STUDY OF TOTAL PHENOLIC CONTENT, TOTAL FLAVONOID CONTENT, ANTIOXIDANT, ANTIBACTERIAL ACTIVITIES AND PHYTOCHEMICAL SCREENING OF MEDICINAL PLANTS (Aegle marmelos, Cuscuta reflexa, and Pityrogramma calomelanos)

ABSTRACT

 

Natural products are the compounds or substances produced by living organism that are found in the nature. Phytochemistry or Plant chemistry is the branch of chemistry, deals with chemical nature of the plant or plant products, also called a chemistry of natural products. Natural products of plants may provide a new source of antimicrobial agents with possibly novel mechanism of action. Antibacterial potency of medicinal plant extracts has been tested against Bacillus subtilis ATCC 3026 and Enterococcus faecalis ATCC 32026 by disc diffusion assay. TPC, TFC and antioxidant was calculated by taking gallic acid, quercetin and ascorbic acid as standard respectively. Antibacterial analysis identified with ZOI for ethyl acetate extract of leaves of A. marmelos (14mm, 12mm), fruits of A. marmelos (13mm, 11mm), hexane extract of stem of C. reflexa (13mm, 12mm), leaves of P. calomelanos (15mm, 12mm)  and chloroform extract of C. reflexa (13mm, 10mm) were active for the inhibition of the growth of both (Enterococcus faecalis and Bacillus subtilis) bacteria used. The methanol extract of Aegle marmelos fruits showed the strongest DPPH radical scavenging activity with IC₅₀ values 28.093 µg/mL very close to standard ascorbic acid (22.451 µg/mL). The result demonstrated that the total phenolic content was highest in methanolic extract of C. reflexa (88.1666 mg GAE/g extract) and the total Flavonoid content was highest in methanol extract of P.calomelanos leaves (169.61 mg QE/g extract) while the others had significantly lower values. These extracts showed good antibacterial activity, antioxidant, TPC and TFC so could find out the possibility of these medicinally important plants as a potent antimicrobial drug and for other pharmacological properties to develop as cost effective formulation.

 

Keywords: TPC, TFC, Antibacterial activity, Antioxidant, Phytochemical Screening, IC_50, ZOI, Medicinal Plants

 

LIST OF ABBREVIATIONS

 

            μg                  Microgram

            BHT                 Butylated Hydroxytoluene

            DMSO                Dimethyl Sulfoxide

           DPPH                 1, 1-Diphenyl-2-picryl-hydrazyl

            FCR                 Folin-Ciocalteu reagent

            GAE                 Gallic Acid Equivalent

            MeOH                Methanol

            MHA                 Muller Hilton Agar

            mL                  Mililitre

            mM                  Milimeter

            nm                   Nano Meter

            NMR                  Nuclear Magnetic Resonance

            QE                   Quercetin Equivalent

       TFC                     Total Flavonoid Content

       TLC                 Thin Layer Chromatography

       TPC                Total Phenolic Content

       UV                 Ultra Violet

       UV-Vis             Ultra Violet Visible Spectroscopy

       ZOI               Zone of Inhibition

FIRST CHAPTER: INTRODUCTION

1.1 Biodiversity in Nepal

Nepal is richly endowed with natural resources across a variety of ecosystems such as forests, grasslands, wetlands, high mountains, the himalayas, and the lowland plains which provide invaluable habitats for flora and fauna. Most of the ecosystem, besides deserts and oceans are found in Nepal, it is recorded that a total of 118 types of ecosystem, 75 types of vegetation and 35 types of forests. There are different animals and plants found in Nepal. The country’s 118 ecosystem harbours over 2% of flowering plants, 3% of pteridophytes and 6% of bryophytes in world. Similarly, the country harbours 3.9% of mammals, 8.95% of the birds and 3.9% of the worlds fauna of butterfly.¹

Nepal is known as one of the richest country in forest of the world. Since there is diverse medicinal herbs, shrubs & tree located in mountainous, hilly & terai regions. Nepalese medicinal plants are considered a vast source of several pharmacologically active principles and compounds, which are commonly used in home remedies against multiple ailments. Over the last few years, researchers have aimed at identifying and validating plant derived substances for the treatment of various diseases. Similarly it has been already proved that various parts of plants such as Leaves, fruits, seeds, barks etc. provide health and nutrition promoting compounds in human diet.^1,2

Human is using numerous plants and plant derived products to cure and for relief from various physical and mental illness since ancient time. Chemicals isolated from the plants are the potential source for bioactive compounds. Chemicals obtained from the medicinal plants are phytochemicals and also the main constituent compounds of the drugs. Medicinal plant chemicals and its parts are important in making drugs; they have been using as drugs ingredients from the traditional ayurveda, herbal system of medicine to the modern synthetic system of medicine.³

A natural product is a chemical organic substance which is produced by the living organisms found in the nature that are produced by the pathways of primary and secondary metabolism. It can be synthesized by the chemical synthesis by semi synthesis and total synthesis and responsible for the development in the field of organic chemistry by providing challenging synthetic targets.⁴

1.2 Natural Product

Natural products are the compounds or substances produced by living organism that are found in the nature. The term natural product can also be extended for organic chemistry to refer the mean purified organic compounds isolated from the natural sources that are produced by the pathway of primary or secondary metabolism.⁵

Natural products chemistry is a distinct area of chemical research plays an important role in the chemistry in the pre-clinical and clinical studies for drug discovery research. It is associated with the isolation and purification of the chemical products and are developed by using various methods in determination of chemical structure by NMR studies and identification of the pharmacological areas of the chemical product.⁶

Natural product compounds are widely distributed in the different plants with their structural complexity. The major classes of these compounds are alkaloids, flavonoids, terpenoids, steroids, polyphenols, glycosides and many more which are phamacologically active so widely used in different purposes.

Natural products include;

1. An entire organism (eg plant, animal or microorganism) that can be used by simple process of preservation like drying,

2. Part of an organism (eg leaves, steam, fruits and flowers or an isolated animal organ),

3.  An extract of an organism or part of the organism,

4.Pure compounds (like alkaloids, flavonoids, glycosides, terpins, phenolics etc).⁷

 

Uses of natural products

Natural products have played a very important role in health care and prevention of diseases. The general uses of natural products are:

1.                  Used in traditional as well as modern system of medicine.

2.                  They can be treated as foods, shelters, clothes etc.

3.                  They can be used in controlling of pests.

4.                  These can be used in dying and cosmetics.

5.                  They can be used for life saving as well as life taking agents.⁸

Some examples of natural products include; 

1.3 Phytochemical as a source of drugs

Phytochemistry or Plant chemistry is the branch of chemistry, deals with chemical nature of the plant or plant products, also called a chemistry of natural products. The word “Phyto” is a Greek word which means plant. Phytotherapy acts as a source of treating and improving certain deformities by using the beneficial effects of medicinal plants. Phytochemicals are the bioactive, natural chemical compounds, present in plants.⁹ The plant contains a wide variety of chemical compounds and they are broadly classified into two types, primary and secondary metabolites. Primary metabolites synthesized by basic metabolic pathway are required for survival. They are mainly involved in the primary metabolic processes such as building and maintaining plant cell. They are directly involved in normal growth, development, and reproduction in plant.¹⁰ Primary metabolites includes carbohydrates, lipids, proteins, amino acids, chlorophyll etc. Secondary metabolites are the organic compounds which are not absolutely required for survival. Unlike primary metabolites, absence of secondary metabolites does not result in death, but rather in long term impairment of the organism survivability.  The secondary metabolites are naturally synthesized in all parts of the plant body especially bark, leaves, stems, roots, flowers, fruits, seeds, etc. Secondary metabolites often play an important role in plant defense against herbivores and other interspecies defenses. Secondary metabolites include flavonoids, tannins, alkaloids, essential oils, saponins, terpenoids and phenolic compounds etc. Secondary metabolites have some valuable biological properties like antimicrobial activity, antioxidant activity, antifungal, antibacterial, anticonstipative, spasmolytic, anticancer, modulation of detoxification enzymes, decreased platelet aggregation, immune system stimulation, and hormone metabolism modulation etc.¹¹

In pharmaceutical industries plants are recognized for their broad structural diversity as well as their broad range of pharmacological activities. Phytochemicals are the biologically active compounds present in plants. These phytochemicals are derived from various parts of plants such as leaves, flowers, seeds, barks, roots and pulps. Those phytochemicals can directly be used as the source of medicinal agents. They serve as raw materials base for an elaboration of more complex semi-synthetic chemical compounds.¹²

The common techniques used in the field of phytochemistry are extraction, isolation, purification, and structural elucidation of natural products including their characterization. Phytochemicals are chemical compounds that occur naturally in plants and the terms used to refer those chemicals that may affect the health. These chemicals are related to various classes of natural products such as alkaloids, flavonoids, saponins, tannins, anthraquinones, coumarins, terpenoids etc. The complete phytochemical screening of plant can be performed by taking the extract of the plant parts in different solvents with different polarity and various tests should be performed with these extracts. Extraction is the crucial first step in the analysis of medicinal plants because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization. The fact is that the plant extracts mainly contain combination of various types of bioactive compounds or phytochemicals with different polarities, their separation still remains a big challenge for the process of identification and characterization of bioactive compounds.¹³

1.4 Antimicrobacterial Activity

Biosynthetically or synthetically produced chemical compounds which either destroy or usefully suppress the growth and metabolism of variety of microscopic or submicroscopic forms of life are called antibiotics. These are able to destroy or suppress the bacteria and so called bactericides or germicides. Of thousands of antimicrobial agents only a small numbers are safe chemotherapeutic agents, effective in controlling infectious disease in plants, animals and humans.¹⁴

The fresh and dry powder of plant material can be extracted in water or organic solvents. The antibacterial testing may be carried out either on these crude extract or the pure compounds. The mainly used antibacterial assays are described below;

1.4.1 Disc Diffusion Method

The disc diffusion test, or agar diffusion test, or Kirby–Bauer test (disc-diffusion antibiotic susceptibility test, disc-diffusion antibiotic sensitivity test, KB test), is a test of the antibiotic sensitivity of bacteria. It uses antibiotic discs to test the extent to which bacteria are affected by those antibiotics. In this test, wafers containing antibiotics are placed on an agar plate where bacteria have been placed, and the plate is left to incubate. If an antibiotic stops the bacteria from growing or kills the bacteria, there will be an area around the wafer where the bacteria have not grown enough to be visible. This is called a zone of inhibition.¹⁵

In this method, the medium is inoculated with the test organism and the samples to be tested are mixed with the inoculated medium. The material is inoculated and growth of microorganism is viewed and compared with the controlled culture which doesn’t contain the test samples. The experiment is repeated at various dilutions of the test samples in the culture medium and highest dilution at which the sample just prevents the growth of microorganism is determined.¹⁶

1.4.2 Well Diffusion Method

In this method, wells are cut in seeded agar and the test sample is then introduced directly into these wells. After incubation the diameter of the clear zone around the well is measured and compared against zone of inhibition produced by solution of known concentration of standard antibiotics.¹⁸

1.5 Antioxidants

Antioxidants are considered important nutraceuticals that can inhibit oxidation, on account of many health benefits. Oxidation is a chemical reaction that can produce free radicals, thereby leading to chain reactions that may damage the cells of organisms. Compounds that protect cells against the damaging effects of reactive oxygen species and prevent the production of such highly reactive free radicals, aids in repairing from oxidative damage and have effective functioning of naturally preventing in our body are antioxidants. 1, 1-Diphenyl-2-picryl-hydrazyl (DPPH) is a stable free radical which has an unpaired valence electron at one atom of Nitrogen Bridge. Scavenging of DPPH radical is the basis of the popular DPPH antioxidant assay. Various research groups have used widely different protocols which differed in the concentration of DPPH, incubation time, reaction solvent and pH of the reaction mixture. High concentrations of DPPH in the reaction mixture give absorbance beyond the accuracy of spectrophotometric measurements. As a result of these differences in reaction conditions, the IC₅₀ values for even the standard antioxidants like ascorbic acid and butylated hydroxytoluene (BHT) vary a lot. Thus, it is not possible to compare the results of different laboratories. Light, oxygen and pH of the reaction mixture also affect the absorbance of DPPH. The present investigation on the DPPH antioxidant assay was carried out for developing a standard protocol within the sensitivity range of spectrophotometric assays.¹⁸

The DPPH free radical scavenging activity assay has been largely used by various researchers as a quick assay, reliable and reproducible parameter in search of in vitro antioxidant activity of pure compounds as well as plant extracts. This method is based on the ability of DPPH free radical to reduce and decolorize in the presence of antioxidants. Antioxidants either transfer a hydrogen atom or electron to DPPH free radical to neutralize its free radical character and become a stable diamagnetic molecule. Scavenging of free radicals determines the free radical scavenging capacity or the antioxidant potential of the test sample which shows its effectiveness, prevention, interpretation and repair mechanism against injury in biological system.¹⁹

1.5.1 Mechanism of DPPH with Antioxidants

The proposed mechanism involves the transfer of hydrogen atom from an antioxidant to the DPPH free radical to form stable DPPH-H molecule with the loss of violet color and loss of its paramagnetic resonance and doesn’t absorb 517 nm. DPPH solution shows a strong absorbance band at 517 nm due to its odd electron appearing a deep violet color; the absorption vanishes electron pairs off. The resulting decolorization is stoichiometric with respect to the number of hydrogen taken up.

                

The antioxidant activity of plant extracts or its compounds is evaluated by comparing with standard antioxidant. Some antioxidants are ascorbic acid, cysteine, etc.   

1.5.2 Mechanism of DPPH with Ascorbic acid                  

Figure 1. 1 : Mechanism of DPPH and ascorbic acid

Two molecules of DPPH are reduced by one molecule of Ascorbic acid. Similarly, gallic acid and quercetin can donate a pair of hydrogen atoms to DPPH molecule and thus get oxidized acting as an antioxidant.

DPPH radical scavenging properties of antioxidants are generally evaluated by determining absorbance at 517 nm wavelength. The radical scavenging mechanisms of ascorbic acid on DPPH radicals were investigated by UV-Vis spectrometric, electrochemical and mass spectrometric determinations. It was found that DPPH existed in two different forms, radical form (DPPH·) and highly conjugated form (DPPH+), and ascorbic acid could scavenge both of them in the solution. Generally, the two forms of DPPH were at equilibrium in the solution. This makes it possible to indirectly evaluate DPPH radical scavenging properties of the same kinds of antioxidants based on A517 nm changes of DPPH+, but for different kinds of antioxidants, it is impossible to correctly evaluate radical scavenging properties because of their different relative scavenging effects on the two forms of DPPH.

1.5.4 Phenolic Compounds

Plants produce an extraordinary diversity of phenolic metabolites that contain one or more acidic hydroxyl residues attached to an aromatic (phenyl) ring. Polyphenols are naturally occurring secondary metabolites found largely in the fruits, vegetables, cereals, and beverage etc, generally involved in defense against UV radiations or aggression by pathogens. Hydroxycinnamic acids, flavonoids, anthocyanins and tannins represent the major classes of phenolics, which collectively account for approximately 40% of the organic carbon in the biosphere. Although structural phenolic compounds such as lignin, suberin and other structural polymers comprise much of this carbon pool, the amazing array of non-structural phenolic shave many functions in plants, including acting as antioxidants. Phenolic compounds are excellent oxygen radical scavengers because the electron reduction potential of the phenolic radical is lower than the electron reduction potential of oxygen radicals and also because phenoxyl radicals are generally less reactive than oxygen radicals. Therefore, phenolic compounds can scavenge reactive oxygen intermediates without promoting further oxidative reactions. It follows that many environmental stresses that cause oxidative stress often induce the synthesis of phenolic metabolites. Many available methods of quantification of total phenolic content in food products or biological samples are based on the reaction of phenolic compounds with a colorimetric reagent, which allows measurement in the visible portion of the spectrum.²⁰

The Folin–Ciocalteu (F–C) assay is such a method and has been proposed as a standardized method for use in the routine quality control and measurement of antioxidant capacity of food products and dietary supplements. The F–C assay relies on the transfer of electrons in alkaline medium from phenolic compounds to phosphomolybdic/phosphotungstic acid complexes to form blue complexes that are determined spectroscopically at approximately 760 nm. Although the exact chemical nature of the F–C reaction is unknown, it is believed that sequences of reversible one or two electron reduction reactions lead to blue species. Major considerations in the interpretation of the F–C assay are that the chemistry is non specific and that other oxidation substrates in a given extract sample can interfere in an inhibitory, additive or enhancing manner. Inhibition could occur as a result of oxidants competing with the F–C reagent or air oxidation after the sample is made alkaline, the F–C reagent is added before the alkali. Additive effects occur from unanticipated phenols, aromatic amines, high sugar levels or ascorbic acid in the extract.²¹

Ascorbic acid readily reacts with the F–C reagent and therefore must be considered. It can be measured before adding the alkali or by a more specific assay and then subtracted from the F–C value. Sulfites and sulfur dioxide also react with the F–C reagent, and this has been a problem in wines, where SO₂ is a common additive. Owing to the general nature of the F–C chemistry, it is indeed a measure of total phenols and other oxidation substrates. However, the F–C assay is simple and reproducible and has been widely used for studying phenolic antioxidants. This protocol describes a rapid, small-scale, high-throughput assay for approximating the total phenolics and other antioxidant substrates on the basis of the improved F–C assay of Singleton and Rossi, using gallic acid as a standard. This protocol is provided along with an oxygen radical absorbance capacity protocol and a rapid protocol for measuring ascorbate content in plant tissues. Overall, these assays provide a general diagnostic tool of the antioxidant capacity of the extracts.²²

1.5.6 Flavonoid Compounds

Flavonoids comprise the most studied groups of polyphenols. The common basic structure consisting of two aromatic rings bound together by three carbon atoms that form an oxygenated heterocycle are actually flavonoids. There have been more than 4,000 varieties of flavonoids are reported, many of which are responsible for attractive colors of the flowers, fruits and leaves. Based on the variation in the type of heterocycle involved, flavonoids may be divided into six subclasses: flavonols, flavones, flavonones, flavanols, anthocyanins, and isoflavones. The individual groups within each group arise from variation in number and arrangement of the hydroxyl groups and their extent of alkylation or glycosylation. Quercetin, myricetin, catechins etc are the most common flavonoids.²³

The analysis of flavonoids in propolis has been done by colorimetric methods, thin layer chromatography, gas chromatography, gas chromatography-mass spectrometry and high performance liquid chromatography. Although chromatographic techniques in combination with absorption spectrum analysis and mass spectrometry provide definitive information for identification and quantification of flavonoids, these methods usually require advanced instruments, various authentic standards and are time-consuming. On the other hand, colorimetric methods targeting flavonoids of similar structures are convenient and appropriate for routine analyses.²³ However, none of the colorimetric methods can detect all kinds of flavonoids. Groups of flavonoids were found to complex stability with aluminum chloride and 2,4-dinitrophenylhydrazine. To determine the total flavonoid content of the extracts, aluminum chloride colorimetric method is most common and reachable one. In this work, different extracts of samples were investigated by AlCl₃ colorimetric analysis.²⁴

In the present work, the medicinal plants Aegle marmelos, Cuscutta reflexa and Pityrogramma calomelanos  have been selected on the basis of its traditional uses in the local area and its users, healers and the old persons having indigenous knowledge to screen its phytochemicals and biological properties from the Nepalese origin that has not been studied before this work.

1.6 Introduction of Some Medicinal Plants Found in Nepal:

Medicinal plants are rich source of antibacterial agents. Ancient people used plants as a medicine to treat various infections, among various infections, bacterial and fungal infections falls under the most common infection especially in developing countries like Nepal. Increment of scope in antimicrobials from natural sources is because of increasing rate of multidrug resistant strains of microorganisms as well as newly developed strains with reduced susceptibility to available antimicrobial agent due to haphazard use of broad spectrum antibiotics, immunosuppressive agent, intravenous catheters, organ transplantation and ongoing epidemics of HIV infection.²⁵ Natural products of plants may provide a new source of antimicrobial agents with possibly novel mechanism of action. The search of biologically active compounds from plant extracts has always been of great interest to scientists for the discovery of new drugs effective in the treatment of several diseases. The systematic screening of antimicrobial plant extracts represents a continuous effort to find new compounds with the potential to act against multi-resistant pathogenic bacteria and fungi. Therefore, it is imperative to investigate plants to better understand their properties, safety and efficacy.²⁶

1.6.1 Description of Aegle marmelos (Beal)

Figure 1. 2 : Fruit and Leaf of Aegle marmelos Plant

Aegle marmelos belongs to the Rutacae family and Aurantioideae sub-family, is a medium- sized deciduous tree. Its local name is Bael in Nepali. It is also known as ‘Bengal quince’, Indian quince’, ‘Golden apple’, ‘Holy fruit’, ‘Stone apple’, ‘Bel; Bela; ‘Sriphal’, ‘Belgeri’, ‘Baelpatra,’ Bilva’ ‘Maredoo’ and finds mention in Ramayana, Yajurveda, Buddhist and Jain literature. It has been great mythological significance and found near every temple in Nepal. According to Hindu custom, the trifoliate aromatic leaves of the tree traditionally used as sacred offering to 'Lord Shiva'. It grows throughout Nepal peninsular as well as in India, Srilanka, Pakistan, Bangladesh, Burma, Thailand and most of the South - East Assian countries. These species are  normally characterized by a thick trunk with spiny branches and a soft, flaky bark, pale-green leaves with 3-5 leaflets, grows to a height of 7 to 8 m.²⁷ The plant has greenish-white flowers that bloom in late spring and produce an oval-shaped, sweet-tasting, soft fruit covered in a woody shell that turns yelow when the fruit ripens.²⁸

The Bael (Aegle Marmelos) (L.) Corr.) has enormous traditional uses against various diseases. The present dissertation work aims to compile medicinal values of Aegle Marmelos generated through the research activity using modern scientific approaches and innovative scientific tools. which has enormous traditional values against various diseases and many bioactive compounds have been isolated from this plant.

The bael (Aegle marmelos Correa) is an important indigenous fruit of Nepal. The tree is deciduous, 6-8 meter in height, flowering start in the month of May -June and fruit are available from March to June. The importance of bael fruit also lies in curative properties which makes the tree one of the useful medicinal plants of Nepal. The fruit is hard shelled berry and well known for its medicinal properties due to marmelosin content. It has been known in Nepal from prehistoric times. It is very popular because of its rich nutritive, sweet, aromatic mucilage and pectin contents and good for all kinds of stomach disorders. Bael fruits are very useful in chronic diarrhea and dysentery, particularly in the case of patients having diarrhea, alternating with the spells of constipation.

1.6.2 Distribution of Aegle marmelos 

A. marmelos trees are generally found in the outer Himalayas, Siwaliks and Tarai with altitudes upto 1500 m. In Nepal, A. marmelos is distributed abundantly in the Siwalik region, Inner Tarai, and lower valley region mostly at riverside having sandy soil at 150–1,220 m altitude.²⁹

1.6.3 Ethnomedicinal uses of Aegle marmelos

A. marmelos has both medicinal and religious values. The plant is often seen growing in temple gardens of Nepal where the leaves are used in prayers to Lord Shiva and an important fertility ritual, Bel biha.³⁰ This is generally considered as sacred tree by the Hindus. Leaves, fruits, stems and roots of this tree at all stages of maturity are used as ethno medicine against various human ailments. The ripe fruit is taken scientifically as brain tonic and energetic, and it improves eternal power and longevity also used for preparation of high quality soft drink and beverages. It has very cooling effect and protects the body system for maintaining of body temperature during hot summer. The mature fruit is used for preparation of murrabba, candy, toffee and bael powder. Moreover, it is considered as a very good medicine for the patients suffering from constipation. Unripe A. marmelos fruit is prescribed for curing cholera, diarrhea, worms and other stomach diseases because of its antibacterial, antiviral and anthelmintic properties. The leaves are excellent medicine for the diabetic patients.³¹

1.6.4 Phytochemicals of Beal

Aegle marmelos has been known to be one of the most valuable medicinal plants of Nepal. More than 100 phytochemical compounds have been isolated from various parts of Aegle marmelos, namely phenols, flavonoids, alkaloids, cardiac glycosides, saponins, terpenoids, steroids and tannins.³² Bael fruit contains volatile compounds like hexanal, isoamyl acetate, limonene, β-phellandrene, p-cymene, acetoin, (E)-2-octenal, (E,E)-2,4-heptadienal, dehydro-p-cymene, linalool; 3,5-octadiene-2-one, α-cubebene, trans-p-mentha-2,8-dienol, citronellal, cineole, p-cymene, citronella, citral, cuminaldehyde, β-cubebene, β-caryophyllene, hexadecane, pulegone, α-humulene, verbenone, carvone, carvyl acetate, dihydro-β-ionone, (E)-6,10-dimethyl-5,9-undecadien-2-one, β-ionone, caryophyllene oxide, humulene oxide and hexadecanoic acid. They also contain coumarins like aegeline, aegelenine, marmelin, o-mtheyl halfordinol, alloimperatorin, furocoumarins, psoralen, o-isopentenyl halfordinol and marmelosin. They also contain tartaric acid, linoleic acid, tannins, phlobatannins, flavon-3-ols, leucoanthocyanins, anthocyanins, and flavonoid glycosides. (Rastogi & Mehrotra, 1990; Parmar & Kaushal, 1982; Roy & Khurdiya, 1995; Suvimol & Pranee, 2008; Maity, 2009).

The fruit is rich in Vitamin B (Riboflavin) and also has fair amount of Vitamin ‘A’ , Vitamin ‘C’ and minerals such as calcium, phosphorus, potassium and protein content in ripe fruit is very high.

Figure 1. 3 : Some phytochemical constituents present in Aegle marmelos

These compounds are well known to possess biological and pharmacological activity against various chronic diseases. Antioxidant, antiulcer, antidiabetic, anticancer, antihyperlipidemic, anti-inflammatory, antimicrobial and antispermatogenic effects have also been reported on various animal models by the crude extracts of this plant.³³

1.6.5 Taxonomy of Beal

Domain: Eukaryota 

  Kingdom: Plantae

    Phylum: Spermatophyta

      Subphylum: Angiospermae

        Class: Dicotyledonae

          Subclass: Rosidae

            Order: Sapindales

              Family: Rutaceae

                Genus: Aegle

                  Species: marmelos

 

1.6.6 Description of Cuscuta reflexa

Figure 1. 4 : Whole plant of Cuscutta reflexa

Around 200 members of genus Cuscuta are found world widely, many of them are regarded as medicinal plants. The parts of some Cuscuta species such as C. chinensis and C. australis are used as supplements in the pharmaceutical market.³⁴ Cuscuta reflexa is commonly called as dodder plant, and also known as devil’s hair, witch’s hair, love vine, amarbel or akashabela etc.³⁵ Cuscuta sp. is a beneficial plant which appears as leafless and yellowish green in appearance; also characterized as a twining herb, which belongs to the family Convolvulaceae.³⁶ It has no root under the ground, is a homoparasitic weed plant and also an extensive climber. It has no chlorophyll and cannot make its own food by photosynthesis. Dodder plant has the ability not only to recognize its host plant but also to move towards its prey with significant precision and efficiency. Dodder plants choose an appropriate host between many plants on the basis of volatile compounds release by the host plant as their normal process of transpiration.³⁷ Cuscuta sp. absorbed plants water and other essential inorganic nutrients through conducting cells. The xylem of host and parasite have connection between them, thus organic substances transported easily from phloem tissue of host plants to parasite.³⁸

1.6.7 Distribution of Cuscuta reflexa

Cuscuta is found in the temperate and tropical regions of the world with huge species diversity in tropical and subtropical regions. This plant is distributed throughout Nepal, and is used for medicinal purpose in India, China, Indochina, Madagascar and South Africa. It is usually found in India and Ceylon up to an altitude of 2348 m. It is also found in Afghanistan, Malaysia, Nepal and Thailand. It grows on thorny, non thorny and other shrubs, sometimes completely covering bushes and trees.

Cuscuta reflexa is a parasitic plant which belongs to family Convolvulaceae. It is commonly known as dodder plant, amarbel, akashabela. Traditionally it is called miracle plant. It is rootless, perennial, leafless climbing parasitic twining herb which takes food from host plant with help of special organ called haustorium.³⁰ The stem of cuscuta sp. is a long branched, glabrous succulent, densely interlaced, pale greenish yellow, sometimes dotted with red. They possesses waxy white flowers, which are regular, bisexual, small, scented, solitary or in umbellate clusters of 2-4 or in short  racemes; pedicels short, glabrous, usually curved (rarely 0); bracts 1.5 mm. long, ovate-oblong, obtuse fleshy.³⁹

1.6.8 Ethnomedicinal uses of Cuscuta sp.

Cuscuta reflexa Roxb. is a valuable medicinal herb. Stem of this plant is antibacterial and used externally to treat itching and internally in fever.⁴⁰ Stems of Cuscuta reflexa is also used in constipation, flatulence, liver complaints and bilious affections. The juice of plant mixed with the juice of Saccharum officinarum is used in the treatment of jaundice. Cuscuta reflexa is also applied as a hair growth promoter.⁴¹ Seeds are said to be tonic, diaphoretic and demulcent and are used to purify the blood. The cold infusion of seeds is given as a depurative and carminatives in pain and stomach ache. It also gives anti-inflammatory and anti-cancer activity. The aqueous and alcoholic extract of C. reflexa has diuretic property.⁴² The crude water extract of C. reflexa also shows anti HIV activity.⁴³

Cuscuta reflexa is investigated for Antihypertensive, Antidiabetic, Antioxidant, Hair growth promoting, Antimicrobial, Spasmolytic, Antitumor, Antiviral, Anti-inflammatory, Antipyretic effect etc.⁴⁴

1.6.9 List of chemical constituents isolated from species of Cuscuta

Kaempferol, Kaempferol–3-O-glucoside (Astragalin), Myricetin, Myricetin glucoside, Quercetin, Kaempferol -3- O-galactoside, Quercetin -3-O- galactoside, Quercetin -3-Oglucoside Isorhamnetol, Azaleatin Cuscutalin, Cuscutin, Linolenic acid, Linoleic acid, Oleic acid , Stearic acid, Palmitic acid, Beta sitosterol , Bergenine, Amarbelin, Dulcitol, Myricetin, Myricetin glucoside , Luteolin,Coumarin, Maragenin, n-Heptacosane, Alpha – Amyrin, Cusctutamine , nOctacosane, n-Nonacosane ,n-Triacontane ,n-Hentriacontane ,1- Triacontane, n-Pentacosane Cuscutoside-A, Cuscutoside-B, Arbutin Chlorogenic acid, Caffiec acid, p-Coumaric acid , Stigmasterol , Avenasterol, Matrine , Saphoronal, Cuscutic acid C, Campesterol, Methylcytisine ,Cus-1,Cus – 2 ,3,5 Dicaffeoyl quinic acid, 4,5 Dicaffeoyl quinic acid, Laceeroic acid Australiside A, Cuscutic acid A, Cuscutic acid B, Cuscutic acid D, Hydroxyoleanane, 6,7,8- Trimethoxy -2H-1- benzopyran-2-one Lupeol, Beta – Amyrin, Alpha Amyrin Acetate, Beta Amyrin Acetate , Oleanolic acetate , Oleanolic acid , 7-Propenamide, Sesamin ,Trihydroxy auran, Propenamide, 6,7-Dimethoxy-2H1-benzopyran-2-one, Daucosterol.⁴⁵

 

Figure 1. 5 : Major phytochemical constituents present in Cuscuta reflexa

1.6.10 Taxonomy of Cuscuta reflexa

Kingdom : Plantae

  Subkingdom: Tracheobionta

    Superdivision: Spermatophyta

      Division: Angiospermes

        Class: Eudicots

          Subclass: Asterids

            Order: Solanales

              Family: Cuscutaceae

                Alternate: Convolvulaceae

                  Genus: Cuscuta

                    Species: reflexa

 

 

1.6.11 Discription of Pityrogramma calomelanos

Figure 1. 6 : Leaves of Pityrogramma calomelanos

Genus Pityrogramma is derived from the Greek word "pitura-gramma" and refers to the lower surface of the fronds becoming obscured by rod-like secretions. Species calomelanos is derived from the Greek word "kalo-melanw" and it means "beautifully dark".

Although we have found no reports of toxicity for this species, a number of ferns contain carcinogens so some caution is advisable. Many ferns also contain thiaminase, an enzyme that robs the body of its vitamin B complex. In small quantities this enzyme will do no harm to people eating an adequate diet that is rich in vitamin B, though large quantities can cause severe health problems. The enzyme is destroyed by heat or through drying, so cooking the plant will remove the thiaminase.

1.6.12 Distribution of Pityrogramma calomelanos

Pityrogramma calomelanos is a fern growing about 40 cm tall. The leaves have been used locally for medicinal purposes.

1.6.13 Ethnomedicinal uses of Pityrogramma calomelanos

The leaves are used externally to heal wounds and stop bleeding. An infusion of the whole plant is used to 'strengthen men's backs', i.e., to increase male sexual stamina, and to treat female haemorrhaging. An aqueous extract is drunk or applied locally to treat venereal disease in Guyana. It is also used for asthma, cough, cold, pneumonia, tuberculosis, and whooping cough. The root is bechic. An infusion is used to treat pulmonary conditions. An infusion of the leaves is used in the treatment of bronchitis, colds and stomach pains. The leaves are crushed and used as a poultice to promote the healing of ulcers, wounds and cuts. They (or the leaf juice) are also placed on wounds to stop bleeding. The plant contains the sesquiterpene lactones pterosin (which is reportedly antibiotic) and calomelanolactone.

1.6.14 Lists of chemical constituents Isolated from species of Pityrogramma calomelanos

Chalcones are abundantly present in nature starting from ferns to higher plants.⁴⁶ Chemically they are 1,3-diphenyl-2-propen-1-ones and are often cytotoxic in vitro⁴⁷ and some of their derivatives are reported to be antimutagenic.⁴⁸ A hydroxychalcone, isolated from Pityrogramma calomelanos was found to be a cytotoxic and tumor reducing agent.⁴⁹ Among flavonoids, chalcones have been identified as interesting compounds that are associated with several biological activities.⁵⁰ With the exception of kaempferol 7-methyl ether, 2’,6’-dihydroxy-4’-methoxydihydrochalcone is identified in white-backed fronds of P. calomelanos.⁵¹

Figure 1. 7 : Phytochemical constituents present in P. calomelanos

1.6.15 Taxonomy of Pityrogramma Calomelanos :

Kingdom : Plantae 

  Subkingdom : Tracheobionta 

    Division : Pteridophyta 

      Class : Filicopsida

        Order : Polypodiales

          Family : Pteridaceae 

            Genus : Pityrogramma

              Species : calomelanos (L.) Link – Dixie silverback fern

SECOND CHAPTER: LITERATURE REVIEW

The scope and impact of natural products will be greatly increased by conducting research on different areas and continue to expand by providing opportunities for new discoveries for dissemination of high quality work in this field. Medicinal plants used as herbs as a traditional medicine for various types of diseases. These are used for the preparation of modern drugs or used as a source for raw material. It lies in the bioactive phytochemical constituents like alkaloids, saponins, flavanoids, tannins, terpenoids, phenolic compounds, steroids and glycosides. These form the backbone of the drugs.

Literature review of the work done on the selected plant materials were composed from information published on some books, journal articles (Scholarly articles), websites (Sci-hub.tw, Google, Google scholar) and are discussed on first come first basis.

Medicinal plants have an immeasurable ability to produce phytochemicals; most of them are phenols, aromatic substances and their oxygen derivatives. These secondary metabolites produced from the plants are enormous in the number, of which approx 15000 have been isolated, that is less than 10% of the total secondary metabolites produced by plants (Schultes, 1978). Since prehistoric era of mankind, treatment and cure of the diseases was one of the main concerns of human beings. Ayurveda completely depends on the plant systems for the evaluation of new chemical entities having therapeutic potentials (Dev, 1999). Some secondary metabolites have some other functions also, such as terpenoids which gives plant their odour, tannins and quinines are responsible for plant pigmentation, some of them are responsible for plant flavour (e.g. terpenoids capsaicin from chilli peppers) and some of them are used as herbs and spices as seasonal food yield using medicinal compounds (Cowan, 1999). Medicinal properties of the plants are due to the active phytoconstituents present in the plants; these phytoconstituents are alkaloids, flavonoids, glycosides, saponins, tannins, terpenoids, steroids etc. These phytochemicals posses potential health benefits, contributes in the prevention of cardiovascular diseases, cancer, osteoporosis, antioxidant activity and many more. Polyphenols have advantageous effects on cardio vascular system and plays an important role in the prevention of neurodegenerative diseases and diabetes mellitus (Vita, 2005). Medicinal plants are the treasure of various hidden chemicals. In the traditional system of medicines, plant sources are major resource to cure diseases. Medicinal plants are getting attraction of most of the researches for the evaluation of new drugs, because of the polyvalent action and lesser side effects of plant products (Mahesh, 2008). Secondary metabolites produced from the plants serves as plant defence mechanism and protect plant from microorganisms, insects and herbivores (Abdul, 2012). The promising potential of plant derived substances, plant oils and plant extracts become the basis of many applications as food preservation, pharmaceuticals, novel medicines and natural therapies. Plants are wealthy in secondary metabolites, and are major source of new chemical molecules; therefore they are a potential source of new drugs to cure various life threatening diseases (Savoia, 2012).

The history of Bael has been traced to Vedic period (2000-800 BC).⁵² Bael tree has great mythological significance and abounds in the vicinity of temples. Bael tree is held sacred by Hindus. Leaves of A. marmelos are used to worship Lord Shiva. It is considered as an emblem of fertility.⁵³ Vedic literate depicts that leaf extract is used to cure asthmatic complaints. Fresh leaf juice along with honey has been reported to serve as a laxative and febrifuge.⁵⁴ The potential use of leaf of A. marmelos in the aliment of backache, eye complaints, vomiting, cut and wound, animal injuries, ulcer, dropsy, beriberi, weakness of heart, cholera, diarrhoea, diabetics, nervous disorders, acute bronchitis and complications associated with child birth are reviewed and mentioned.⁵⁵ Leaves of A. marmelos contain alkaloids, phenylpropanoids, terpenoids and other miscellaneous compounds with potential hypoglycemic, anti-inflammatory, antimicrobial, anticancer, radioprotective, chemopreventive and anti-oxidative activity was already reported.⁵⁶ Studies have shown that the bael fruit pulp contains important bioactive compounds such as carotenoids, phenolics, alkaloids, pectins, tannins, coumarins, flavonoids and terpenoids (Parmar & Kaushal, 1982; Roy & Khurdiya, 1995; Suvimol & Pranee, 2008; Maity, 2009). Bael fruit contains volatile compounds like hexanal, isoamyl acetate, limonene, β-phellandrene, p-cymene, acetoin, (E)-2-octenal, (E,E)-2,4-heptadienal, dehydro-p-cymene, linalool; 3,5-octadiene-2-one, α-cubebene, trans-p-mentha-2,8-dienol, citronellal, cineole, p-cymene, citronella, citral, cuminaldehyde, β-cubebene, β-caryophyllene, hexadecane, pulegone, α humulene, verbenone, carvone, carvyl acetate, dihydro-β-ionone, (E)-6,10-dimethyl-5,9-undecadien-2-one, β-ionone, caryophyllene oxide, humulene oxide and hexadecanoic acid (Suvimol & Pranee, 2008). They also contain coumarins like aegeline, aegelenine, marmelin, o-metheyl halfordinol, alloimperatorin, furocoumarins, psoralen, o-isopentenyl halfordinol and marmelosin. They also contain tartaric acid, linoleic acid, tannins, phlobatannins, flavon-3-ols, leucoanthocyanins, anthocyanins, and flavonoid glycosides (Rastogi & Mehrotra, 1990; Parmar & Kaushal, 1982; Roy & Khurdiya, 1995; Maity, 2009). Further, it has been emphasized that anhydroaegeline obtained from A. marmelos could be used as marker to standardize the plant material with potential anti diabetic activity.

Figure2. 1 : Some structure of compounds present in Bael

Invariably, all part of the tree viz. stems, barks, roots, leaves, flowers and fruits have been described for their use in Ayurveda and Siddha for the treatment of respiratory disorders, constipation, ulcer, diarrhea, dysentery.⁵⁷ However, only few reports pertinent to the antimicrobial potential of A. marmelos is available. Pattnaik et al using disc diffusion method reported that essential oils of A. marmelos exhibit significant antibacterial and antifungal activities.⁵⁸ Similarly, Rana et al., using spore germination assay demonstrated that essential oil in the leaves of A. marmelos exhibit significant antifungal activity.⁵⁹ Scientific studies have validated many of the ethnomedicinal uses of bael leaf. Studies have shown that the leaf possess antimicrobial effects (Rana, Singh, & Taneja, 1997), anti-inflammatory (Arul, Miyazaki, & Dhananjayan, 2005), antipyretic (Arul, 2005), analgesic (Arul, 2005), to decrease the thyroid hormone concentration (Kar, Panda, & Bharti, 2002), anti-fertility effects (Chauhan, Agarwal, Kushwaha, & Mutreja, 2007; Chauhan & Agarwal, 2009), antidiabetic effects (Ponnachan, Paulose, & Panikkar, 1993; Upadhya, Shanbhag, Sunethea, Balachandra Naidu, & Upadhya, 2004), anti-lipidemic (Vijaya, Ramanathan, & Suresh, 2009), cardioprotective effects (Rajadurai & Prince, 2005), antineoplastic effects (Khan, 2002; Lampronti, 2003; Lambertini, 2004; Jagetia, Venkatesh, & Baliga, 2005), radioprotective effects (Baliga, Bhat, Pereira, Mathias, & Venkatesh, 2010) and chemoprotective effects against doxorubicin-induced clastogenesis (Venkatesh, Shantala, Jagetia, Rao, & Baliga, 2007). With regard to bael, studies by Kamalakkannan and Prince (2003); Kamalakkannan and Prince (2003) have shown that the aqueous extract of the bael fruit pulp possess potent antioxidant effects. Subsequently, the hydroalcoholic extract of bael pulp is also shown to possess nitric oxide scavenging activities in vitro (Jagetia & Baliga, 2004). Additionally, the bael fruit drink was also reported to possess high quantities of total phenolic compounds (83.89/37.6 mg gallic acid equivalents/100 ml) and was also a good antioxidant in both DPPH (photochemiluminescence) and PCL (photochemiluminescence) assays (Abdullakasim, 2007). Singanan, (2007) worked on Aegle Marmelos leaf extract on alcohol induced liver injury in albino rats and presented data of excellent hepatoprotective effects.⁶⁰  Maheshwari, (2009) studied on ethnolic ectract of dried fruit pulp of Aegle Marmelos against various intestinal pathogens i.e. Shigella boydii, S. sonnei & S. Flexneri and proposed that certain phytochemicals including Phenols, Tannins and Flavonoids were effective against all.⁶¹ It was also confirmed by Kaur, (2009) by getting treat E. Coli with Aegle Marmelos fruit extract.⁶² In consonance, Citarasu, (2003) also experimented Aegle Marmelos on certain pathogenic bacteria like Salmonella typhi, Pseudomonas aeruginosa, Aeromonas hydrophyla & Vibrio sp., and concluded its positive bactericidal effects.⁶³ Ghangale G. R (2008) also evaluated aqueous extract of Aegle mannelos for anti inflammatory activity by using rat paw oedema model and proposed that Aegle mannelos posseses anti-inflammatory activity.⁶⁴ Patil R. H ( 2009) reported the antifungal activity of ethanolic extract of the Aegle marmelos leaves including antidiarrhoeal, and antimicrobial, activities.⁶⁵ Rana B. K. ( 1997) evaluated anti fungal activity of essential oils isolated from the leaves of Bael using spore germination assay. Aqueous extract of Aegle marmelos leaves, was evaluated for hypoglycemic and antioxidant effect by Upadhya ( 2004), by using alloxon induced diabetes in male albino rats and proposed AML may be useful in the long-term management of diabetes.⁶⁶ Similarly, The anti hyperlipidaemic activity of aqueous extract of Aegle marmelos fruits was demonstrated by P.S. Marinzene (2005), using the streptozotocin-induced diabetic wistar rats.⁶⁷ Sunderam, (2009) worked on alcoholic extract of Aegle Marmelos, Momordica Charantia and Eugenia Jambolana separately; against Streptozotocine induced diabetic rats and confirmed their protective activity against laboratory induced cell necrosis. Beside of all above cited work, Hema & Lalithakumari (1999) had presented a tremendous results of Aegle Marmelos and documented its hypoglycemic action along with other pharmacological actions on molecular level.⁶⁸

Cuscuta reflexa  Roxb.  (Cuscutaceae a division of Convolvulaceae) is an extensive climber parasite. It has no chlorophyll and cannot make its own food by photosynthesis.  Some  research  studies  say  that  the  plant  has  very low  levels  of chlorophyll and can slightly photosynthesis. But other species of Cuscuta are entirely dependent on the host plants for nutrition. The stem is thread like filaments. It begins to grow and attach themselves to nearby host plants. The nature plants lives its entire life without attachment to the ground. It has long history of ethnomedicinal use. Cuscuta is a genus of about 100 – 170 species.

Cuscuta is a group of 100- 170 species of yellow, orange, red or rarely green parasitic plants. Cuscuta belongs to the Cuscutaceae family and now on the basis of Angiosperm phylogeny group it is accepted as belonging to morning glory family, Convolvulaceae (Story, 1958). Cuscuta is found at the temperate and tropical regions of the world with huge species diversity in tropical and subtropical regions. Cuscuta reflexa is commonly called as dodder plant, and also known as devil’s hair, witch’s hair, love vine, amarbel or akashabela etc. Cuscuta reflexa is a parasitic weed plant and also an extensive climber. Cuscuta grows as homoparasite, it has very low level of chlorophyll and photosynthesis activity; completely depends over the host plant for its survival. Dodder plant sucks nutrient sap from the host plant via vascular tissue of the host plant and grows itself. This plant has no roots in the ground and it grows over the host body without touching the ground surface in its complete life span (Dawson, 1994). Dodder plant has the ability not only to recognize its host plant but also to move towards its prey with significant precision and efficiency. Dodder plant can also choose an appropriate host between many plants on the basis of volatile compounds release by the host plant as their normal process of transpiration (Kapoor, 2008). Parasitism of Cuscuta reflexa is wrapping around itself over the host plant after attachment with host. Cuscuta makes haustorial connection with the vascular tissue of the host plant. This haustorium is able to penetrate the xylem and phloem of the host plant and attached with tissues of the host plant. Cuscuta reflexa varies in the colour of flowers produced from white to pink. Flowers generally produced in the early summer and autumn but also depend on the species. Seeds are produced in the large quantities. Seeds of Cuscuta reflexa can survive in the soil for many years in the search of appropriate host, at this time it depends on the food reserve in endosperm of the seed (Sarma, 2008).

In Ayurvedic medicine, the plant is said to be useful in diseases of eye and heart (Chopra, Chopra Handa and Kapoor, 1958). The chemical examination of the plant has been done by Aggarval and Dutt (1935). Some pharmacological studies on this plant were conducted by G.S Singh and K.W. Garg (1973). Their research studies are found to have anti histamines action in this plant. Some recent studies show that the following chemical constituents are identified i.e. Quercetin, Cuscutine, and Cuscutamide etc. Various studies and reports have confirmed that C. campestris Yuncker has shown anti-inflammatory effect,⁶⁹ C. reflexa Roxb as antifungal activity,⁷⁰ insecticidal effects,⁷¹ anti-bacterial and free radical scavenging activity.⁷² It has also been reported that C. reflexa Roxb. Shows anti-analgesic, anti-pyretic and anti- inflammatory activity and has been used to treat rheumatism⁷³ and paste of whole plant traditionally has been used for the treatment of headache.⁷⁴ The Tripura tribe of Bangladesh and Satar tribes of Nepal has attributed amultitude of functions to this plant – for the treatment of oedema and body ache, as an aphrodisiac, maintenance of hepatic system, for alleviation of skin infections and also for curing jaundice (Siwakoti and Siwakoti, 1999; Hossan, 2009). Based on this tribal information, this plant was analysed for its anti-inflammatory and anti-cancer activities in vitro. Since this plant is used to cure inflammation, cancer and liver disorders, we have used in vitro models for hepatic inflammation and hepatic cancer for the study. The macrophages in liver (also called Kupffer cells) have important roles in mediating the inflammatory response in liver (Knolle and Gerken, 2000). According to a study cuscuta as an ingredient of medical wine is used for treating arthritis, scapulohumeral periarthritis, rhinitis, lumbar disk herniation, lumbar and leg pain and toothache with good therapeutic effect.⁷⁵ A new flavanone- reflexin (Tripathi, 2005), tetrahydrofuran derivatives and a coumarin (Uddin, 2007) have been isolated from stems of the plant. Methanol extracts of the stem reportedly demonstrated anti-steroidogenic (Gupta, 2003), and antibacterial activities (Pal, 2006). Cuscuta reflexa contains a number of compounds like flavonoids (kaempferol, quercitin), coumarins, and flavonoid glycosides (Ghani, 2003). Earlier studies have shown that both kaempferol and quercetin could significantly improve insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes. It was further reported that these two compounds act at multiple targets to ameliorate hyperglycemia (Fang, 2008). Cuscuta species have been reported to contain several phytoconstituents including flavonoids, glycosides, alkaloid, tannins, lignins and other organic substances. Besides them also conatins essential oil and trace elements.⁷⁶ The literature demonstrated that the Cuscuta spp. are considered as the good source of nature for antioxidant property. According to the recent study it was confirmed that aerial parts of three Iranian species of Cuscuta (C. campestris, C. monogyna and C. approximata) were examined the Polyphenolic compound, total phenolic content (TPC) and antioxidant activity by using HPLC analysis, microplate reader and DPPH microplate method. The HPLC analysis revealed the presence of various compounds in Cuscuta. Phenolic compounds were found high in C. monogyna and C. approximata, respectively. The dilute methanolic extract of C. approximata and C. monogyna had the highest total phenolic (56.67 mg/g and 49.59 mg/g), respectively. This study confirms the potential of antioxidant activity of Cuscuta extract.⁷⁷ Alcoholic extracts of C. reflexa assessed as the good scavenger of superoxide radical and DPPH radical. C. reflexa contain the highest total phenol content this property could possibly related to its higher polyphenol content. It has been analysed for the free radical-scavenging activity by using (1,1-diphenyl-2-picrylhydrazyl) DPPH radical, inhibition of lipid peroxidation induced by FeSO₄ in egg yolk, presence of phenolic compound using the Folin-Ciocalteu method and identification of antioxidant compound in bio-autographic analysis using DPPH agent.⁷⁸ C. epithymum (L.) was also investigated for antioxidant property.⁷⁹ Methanolic extracts exhibited good antioxidant activities of C.reflexa and C.europrea evaluated by the measurement of total phenolic contents (TPC), total flavonoid contents (TFC), DPPH radical scavenging (IC₅₀). Zn, Fe, Cu, Co and Pb are analyzed by using Atomic Absorption Spectrophotometer. The plant materials are contained the TFC (13.91–68.13 CE mg/100g of dry extract), TPC (35.18-189.68 GAE mg/100 g of dry extract), DPPH radical scavenging activity, IC₅₀ (669.37–88.85%), Respectively.⁸⁰ Although several claims of anti-inflammatory, analgesic, antioxidant, hepatoprotective and other activities of the plant have been reported in literature there is still need of scientific validation of these claims by the using systematic and good experimental approach.

Pityrogramma, or the gold- and silver-backed ferns, consists of about 16 tropical species, which are occasionally cultivated in green-houses for the colourful yellow or white farina found on the lower leaf surfaces of most species. Pityrogramma is a genus with about 17 species occurring mainly in tropical America (Smith, 2006). The species Pityrogramma calomelanos (L.) is used as an ornamental and medicinal plant (Ambrósio and Barros, 1997; Corrêa, 1984). Phytochemical studies on ferns have revealed that they contain a wide range of alkaloids (Dong, 2012), flavonoids (Xia, 2014), polyphenols (Socolsky, 2012), terpenoids (Socolsky, 2007), and steroids (Ho, 2012). The structures of these compounds usually differ from those of related secondary metabolites produced by other higher plants, making them a potentially valuable source of chemical diversity. Several reviews on the ferns have been published since 2012: Liu, (2012) reviewed ferns eaten in China, Christenhusz and Chase (2014) highlighted recent trends and concepts in fern classification, and Plackett, (2015) discussed missing links in shoot evolution and the development of ferns. However, there have been no review articles covering studies on the phytochemistry and pharmacology since 1985, when Soeder (1985) summarized the occurrence, chemotaxonomy and physiological activity of ferns’ chemical constituents. Since then, the number of known phytochemicals from ferns has increased dramatically, as has their range of potential pharmacological applications. This review summarizes current knowledge regarding the phytochemistry and pharmacology of fern species. Chalcones are abundantly present in nature starting from ferns to higher plants.⁸¹ Chemically they are 1,3-diphenyl-2-propen-1-ones and are often cytotoxic in vitro⁸² and some of their derivatives are reported to be antimutagenic.⁸³ A hydroxychalcone, isolated from Pityrogramma calomelanos was found to be a cytotoxic and tumor reducing agent.⁸⁴ Among flavonoids, chalcones have been identified as interesting compounds that are associated with several biological activities.⁸⁵ The most common chalcones found in foods are phloretin and its glucoside phloridzin (phloretin 2´-0-β- glucopyranoside), and chalconaringenin. Studies on the bioavailability of chalcones from food sources are limited, but synthetic chalcones have been reported to have a wide range of biological properties. In an effort to develop a potent anti-inflammatory and cancer chemopreventive agents a series of chalcones were synthesized.⁸⁶  These compounds were tested for their inhibitory effects on the activation of mast cells, neutrophils, macrophages, and microgial cells. It is conceivable that mast cells, neutrophiles, and macrophages are important players in inflammatory disorders.⁸⁶ Activation of microglial cells also plays a crucial role in inflammatory diseases of the Central Nervous System. Thus, inhibition of the activation of these inflammatory cells appears to be an important therapeutic target for the design of new drugs for the inflammatory diseases treatment. Particularly interesting are the properties of chalcones in the induction of apoptosis^87,88 and their ability to change mitochondrial membrane potential.⁸⁹ These authors noted that chalcones with fewer hydroxyl groups on rings A and B were more effective in this regards, as compared to chalcones containing more hydroxyl groups. This difference was attributed to the acidity of the phenolic hydroxyl groups. One of the most widely cited mechanisms by which chalcones exert their cytotoxic activity is that of interference with the mitotic phase of the cell cycle. Edwards et al ⁹⁰ proposed a hypothetical basis for the anti-mitotic activity of chalcones. Indeed, they found a large number of methoxylated chalcones with antimitotic activity against HeLa cells. In the present work we evaluated the capacity of 2-hydroxychalcones with different methoxy subtitutions on ring B to inhibit cellular proliferation and induce apoptosis and correlate it with the chemical reactive indexes in HepG2 hepatocellular carcinoma cells.

 

2.1 Objectives of Study

Although a lot of work was done on the ethnobotanical uses of the medicinal plants of Nepal. But only few scientific studies were done on the biological activities of indigenous medicinal plants of this region. That’s why this study was designed to study the TPC, TFC, antioxidant and antimicrobial activities of selected medicinal plants. Following are the major objectives of the proposed study: 

General objectives:

Phytochemical and biological study of fruits and leaves of Aegle marmelos, Stem of Cuscuta reflexa Roxb and leaves of Pityrogramma calomelanos in hexane, ethyl acetate, Chloroform and methanol extracts.

 

Specific objectives:

v  Phytochemical screening test of some medicinal plant in hexane, ethyl acetate, Chloroform and methanol extracts.

v  Determination of anti-bacterial activity of Aegle marmelos, Cuscutta reflexa Roxb and Pityrogramma calomelanos in hexane, ethyl acetate, Chloroform and methanol extracts.

v  Determination of antioxidant activity of extracts using DPPH free radical scavenging assay.

v  To determine the total phenolic content of methanol extract of all plants at different concentration of extracts.

v  To determine the total flavonoid content of methanol extract of all plants at different concentration of extracts.

THIRD CHAPTER: EXPERIMENTAL SECTION

3.1 Materials :

3.1.1 Plant Materials

Plant materials were collected from local area of Sindhupalchok, Jhapa and Rukum district of Nepal on December 2018. They were authenticated by comparison with herbarium, species deposited at Central Department of Botany. Voucher specimens were deposited at MSc Lab of Tri-Chandra College. The names of the plants, local names, collected parts & voucher number are shown in table 1.

Table 3. 1 : Plants name with collection site, local name, collected parts and their voucher number

SN	Name of Plants	Collection Site	Local Names	Collected Parts	V. No.
1.	Aegle marmelos	Jhapa	Bael	Fruits	BF
2.	Aegle marmelos	Sindhupalchok	Bael	Leaves	BL
3.	Cuscuta reflexa	Rukum	Aakash Beli	Whole plants	AB
4.	Pityrogramma calomelanos	Rukum	Rani Sinka	Leaves	F

 

3.1.2 Instruments

        i.            Soxhlet extractor

      ii.            Rotavapour

: Rotavapour

: Heating bath (labtherm LT-40)

: Vacuum controller pump (Rocker 400)

    iii.            UV lamp

    iv.            UV Spectrometer (ELICO SL 177)

      v.            Digital balance (KERN PCB)

    vi.            Hot air oven (AccumaX India)

  vii.            Water Bath (Cifton, victo)

viii.            Shaker (Stuart Scientific)

    ix.            Refrigerator (Whirlpool, DC 260 5S/2014)

 

3.1.3 Solvents and Chemicals

        i.            Solvents like hexane, Ethyl acetate, methanol, Chloroform (Thermo Fisher Scientific India Pvt. Ltd., Mumbai) were of Laboratory grade.

      ii.            Distilled water (Ultra super TM, Marech Pvt. Ltd., Lalitpur, Nepal)

    iii.            Aluminium Chloride

    iv.            Ethanol (Bangal chemicals and pharmaceuticals Ltd. Calcutta, India)

      v.            Sodium hydroxide (Thermo fisher)

    vi.            Gallic acid (Thermo Fisher scientific India Pvt. Ltd. Mumbai)

  vii.            DPPH (Sigma Chemical Company, USA)

viii.            Quercetin (Sigma Chemical Company, USA)

 

3.2 Methods

3.2.1 Soxhlet Extraction of Plant Materials

Leaves and fruits of Aegle marmelos, stem of Cuscuta reflexa and leaves of Pityrogramma calomelanos were washed with distilled water and kept in incubator at 37°C for 3-4 days and grinded into fine powder. Finally crushed 150 g of leaves and fruits of A. marmelos, stem of C. reflexa and leaves of P. calomelanos were taken and extracted in a soxhlet extractor with 500 mL hexane, chloroform, acetone, ethyl acetate and methanol as solvent for 7-8 hours. Then hexane, chloroform, acetone, ethyl acetate extract of  A. marmelos, C. reflexa and P. calomelanos plants were concentrated under reduced pressure in a rotavapour to get viscous liquid.

 

Flow chart for extraction, screening and analysis

 

Where,

 Bioassay = Antimiocrobial, Antioxidant, TPC and TFC

Figure 3. 1 : Flow chart diagram for extraction

3.2.2 Phytochemical Screening Test of Plant extract

Phytochemical screening of plant extracts were carried out according to the standard protocol. In this method, all plant material were completely extracted by soxhlet extractor with increasing polarity (Hexane, ethyl acetate, chloroform and methanol) and subjected to phytochemical screening. The presence of main groups of natural constituents in different extracts were analysed by using different specific reagents.⁹¹ Phytochemical screening of the selected plant materials involves the selective and successive extraction and the method employed is primarily based on the standard procedure put forward by Dhote, (2015).⁹² The detail procedure is given in Annex 1.

3.2.3 Thin layer chromatography

The hexane, ethyl acetate and chloroform extracts of different parts of plants were examined by TLC in solvents mixture (hexane and ethyl acetate) at different ratios and the spots were visualized in day light as well as under UV light, both at 254 nm and 366 nm. The chromatogram was further visualized by spraying with 1% ferric chloride solution in methanol. The solvent systems used for the development of the chromatogram are :

Hexane : Ethyl acetate                                    2.5 : 7.5

Hexane : Ethyl acetate                                    5 : 5

Hexane : Ethyl acetate                                    7.5 : 2.5

3.2.4 Determination of Antimicrobial activity

Screening of the various bioactive compounds from the plants has led to the discovery of new medicinal drugs. Antibacterial screening of the plant extracts was performed by agar well diffusion method. Effectiveness of antimicrobial substance was evaluated by determination of Zone of inhibition (ZOI) as given by Cavalieri S. J.¹⁴

Antibacterial assay of plant extracts were performed by agar disc diffusion method in Muller Hinton agar (MHA) and the minimal bacterial concentration of the extracts were determined by micro dilution method using plant fraction serially diluted in sterile NB (NCCLS, 1999).

A. Collection of Test Organisms           

The microbial strains were identified strains that were obtained from Polytechnic Research Institute of Nepal (PORIN), Kathmandu, Nepal. The studies of microbial strain included Enterococcus faecalis (ATCC 29212) and Bacillus subtilis (ATCC6051).

B. Preparation of solution

100 mg/ml of working solution was made by transferring 0.01 g of each crude extract to sterile vial aseptically containing 1 ml of DMSO solvent. The extract was dissolved in DMSO. After making up solution, the test tubes were capped, sealed and stored at 4 ºC until assay was completed.

C. Muller Hilton Agar (MHA) Plates

The Muller Hilton Agar medium was prepared according to the manufacturers recommendation. For this the MHA was dissolved in distilled water in a conical flask by warming on hot plate. The conical flask was cotton plugged, sealed with the aluminium foil and sterilized in an autoclave. After cooling the media to room temperature, it was poured into the clean, dry and autoclaved petridishes approximately equal in amount and allowed to set at room temperature. When the agar solidified, the plates were sealed with parafilm and stored in refrigerator until futher use.

D. Nutrient Broth Solution

Nutrient Broth solution was prepared according to the manufactures recommendation. The nutrient broth was dissolved in the distilled water in a conical flask by warming on the hot plate. The conical flask was cotton plugged, sealed with aluminium foil and sterilized in an autoclave. The solution was then allowed to cool down at room temperature and stored in the refrigerator until use.

E. Preparation of Standard Culture Inoculums

The test organisms to be tested were aseptically touched with the help of inoculating loop from primary culture plate. Then it was transferred into a test tube 10 ml of sterile liquid media of nutrient broth and incubated overnight at 37 ºC in incubator.

F. Transfer of Bacteria in Petri Plates

The agar plates for the assay were prepared by labelling them with the name of the bacteria and the name code of discs. The inoculums of bacteria were transferred into petridish containing solid nutrient media of agar using sterile swab. The sterile cotton swab was dipped into well mixed saline test culture and removed excess inoculums by pressing the saturated swab against the inner wall of the culture tube. The swab was used to spread the bacteria on the media in a confluent lawn. It was done by rotating the petridish at 90 degree and continuing the spread of bacteria. One swab was used for one species of bacteria. The culture plates were allowed to dry for 5 minutes.

G. Screening and Evaluation of Antibacterial Activity

The wells were made in the incubated media plates with the help of sterile cork borer of diameter of 4 mm and labelled properly. Then, 15 μl of the working solution of the plant extracts and essential oil were loaded into the respective wells with the help of micropipette. Ofloxacin was used as a control in the separate well. The plates were then left for half an hour with the lid closed so that extracts diffuse into the media. The plates were incubated overnight at 37 ºC. The plates were then observed for zone of inhibition (ZOI) produced by antibacterial activity of plant extracts and essential oil and the inhibition zones were measured by the use of a scale.

3.2.5 Determination of Antioxidant Activity

DPPH radical scavenging activity was assessed for the determination of antioxidant power of any medical plant.¹⁹ The free radical scavenging activity of methanol extracts of A. marmelos, C. reflexa and P. calomelanos plants and ascorbic acid was measured in terms of hydrogen donating ability using the stable radical DPPH. About 0.2 mM solution of DPPH in 100 % methanol was prepared by dissolving 78 mg of DPPH 1000 mL methanol and stirred and kept overnight at 4 ^oC. Thus, prepared purple colour of DPPH free radical solution was stored at -20 ^oC for further use. The different concentrations 20, 40, 60, 80, 100 μg/mL of each extracts i.e acetone and methanol fraction of fruit, leaf and stem and stem bark, were prepared by the serial dilution of the stock solution of respective extracts. To each 2 mL of each concentration of the extracts, 2 mL of 0.2 mM methanolic DPPH solution was added. A control was prepared by mixing 2 mL of Distilled water and 2 mL of 0.2 mM methanolic DPPH solution. The mixture was shaken and allowed to stand at room temperature for 30 min. The absorbance was measured at 517 nm using a spectrophotometer against the blank solution. The radical scavenging activity was expressed as percentage radical scavenging using the following equation.

Percentage radical scavenging activity is calculated by using formula:

% radical scavenging activities=(Abs_control-Abs_conc)/Abs_control ×100%

Where,

            Abs _control = absorbance of control

            Abs _conc = absorbance of solution of each concentration

The IC₅₀ value is the concentration of sample required to scavenge 50% of DPPH free radical. The IC₅₀ value of the crude extract was compared with that of ascorbic acid, which was used as the standard. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity.

A. Preparation of the 0.2 mM DPPH solution

DPPH has the molecular weight of 394.32 g/mol. Thus 100 mL of 0.2 mM solution of DPPH was prepared by weighing the 0.007886 g of the DDPH carefully and dissolving it on methanol and finally maintain the volume to 100 mL and was kept in dark place until the used.

B. Measurement of DPPH free radical scavenging activity

Firstly, 10 mg of the samples to be tested was dissolved in 10 mL methanol to get the stock solution of concentration of 1 mg/ml (1000 μg/mL). Stock solution of each extracts were screened preliminary for their antioxidant activity by adding 0.5 mL of 0.2 mM DPPH in 1 mL of extract solutions differently and was left for 30 minutes in dark. After

30 minutes, the sample with the color of DPPH (purple) was discarded for the final test and the samples with the pale yellow color (color change form purple to yellow) was taken for further testing as they were expected to be the potential antioxidants.

Different concentrations of test samples of 20, 40, 60, 80 and 100 μg/mL were made from stock solutions. Then 2 ml of all the concentration of test solution were mixed with 2 ml of DPPH solution. The test tubes were shaken vigorously for the uniform mixing then the solutions was kept for 30 minutes in a dark place at room temperature. The control was prepared as above but without the plant extracts (methanol + DDPH). After 30 minutes absorbance of all the samples was measured at 517 nm using a UV-visible spectrophotometer. Ascorbic acid of same concentrations was prepared as a standard and its absorbance was also taken spectrophotometrically at 517 nm. Calibration curve was prepared. Percent radical scavenging activity by sample treatment was determined by comparison with methanol treated control group; ascorbic acid was used as positive control.

3.2.6 Total Phenol Content (TPC) Determination

The total Phenol content of all selected plant extracts were estimated using Folin Ciocalteu reagent involving gallic acid standard based on oxidation-reduction reaction. The procedure carried out for the total phenol content was based on the standard procedure put forwarded by Waterhouse.⁹³

 

A. Preparation of Standard Gallic Acid Stock Solution

Various concentrations of Gallic acid- 20, 40, 60, 80 and 100 μg/ml were prepared in methanol by serial dilution method. Then aliquot of 1 ml gallic acid of each concentration was transferred into the corresponding test tubes and to it 5 ml FCR (10 % in distilled water) and 4ml Na2CO3 (7 % in distilled water) was added to get total volume of 10ml. The blue colored solution was shaken well and incubated for about 30min in 40 ⁰C in water bath. Then the absorbance was measured at 760 nm against blank containing all reagents except gallic acid. Absorbance was measured thrice. The average absorbance values obtained at different concentration of gallic acid was used to plot calibration curve.

 

B. Preparation of Sample

The stock solution of all the extracts was prepared by dissolving 10 mg in 1 mL of methanol (10 mg/mL). Serial dilutions were carried out to get the concentrations of 0.20, 0.40, 0.60, 0.80 and 1.0 mg/mL. To these diluted solutions, 10% FCR and 7% Na2CO3 were added and incubated for 30 minutes as in the case of gallic acid and absorbance was measured at 760 nm against blank for each concentration.

C. Calculation of Total Phenolic Content (TPC)

TPC content compound concentration in extract was expressed as milligrams of gallic acid equivalent per gram of dry weight (mg GAE/g) of extract which was calculated in all the extracts separately using the following formula;

C=cV/m ...……………………………………………  (1)

Where,

            C = Total phenolic content compounds in mg/g, in gallic acid equivalent (GAE)

            c = Concentration of gallic acid established from the calibration curve in mg/mL

            V = Volume of extract in mL

            m = Weight of plant extract

 

D. Statistical Analysis

Data were recorded as a mean of three determinations of absorbance for each concentration, from which linear correlation coefficient (R²) value was calculated. The regression equation is given as;

                        𝑦 = 𝑚𝑥 + 𝑐  …………………………………… (2)

Where,

            y = Absorbance of extract

            m = Slope from the calibration curve

            x = Concentration of extract

            c = Intercept

Using this regression equation concentration of extracts was calculated. Thus, with the calculated value of concentration of each extract, the total polyphenolic content calculated by the equation. The results of phenolics are expressed in terms of gallic acid mg/g of extracts (2).

 

3.2.7 Total Flavonoid Content (TFC) Determination

TFC of the selected plant extracts were determined according to aluminum chloride colorimetric method involving quercetin as standard as described by Kalita, P., (2013).⁹⁴

 

A.    Preparation of Standard Quercetin Stock Solution:

Quercetin stock solution was prepared by dissolving 10 mg of quercetin in 10 mL of methanol (1 mg/mL). Various concentrations of quercetin such as 20, 40, 60, 80 and 100 μg/mL was prepared. An aliquot of 1 mL quercetin of each concentration in methanol was poured to 20 mL test tube containing 4 mL of double distilled water. At the zero time, 0.3 mL of 5% NaNO₂ was added to the test tube. After 5 minutes, 0.3 mL of 10% AlCl₃ was added to the test tube. After 6 minutes, 2 mL of 1M NaOH was added to the mixture. Immediately, the total volume of the mixture was made upto 10 mL by adding 2.4 mL double distilled water and mixed thoroughly. Absorbance of the pink colored mixture was determined at 510 nm versus blank containing all reagents except Quercetin. All the experiments were carried out in triplicate. The average absorbance values obtained at different concentrations of quercetin were used to plot calibration curve.

B.     Preparation of Sample:

The stock solutions of all the extracts were prepared by dissolving 10 mg in 1 mL of methanol. Serial dilution was carried out to get the concentrations of 20, 40, 60, 80 and 100 μg/mL. Following the procedure described above, absorbance for each concentration of each extracts was recorded.

C.    Calculation of Total Flavonoid Content:

The total flavonoid content was calculated in all the extracts separately using the formula below.

C=cV/m ...… … … … … … (3)

 

Where,

C = total content of flavonoid compounds in mg/g, in Quercetin equivalent (QE)

c = concentration of Quercetin established from the calibration curve in mg/mL

V = the volume of extract in mL

m = the weight of plant extracts

 

D.    Statistical Analysis:

Data were recorded as a mean of three determinations of absorbance for each concentration, from which linear correlation (R²) value was calculated. The regression equation is given as;

            𝑦 = 𝑚𝑥 + c  … … … … … … (4)

 

Where,

Y = absorbance of extracts

m = Slope from the calibration curve

x = concentration of the extracts

c = intercepts

Using this regression equation, concentration of extracts was calculated. Thus, with the calculated value of concentration of each extracts, the flavonoid content was calculated by the equation (1). The results of flavonoids are expressed in terms of Quercetin mg/g of extracts.

FORTH CHAPTER: RESULTS AND DISCUSSION

4.1 Phytochemical Analysis  

Phytochemical analysis of the leaves and fruits of Aegle marmelos, stem of Cuscuta reflexa and leaves of Pityrogramma calomelanos extract was carried out on the basis of procedure put forward by Dhote, (2015).⁹² The phytochemical screening was done in four different solvents; hexane, ethyl acetate, Chloroform and methanol. The result obtained from phytochemical screening of three different plants of different parts in different solvents is tabulated below.

Table 4. 1 : Phytochemical screening of fruit extracts of Aegle marmelos

SN	Group of Compounds	Name of Tests	Hexane extract	Et. acetate extract	CHCl3 extract	MeOH extract
1	Basic Alkaloids	i.Dragendroff's	+	+	+	+
		ii.Mayer's	+	+	+	+
		iii.Wagner's	-	-	+	+
2	Flavonoids	i.Sinoda's	-	+	+	+
		ii.Sibata's	-	+	+	+
3	Coumarins		+	+	+	+
4	Saponins	Foam test	+	+	+	+
5	Polyphenols	FeCl3 test	-	+	+	+
6	Terpenoids	Salkowski's test	-	-	+	-
7	Cardiac Glycosides	Killer killani's	-	-	-	-
8	Proteins	Xanthoproteic	-	-	-	+
9	Carbohydrates	Molisch test	+	+	+	+

 

Table 4. 2 : Phytochemical screening of leaf extracts of Aegle marmelos

SN	Group of Compounds	Name of Tests	Hexane extract	Et. acetate extract	CHCl3 extract	MeOH extract
1	Basic Alkaloids	i.Dragendroff's	+	+	+	+
		ii.Mayer's	+	+	+	+
		iii.Wagner's	-	-	+	+
2	Flavonoids	i.Sinoda's	+	+	-	+
		ii.Sibata's	+	+	-	+
3	Coumarins		+	+	+	+
4	Saponins	Foam test	+	+	+	+
5	Polyphenols	FeCl3 test	+	+	+	+
6	Terpenoids	Salkowski's test	-	-	+	-
7	Cardiac Glycosides	Killer killani's	-	-	-	-
8	Proteins	Xanthoproteic	-	-	-	+
9	Carbohydrates	Molisch test	+	+	+	+

 

Table 4. 3 : Phytochemical screening of stem extracts of Cuscuta reflexa

SN	Group of Compounds	Name of Tests	Hexane extract	Et. acetate extract	CHCl3 extract	MeOH extract
1	Basic Alkaloids	i.Dragendroff's	-	-	-	-
		ii.Mayer's	-	-	-	-
		iii.Wagner's	-	-	-	-
2	Flavonoids	i.Sinoda's	-	-	-	+
		ii.Sibata's	-	-	-	+
3	Coumarins		+	+	+	+
4	Saponins	Foam test	-	-	-	-
5	Polyphenols	FeCl3 test	+	+	+	+
6	Terpenoids	Salkowski's test	-	+	+	+
7	Cardiac Glycosides	Killer killani's	+	+	+	+
8	Proteins	Xanthoproteic	-	-	-	+
9	Carbohydrates	Molisch test	+	+	+	+

 

Table 4. 4 : Phytochemical screening of leaf extracts of Pityrogramma calomelanos

SN	Group of Compounds	Name of Tests	Hexane extract	Et. acetate extract	CHCl3 extract	MeOH extract
1	Basic Alkaloids	i.Dragendroff's	+	+	+	+
		ii.Mayer's	+	+	+	+
		iii.Wagner's	+	+	+	+
2	Flavonoids	i.Sinoda's	-	+	-	+
		ii.Sibata's	-	+	-	+
3	Coumarins		-	+	+	+
4	Saponins	Foam test	-	-	-	-
5	Polyphenols	FeCl3 test	-	+	-	+
6	Terpenoids	Salkowski's test	+	+	-	+
7	Cardiac Glycosides	Killer killani's	-	+	+	+
8	Proteins	Xanthoproteic	+	+	+	+
9	Carbohydrates	Molisch test	+	+	+	+

Where, (+) = positive response of the test (presence), (-) = negative response of the test (absence)

The results of phytochemical screening shows the presence of many biologically active compounds, such as alkaloids, flavonoids, phenolics, glycosides, terpenoids and saponins etc as the major phytochemicals. The more or less phytochemicals present in the plant extracts may be due to the climatic, geographical, and altitudinal variations. These factors determine the amount of secondary metabolites persistence during adaptation phenomenon. Due to these reasons, the phytochemicals reported in this work are slightly different from the data presented in the literature of selected plant. On the basis of above result, the plant can be attributed to the potential therapeutic and antioxidant properties in the tested samples. Total phenolic and flavonoid content validated the idea behind use of traditional medicinal plants to treat different diseases and could be used sources of active compounds in future study.

4.2 Antibacterial Activity

The antibacterial activity of extract on particular bacteria was measured by viewing the diameter of zone of inhibition (ZOI). The result of antibacterial activity of Hexane, ethyl acetate, chloroform and methanol extracts of Leaves and fruits of Aegle marmelos, stem of Cuscuta reflexa and leaves of Pityrogramma calomelanos has been measured. The methanolic extract of A.marmelos, C. reflexa and P. calomelanos on both bacteria did not show any zones of inhibition at 200 mg/ml. Also, chloroform extract of A.marmelos and ethyl acetate extract of P.calomelanos and C. reflexa did not show any zones of inhibition on both bacteria at 200 mg/ml. Hexane extract of A.marmelos (fruits and leaves) did not show any zones of inhibition on Enterococcus faecalis bacteria at 200 mg/ml.  Results obtained from the antibacterial screening of different extracts are tabulated below.

 

Table 4. 5 : Antibacterial activities of different fruit extracts of Aegle marmelos

SN	Plant Extracts	Bacteria	ZOI (mm) of extracts at concentration 200 mg/ml	ZOI (mm) of Ofloxacin  as control at 200 mg/ml
1	Hexane	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	8 mm	30 mm
2	Ethyl Acetate	i. Enterococcus faecalis	13 mm	32 mm
		ii. Bacillus subtilis	11 mm	30 mm
3	Chloroform	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm
4	Methanol	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm

 

Table 4. 6 : Antibacterial activities of different leaves extracts of Aegle marmelos

SN	Plant Extracts	Bacteria	ZOI (mm) of extracts at concentration 200 mg/ml	ZOI (mm) of Ofloxacin as control  at 200 mg/ml
1	Hexane	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	11 mm	30 mm
2	Ethyl Acetate	i. Enterococcus faecalis	14 mm	32 mm
		ii. Bacillus subtilis	12 mm	30 mm
3	Chloroform	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm
4	Methanol	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm

 

Table 4. 7 : Antibacterial activities of different stem extracts of Cuscuta reflexa

SN	Plant Extracts	Bacteria	ZOI (mm) of extracts at concentration 200 mg/ml	ZOI (mm) of Ofloxacin as control at 200 mg/ml
1	Hexane	i. Enterococcus faecalis	13 mm	32 mm
		ii. Bacillus subtilis	12 mm	30 mm
2	Ethyl Acetate	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm
3	Chloroform	i. Enterococcus faecalis	13 mm	32 mm
		ii. Bacillus subtilis	10 mm	30 mm
4	Methanol	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm

 

Table 4. 8 : Antibacterial activities of different leaves extract of Pityrogramma calomelanos

SN	Plant Extracts	Bacteria	ZOI (mm) of extracts at concentration 200 mg/ml	ZOI (mm) of Ofloxacin as control at 200 mg/ml
1	Hexane	i. Enterococcus faecalis	15 mm	32 mm
		ii. Bacillus subtilis	12 mm	30 mm
2	Ethyl Acetate	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm
3	Chloroform	i. Enterococcus faecalis	11 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm
4	Methanol	i. Enterococcus faecalis	0 mm	32 mm
		ii. Bacillus subtilis	0 mm	30 mm

 

Antibacterial activity of extract is a property of compounds presence in the extracts that destroys bacteria or suppresses their growth or their ability to reproduce. This property of plant extract is shown due to the production of different compounds in the process of secondary metabolism. Tables above indicate that ethyl acetate extract of both leaves and fruits of A. marmelos, hexane and chloroform extract of C. reflexa and hexane extract of P. calomelanos were active for the inhibition of the growth of both (Enterococcus faecalis and Bacillus subtilis) bacteria used. Further, the ethyl acetate leaves extract of A. marmelos was found to be more effective against both types of bacteria. Out of two bacteria used Enterococcus faecalis inhibited effectively than Bacillus subtilis. Among these extracts,  A. marmelos leaves extract have more antibacterial activity than A. marmelos fruits extract. Maximum inhibition occurs in hexane extract of P.callomelanos at Enterococcus faecalis bacteria.

4.3 Antioxidant Activity

4.3.1 DPPH Radical Scavenging Activity

Free radical scavenging activity was determined by using 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH), which is very stable free radical having purple color. When free radical scavengers are added, DPPH is reduced and its color is changed to yellow based on the efficacy of antioxidants. Scavenging of DPPH free radical determines the free radical scavenging capacity or antioxidants potential of the test sample which shows its effectiveness, prevention, interception and repair mechanism against injury in a biological system. Calibration curve was constructed by measuring the absorbance of ascorbic acid in order to calculate the IC₅₀ value. The value obtained from plant extracts was compared with ascorbic acid.

The DPPH free radical scavenging assay was carried out for methanol extracts of fruits and leaves part of Aegle marmelos, stem of Cuscuta reflexa and leaves of Pityrogramma calomelanos were determined with the synthetic antioxidant substance ascorbic acid prepared in methanol at different concentrations of 20, 40, 60, 80 and 100 μg/mL. The plant extract of different concentration with DPPH and ascorbic acid were shaken well and incubated in the dark for 30 minutes at 23^oC temperature. And absorbance was read at 517 nm by the help of spectrophotometer. The absorbance of control was found 0.6965. Calibration curve was constructed by measuring the absorbance of ascorbic acid in order to calculate IC₅₀ value. The value obtained from the plant extract was compared with the standard ascorbic acid. The observed absorbance with the different concentration was plotted in the graph as shown below.

 

Figure 4. 1 : Plot of Absorbance vs the concentration of ascorbic acid

 

 Table 4. 9 : Percentage of radical scavenging with different concentrations

Sample (MeOH Extract)	% of radical scavenging at Concentration (μg/mL)
	20	40	60	80	100
Aegle marmelos Fruits	64.058	76.694	80.887	86.358	87.694
Aegle marmelos Leaves	30.744	38.240	41.399	44.515	47.257
Cuscuta reflexa Stem	32.682	42.231	44.630	47.975	50.345
Pityrogramma calomelanos Leaves	33.487	43.596	48.420	51.206	53.877
Ascorbic acid	72.573	80.615	87.507	93.538	98.277

 

The comparison of percentage radical scavenging at different concentrations between plant extract and ascorbic acid as standard was shown in the graph below.

 

Figure 4. 2 : A plot of percentage radical scavenging activity vs concentration of A. marmelos fruit methanolic extract and ascorbic acid

Figure 4. 3 : A plot of percentage radical scavenging activity vs concentration of A. marmelos leaves methanolic extract and ascorbic acid

 

Figure 4. 4 : A plot of percentage radical scavenging activity vs concentration of C. reflexa stem methanolic extract and ascorbic acid

Figure 4. 5 : A plot of percentage radical scavenging activity vs concentration of P. calomelanos leaves methanolic extract and ascorbic acid

The linear regression of the percentage of radical scavenging versus concentration was used for the calculation of concentration of each plant extract required for 50 % inhibition of DPPH activity (IC₅₀). The antioxidant potential is in an inverse relation with IC₅₀ value, lower value of IC₅₀ indicates high antioxidant potential. The IC₅₀ values of the plant extracts along with the standard ascorbic acid is tabulated below.

Table 4. 10 : Comparision of DPPH antioxidant and IC₅₀ values of different plant extracts with standard ascorbic acid

SN	Sample (Methanolic Extract)	IC50 Values (μg/mL)
1	Standard ascorbic acid	22.451
2	Methanol Extract of Aegle marmelos Fruits	28.093
3	Methanol Extract of Aegle marmelos Leaves	90.656
4	Methanol Extract of Cuscuta reflexa Stem	81.937
5	Methanol Extract of Pityrogramma calomelanos Leaves	74.734

Among the selected plants, methanolic extracts leaves of Aegle marmelos show greater IC₅₀ value as compared to other extracts.

More conveniently, the above table is represented in the bar graph below.

Figure 4. 6 :  Free radical scavenging activity (IC₅₀) in different plant extracts.

The table 4.10 and figure 4.6 showed that the plant extracts were potential antioxidants as their IC₅₀ values were found to be close to the standard ascorbic acid taken. The methanol extract of Aegle marmelos fruits showed the strongest DPPH radical scavenging activity with IC₅₀ values 28.093 µg/mL very close to standard ascorbic acid. The methanolic extract of leaves of Aegle marmelos, stem of Cuscutta reflexa & leaves of Pityrogramma calomelanos showed less antioxidant properties as compared to methanolic extracts of Aegle marmelos fruits. The results reflected that the fruit part of Aegle marmelos plant can act as a very good option in the field of medicine based on antioxidant property of natural products chemistry.

4.4 Estimation of Total Phenolic Content (TPC)

The total soluble phenols present in various extracts of A. marmelos, C. reflexa and P. calomelanos were determined by using Folin-Ciocalteu reagent (FCR) colorimetric method based on oxidation reduction reaction.

4.4.1. Construction of Calibration Curve

Gallic acid was taken as standard for construction of calibration curve. The absorbance value obtained at different concentrations of Gallic acid and the absorbance value are tabulated below.

Table 4. 11 : Absorbance at different concentrations of gallic acid

Concentration of Gallic acid (μg/mL)	20	40	60	80	100
Absorbance	0.231	0.499	0.75	0.997	1.167

                                                                                             

 The calibration curve for standard gallic acid is shown in the figure below.

Figure 4. 7 : Variation of absorbance with concentration for standard gallic acid.

 

4.4.2 Calculation of TPC in Different Plant Extracts

The absorbance values of each extracts at different concentrations (20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL and 100 μg/mL) were recorded at 760 nm by using spectrophotometer. The numerical value of absorbance is tabulated in Annex 3. The TPC in the plant extracts taken under study was calculated by using regression equation y = 0.012x + 0.0085, R² = 0.9966, of the curve obtained from above graph followed by the formula C= cV/m and expressed as mg GAE per g of extract in dry weight. The TPC of different plant extract (mg gallic acid equivalent per g dry extract) is tabulated below.

Table 4. 12 : Total Phenolic Content of methanol extract of Fruits of Aegle marmelos

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	GAE conc. C (μg/mL)	GAE conc. C (mg/ml)	TPC as GAE (=cxV/m)	Mean
1	20	0.001	0.0116	12.302	0.012	12.302	12.2912
2	40	0.001	0.0145	11.905	0.011	11.905
3	60	0.001	0.0183	12.963	0.012	12.963
4	80	0.001	0.0205	11.905	0.011	11.905
5	100	0.001	0.0241	12.381	0.012	12.381

 

Table 4. 13 : Total Phenolic Content of methanol extract of Leaves of Aegle marmelos

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	GAE conc. C (μg/mL)	GAE conc. C (mg/ml)	TPC as GAE (=cxV/m)	Mean
1	20	0.001	0.0131	18.254	0.018	18.254	18.3478
2	40	0.001	0.0177	18.254	0.018	18.254
3	60	0.001	0.0224	18.386	0.018	18.386
4	80	0.001	0.0278	19.147	0.019	19.147
5	100	0.001	0.0313	18.095	0.018	18.095

 

Table 4. 14 : Total Phenolic Content of methanol extract of Stems of Cuscuta reflexa

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	GAE conc. C (μg/mL)	GAE conc. C (mg/ml)	TPC as GAE (=cxV/m)	Mean
1	20	0.001	0.0311	89.683	0.089	89.683	88.1666
2	40	0.001	0.0526	87.5	0.087	87.5
3	60	0.001	0.0751	88.095	0.088	88.095
4	80	0.001	0.0969	87.698	0.087	87.698
5	100	0.001	0.1192	87.857	0.087	87.857

 

Table 4. 15 : Total Phenolic Content of methanol extract of Leaves of Pityrogramma calomelanos

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	GAE conc. C (μg/mL)	GAE conc. C (mg/ml)	TPC as GAE (=cxV/m)	Mean
1	20	0.001	0.0103	7.143	0.007	7.143	7.4046
2	40	0.001	0.0127	8.333	0.008	8.333
3	60	0.001	0.0139	7.143	0.007	7.143
4	80	0.001	0.0155	6.944	0.006	6.944
5	100	0.001	0.0179	7.46	0.007	7.46

More conveniently total phenolic content in different plant extracts are represented in bar diagram too,

Figure 4. 8 : Total phenolic content (TPC) in different plant extracts.

The result demonstrate that the total phenolic content was highest in methanolic extract of C. reflexa (88.1666 mg GAE/g extract) while the others had significantly lower values. The extract which displayed the lowest content of total phenol was methanol extract of leaves of P. calomelanos (7.4046 mg GAE/g extract) and fruits of A. marmelos (12.2912 mg GAE/g extract) respectively.

Phenolic compounds have been known to possess high antioxidant properties due to their free radical scavenging properties. It has been reported that extract containing large amount of polyphenol content possesses a greater antioxidant activity. Although quantitative determination of phenolic compounds in plant extracts are hampered by their structural complexity, diversity, nature of analytical assay method, selection of standard and presence of interfering substances.

Hence, plant contains high phenolics, it can used as food stuffs, preservatives, etc. directly or indirectly by human community. They have also been implicated as anticarcinogenic, antimicrobial, antimutagenic, antiangiogenic and anti-inflammatory agents besides their use in treating critical diseases like depression, cancer, microbial infections, lipid-related diseases, etc.

4.5 Estimation of Total Flavonoid Content

The total flavonoid Contents in the extracts was estimated by using aluminium chloride colorimetric assay. The total flavonoids present in different extracts of different plants were estimated by standard procedure using quercetin as standard. The flavonoids of the plant extracts in the presence of aluminum chloride forms a acid liable complexes, has an intense yellow fluorescence which was observed under UV spectrophotometer at 510 nm. The intensity of light absorption at that wavelength is proportional to the concentration of flavonoids. The numerical data are shown in Appendix 3.

 

4.5.1. Construction of Calibration Curve

For the construction of calibration curve quercetin was used as a standard. The absorbance value obtained at different concentrations of quercetin and the absorbance value are tabulated below.

 

Table 4. 16 : Absorbance at different concentrations of quercetin

Concentration of Quercetin (μg/mL)	20	40	60	80	100
Absorbance	0.231	0.499	0.75	0.997	1.167

                                                                                             

 The calibration curve for standard quercetin is shown in the figure below.

Figure 4. 9 : Variation of absorbance with concentration for standard Quercetin.

 

4.5.2. Calculation of Total Flavonoid Content

The graph shows the linear relationship between absorbance and concentration. The absorbance values of each extracts taken under study at different concentrations (0.20 mg/mL, 0.40 mg/mL, 0.60 mg/mL, 0.80 mg/mL and 1.0 mg/mL) were recorded at 510 nm by using spectrophotometer. The TFC in the plant extracts taken under study was calculated by using regression equation y = 0.013x + 0.0193, R² = 0.9986, of the curve obtained from above graph followed by the formula cV/m and expressed as mg QE per g of extract in dry weight. The TFC of different plant extracts (mg quercetin equivalent per g dry extract) are tabulated below.

 

Table 4. 17 : Total Flavonoid Content of methanol extract of Fruits of Aegle marmelos

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	QE conc. C (μg/mL)	QE conc. C (mg/ml)	TFC as QE (=cxV/m)	Mean
1	20	0.001	0.0215	8.462	0.008	8.462	8.0066
2	40	0.001	0.0231	7.308	0.007	7.308
3	60	0.001	0.0257	8.205	0.008	8.205
4	80	0.001	0.0276	7.981	0.007	7.981
5	100	0.001	0.0298	8.077	0.008	8.077

 

Table 4. 18 : Total Flavonoid Content of methanol extract of Leaves of Aegle marmelos

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	QE conc. C (μg/mL)	QE conc. C (mg/ml)	TFC as QE (=cxV/m)	Mean
1	20	0.001	0.0229	13.864	0.013	13.864	13.251
2	40	0.001	0.0263	13.462	0.013	13.462
3	60	0.001	0.0294	12.949	0.012	12.949
4	80	0.001	0.0332	13.365	0.013	13.365
5	100	0.001	0.0357	12.615	0.012	12.615

 

Table 4. 19 : Total Flavonoid Content of methanol extract of Stems of Cuscuta reflexa

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	QE conc. C (μg/mL)	QE conc. C (mg/ml)	TFC as QE (=cxV/m)	Mean
1	20	0.001	0.0371	68.462	0.068	68.462	69.3412
2	40	0.001	0.0553	69.231	0.068	69.231
3	60	0.001	0.0725	68.205	0.068	68.205
4	80	0.001	0.0915	69.423	0.069	69.423
5	100	0.001	0.1121	71.385	0.071	71.385

 

Table 4. 20 : Total Flavonoid Content of methanol extract of Leaves of Pityrogramma calomelanos

S. N	Sample solution (μg/mL)	Wt. of dry extract per mL m(g)	Absorbance	QE conc. C (μg/mL)	QE conc. C (mg/ml)	TFC as QE (=cxV/m)	Mean
1	20	0.001	0.0635	170.385	0.170	170.385	169.61
2	40	0.001	0.1076	169.808	0.169	169.808
3	60	0.001	0.1517	169.744	0.169	169.744
4	80	0.001	0.1943	168.269	0.168	168.269
5	100	0.001	0.2401	169.846	0.169	169.846

 

More conveniently TFC is represented in bar diagram as;

Figure 4. 10 : Total flavonoid content in different methanolic plant extracts

 

The result demonstrate that the total Flavonoid content was highest in methanol extract of P.calomelanos leaves (169.61 mg QE/g extract) while the others had significantly lower values. The extract which displayed the lowest content of total flavonoid was methanol extract of A. marmelos fruits (8.0066 mg QE/g extract). Flavonoid compounds have been known to possess high antioxidant properties due to their free radical scavenging properties. It has been reported that extract containing large amount of polyphenol content possesses a greater antioxidant activity. Although quantitative determination of flavonoid compounds in plant extracts are hampered by their structural complexity, diversity, nature of analytical assay method, selection of standard and presence of interfering substances.

More conveniently, total flavonoid content (TFC), total phenolic content (TPC) and DPPH radical scavenging activity (IC₅₀ value) in different plant extracts are represented in bar diagram too.

 

Figure 4. 11 : TFC, TPC and IC₅₀ value in different methanolic plant extracts

FIFTH CHAPTER: SUMMARY, CONCLUSION AND RECOMMENDATION

5.1 Summary

For the research work, A. marmelos, C. reflexa and P. calomelanos was collected from Sindhupalchok, Jhapa and Rukum district of Nepal on the basis of its ethno medicinal use. The plant material was successfully extracted with hexane, Chloroform, Ethyl acetate and methanol. The preliminary phytochemical screening of crude extracts of these plants showed that the flavonoids were a major phytochemicals along with the phenolics, glycosides, cumarin and saponins. Among them the most active phytochemical is flavonoid and phenolics. Hence, it can be used in medicine, such as ayurveda, herbal, tribal and folk, in preventing many major diseases. Plant showed little bit different result as observed in literature survey and biological properties of the plants may vary according to the altitude of the collected areas and the time of collection.

 

5.2 Conclusion

Phytochemical screening of methanol fraction of the all four plant extracts (A. marmelos fruits, A. marmelos leaves, C. reflexa stem and P. calomelanos leaves) revealed the presence of alkaloids, flavonoids, polyphenols, glycosides, protein, terpenoids etc. However, in other fractions like ethyl acetate, chloroform extracts of all plants showed alkaloids, flavonoids, saponins etc. as a major phytochemicals. Hexane fraction of all plants showed few phytochemicals.

The hexane extract of P. calomelanos leaves showed significant antibacterial activity against Enterococcus faecalis bacteria with ZOI value 15 mm and also Bacillus subtilis bacteria showed effective antibacterial activity with ZOI 12 mm. likewise, ethyl acetate extract of both leaves and fruits of A. marmelos and hexane and chloroform extract of C. reflexa towards both bacteria (Enterococcus faecalis and Bacillus subtilis) showed good antibacterial activity.

The methanol extract of fruits of A. marmelos (IC₅₀ = 28.093 μg/mL) were found have a very good antioxidant property with IC₅₀ value very close to the standard ascorbic acid (22.451μg/ml) taken. Methanol extract of stem of C. reflexa showed highest amount of phenol content (88.1666 mg GAE/g extract) was determined spectrophotometrically by taking gallic acid as a standard. Total flavonoids content of methanol extract of leaves of P. calomelanos (169.61 mg QE/g extract) and stem of C. reflexa (69.3412 mg QE/g extract) was found to be highest while fruits and leaves of A. marmelos showed significant total flavonoid content which were determined spectrophotometrically by taking quercetin as a standard.

 

5.3 Recommendation

The plant species chosen are said to have a good medicinal value. The methanolic extract of fruits of A. marmelos showed good antioxidant activity so research can be conducted to find out the possibility of this medicinally important plant as a potent antioxidant drug and for other pharmacological properties to develop as cost effective formulation. Phytochemical analysis of the plants showed the presence of many biologically active compounds, such as alkaloids, flavonoids, glycosides, tannins, terpenoids and reducing sugars, which can be attributed to the potential antibacterial and antioxidant properties in the tested samples. Total phenolic and flavonoid content validated the idea behind use of traditional medicinal plants to treat different diseases and could be used sources of active compounds in future study and it could be the potent source for drug discovery process.

Since, extracts of A. marmelos, C. reflexa and P. calomelanos contain a good amount of bioactive chemical constituents. These plant extracts could be used for isolating the active compound and it could be used for discovery process of new drug in future. Further, the in vitro and in vivo bioactivity of these extracts needs to be assessed that can shape to the potential drug discovery. The extract of C. reflexa showed good antibacterial activity and TPC, the extract of A. marmelos showed good antioxidant activity and the extract of P. calomelanos showed high TFC, so research can be conducted to find out the possibilities of this medicinally important plant as a potent antimicrobial drug and for other pharmacological properties to develop as cost effective formulation.

ACKNOWLEDGEMENT 

I would like to express my sincere gratitude to my respectable supervisor Prof. Dr Ram Narayan Jha, Department of Chemistry, Tri-Chandra Multiple Campus, Kathmandu, Nepal, for his encouragement, guidance, suggestions and constructive criticism during the research course that finally took the shape in this form.

 

I am very much thankful to Head of department Prof. Shilakant Lal Karna and co-ordinator of M.sc program, Assist Prof. Dr. Bishan Datt Bhatt, Department of chemistry, Tri-Chandra Multiple Campus, for providing me an opportunity to conduct this dissertation work.

 

I am also thankful to central department of chemistry, Tribhuwan University, kritipur for providing authentic quercetin and also for recording the absorbance values of different plant extracts for calculating flavonoid content.

 

I would like to acknowledge Dr. Laxmi Prasad Thapa, Polytechnic Research Institute of Nepal (PORIN), Kathmandu, Nepal for assisting in antibacterial activity.

 

Special thanks for all teaching and non teaching staffs of Department of Chemistry, Tri-chandra Multiple Campus.

 

Finally but immensely, I would also like to acknowledge with much appreciation the crucial role of my family, Specially Dad Bhim Prasad Ghimire &  Mom Manamaya Ghimire, friends and senior and junior students who helped me directly or indirectly during my dissertation work.

 

 

GYANENDRA GHIMIRE

Date: Dec. 31^st, 2019

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APPENDIX

APPENDIX-I: PHYTOCHEMICAL SCREENING

General Procedure

Extraction:

About 150 grams of each of shade dried A. marmelos fruits,  A. marmelos leaves, C. reflexa stem and P. calomelanos leaf was extracted successively with four different solvents (Hexane, Ethyl acetate, Chloroform and Methanol) regarding their increasing polarity by soxhlet method. The extract obtained from the soxhlet was concentrated by rotatory evaporator and subjected for phytochemical test according to following procedure:

1. Test for Tannins:

10 ml of bromine water was added to the 0.5 g plant extract. Decoloration of bromine water showed the presence of tannins.

2. Test for Saponins (Frothing test):

1 mL of the plant extract was diluted with distilled water to 10 mL and shaken in a graduated cylinder for 15 minutes. The formation of one centimeter layer of foam indicates the presence of saponins.

3. Tests for Flavonoids

i) Shinoda's Test: Pieces of magnesium ribbon and HCl concentrated were mixed with aqueous crude plant extarct after few minutes and pink color showed the presence of flavonoid.

ii) Sibata’s test: The methanol extract (1 mg) was diluted with about 1 mL methanol in a test tube. Then the solution was treated with Zn powder in presence of 5-6 drops of conc. HCl. Presence of orange yellow color indicated the presence of flavonoids.

4. Tests for Glycosides (Keller-Kiliani Test):

A solution of glacial acetic acid (4.0 ml) with 1 drop of 2.0 % FeCl₃ mixture was mixed with the 10 ml aqueous plant extract and 1 ml H₂SO₄ concentrated. A brown ring formed between the layers which showed the entity of cardiac steroidal glycosides.

5. Test for Terpenoids (Salkowaski’s test):

2.0 ml of chloroform was added with the 5 ml aqueous plant extract and evaporated on the water path and then boiled with 3 ml of H₂SO₄ concentrated. A grey color formed which showed the entity of terpenoids.

6. Test for Steroids:

2 ml of chloroform and concentrated H₂SO₄ were added with the 5 ml aqueous plant crude extract. In the lower chloroform layer red color appeared that indicated the presence of steroids.

7. Test for phenols:

A small amount of the ethanolic extract was taken with 1 mL of water in a test tube and 1 to 2 drops of Iron III chloride (FeCl₃) was added. A blue, green, red or purple color is a positive test.

8. Test for alkaloids

i) Dragendorff’s reagent: Two millilitres (2 mL) of acidic solution in the second test tube were neutralized with 10% ammonia solution. Dragendorff’s reagent was added and turbidity or precipitate was observed as indicative of presence of alkaloids.

ii) Mayer’s reagent (Bertrand’s reagent): Drops of Mayer’s reagent was added to a portion of the acidic solution in a test tube and observed for an opalescence or yellowish precipitate indicative of the presence of alkaloids.

iii) Wagner’s test: 2 mg of ethanolic extract was acidified with 1.5% v/v of hydrochloric acid and a few drops of Wagner’s reagent was added. A yellow or brown ppt. indicates the presence of alkaloids.

9. Protein test:

1 ml test solution and 1 ml distilled water was mixed in a test tube and 1 ml of conc. HNO₃ was added. Then it was cooled on a tap water. To this 2 ml 40 % NaOH was added, color developed indicate the presence of protein.

10. Carbohydrate test

i) Fehling’s test: 2 mg of ethanolic extract was shaken with 10 ml of water, filtered and the filtrate was concentrated. To this 1 ml mixture of equal parts of Fehling’s solution A and B were added and boiled for few minutes. Formation of red or brick red colored precipitate indicates the presence of reducing sugar

ii) Molisch test: 2 mg of ethanolic extract was shaken with 10ml of water, filtered and the filtrate was concentrated. To these 2 drops of freshly prepared 20% alcoholic solution of α- naphthol was added. 2 ml of conc. sulphuric acid was added so as to form a layer below the mixture. Red violet ring appeared, indicating the presence of carbohydrates which disappeared on the addition of excess of alkali.

11. Test for Coumarins:

Firstly, single pellets of KOH was taken and dissolved in 1 mL of ethanol. Then 1 mL of extract solution was added. The presence of precipitate indicates the presence of coumarins.

 

APPENDIX-II: PREPARATION OF REAGENTS

 

1. Dragendroff’s reagent

About 1.7 g of bismuth subnitrate was mixed with water (80 ml) and glacial acetic acid (20 ml). To this added potassium iodide solution (50% w/v, 100 ml). Shaken or stirred until dissolved. Solution kept indefinitely when stored in a dark bottle.

2. Mayer’s reagent

Mayer’s reagent is freshly prepared by dissolving a mixture of mercuric chloride (1.36 g) and potassium iodide (5.00 g) in water (100.0 ml).

3. Wagner’s reagent

To prepare Wagner's reagent dissolve 2 g of iodine and 6 g of KI in 100 ml of water.

4. Molisch reagent:

α-naphthol (C10H8OH) dissolved in methanol (CH3OH).

5. Fehling’s reagent

Fehling's A: 7 g CuSO4.5H2O dissolved in distilled water containing 2 drops of dilute sulfuric acid.

Fehling's B: 35 g of potassium tartrate and 12 g of NaOH in 100 ml of distilled water.

6. Ferric chloride solution:

Ferric chloride (1 g) was dissolved in distilled water (100 ml). to this aqueous solution, sodium carbonate is added little by little with stirring until the slight turbidity persisted. The mixture was filtered and the colorless filtrate was used as neutral ferric chloride.

 

APPENDIX-III:

 

Appendix 3.1: Absorption and % Inhibition for standard Ascorbic acid

                                                                                                Control = 0.6964

SN	Concentration of Ascorbic acid (𝛍𝐠/𝐦𝐋)	Mean absorption at 517 nm	% Inhibition
1	20	0.191	72.573
2	40	0.135	80.615
3	60	0.087	87.507
4	80	0.045	93.538
5	100	0.012	98.277

 

Appendix 3.2: Absorption of plant extracts at different concentrations and percentage

radical scavenging

Sample (MeOH Extract)	Absorption at Concentration of
	20 μg/mL	40 μg/mL	60 μg/mL	80 μg/mL	100 μg/mL
Aegle marmelos Fruits	0.2503	0.1623	0.1331	0.0950	0.0857
Aegle marmelos Leaves	0.4823	0.4301	0.4081	0.3864	0.3673
Cuscuta reflexa Stem	0.4688	0.4023	0.3856	0.3623	0.3458
Pityrogramma calomelanos Leaves	0.4632	0.3928	0.3592	0.3398	0.3212

 

 

 

Appendix 3.3: Absorption of plant extracts at different concentrations for TPC

Sample (MeOH Extract)	Absorption at Concentration of
	20 μg/mL	40 μg/mL	60 μg/mL	80 μg/mL	100 μg/mL
Aegle marmelos Fruits	0.0116	0.0145	0.0183	0.0205	0.0241
Aegle marmelos Leaves	0.0131	0.0175	0.0224	0.0278	0.0313
Cuscuta reflexa Stem	0.0311	0.0526	0.0751	0.0969	0.1192
Pityrogramma calomelanos Leaves	0.0103	0.0127	0.0139	0.0155	0.0179

 

Appendix 3.4: Absorption of plant extracts at different concentrations for TFC

Sample (MeOH Extract)	Absorption at Concentration of
	20 μg/mL	40 μg/mL	60 μg/mL	80 μg/mL	100 μg/mL
Aegle marmelos Fruits	0.0215	0.0231	0.0257	0.0276	0.0298
Aegle marmelos Leaves	0.0229	0.0263	0.0294	0.0332	0.0357
Cuscuta reflexa Stem	0.0371	0.0553	0.0725	0.0915	0.1121
Pityrogramma calomelanos Leaves	0.0635	0.1076	0.1517	0.1943	0.2401

APPENDIX - IV

Photographs of A. marmelos, C. reflexa & P. calomelanos

Some pictures taken during laboratory research work