galanga rhizome extracts are tested for phytochemical activity. Ethanolic extract has good number of compounds compared to ethyl acetate, aqueous,methanolic and chloroform extracts.

4.4 CONCLUSION

Phytochemical analysis of rhizome extracts of K. galanga has shown biological compounds like carbohydrates, cholesterol, amino acids, steroids, alkaloids, flavonoids, cardiac glycosides, saponins, tannins, terpenoids, phlobatinins, fatty acids, coumarins and phenols. The result suggests that the phytochemicals present in K, galanga rhizomes may show antimicrobial, anti-inflammatory and antioxidant properties.

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