Isolation of Quercetin from "Allium cepa" Estimation of Total Phenolic and Flavonoid Content in Common Medicinal Plantas "Ficus benghalensis, Elaeocarpus sphaericus, Ipomea carnea and Azeratum conyzoides" and their Antioxidant Activities

Contents

Acknowledgement

Abbreviations

Abstract

CHAPTER ONE
1.1. Introduction
1.2. Objectives of study
1.3 . Literature Survey

CHAPTER TWO
2. Experimental Section
2.1 Materials
2.2 Methods

CHAPTER THREE
3. Result and Discussion
3.1. Sohxlet Extraction of Outer Scale of Allium cepa
3.2. Test for Phenolic and Flavonoid Compounds
3.3. Liquid- liquid Extraction
3.4. Isolation of Quercetin
3.5. Characterization of Quercetin
3.6. Preparation of Extracts for the Estimation of Total Phenolic and Flavonoid Contents
3.7. Estimation of Total Phenolic Content (TPC)
3.8. Estimation of Total Flavonoid Content (TFC)
3.9. Determination of Antioxidant Activity
3.10. Correlation between DPPH Free Radical Scavenging Activity (IC50) and TPC

CHAPTER FOUR
4. Conclusion

References

Appendix

Acknowledgement

I would like to express my profound gratitude to my supervisor Associate Prof. Dr. Meena Rajbhandari, Research Center for Applied Science and Technology (RECAST), TU for her continuous guidance, suggestions and encouragement. Without her continuous supervision this research work would be incomplete. Thus all the credit of this work goes to her.

I gratefully acknowledge Prof. Dr. Mohan Bikram Gewali, Central Department of Chemistry, TU, for his advice and guidance.

I would like to express my sincere gratitude to Prof. Dr. Kedar Nath Ghimire, Head of Department, Central Department of Chemistry, TU for providing me this opportunity to conduct the research work.

I would like to express my sincere thanks to Prof. Dr. Ram Prasad Chaudhary, Executive Director of RECAST for providing laboratory facilities and support throughout the work and also for the identification of plant materials. Also special thanks go to Prof. Dr. S. M. Tuladhar for providing authentic quercetin and for his kind moral support.

My hearty thanks go to Assistant Prof. Mr. Santosh Khanal for his kind co-operation, suggestions and help to operate UV spectrophotometer.

I like to express my sincere thanks to all my respectable teachers and all staffs of Central Department of Chemistry.

I am thankful to Department of Plant Resources Thapathali for recording the IR spectra and Central Department of Biotechnology for recording UV spectra.

I am also thankful to NAST for partial financial support and Dr. Basant Bikram-Gyanu Shah Research Fund Award committee for providing me award.

My sincere thanks go to my senior colleagues Giri Raj Gnawali and Padam Prasad Acharya and to my friends Laxman Bhandari, Harish Chand Thakuri, Keshav Paudel, Hiramani Trital, Keshav Shrestha, Amit Dhungana, Prakash Bhandari, Krishna Chataut and Tulsi Bhandari for their encouraging support.

Finally but immensely, my most sincere obligation goes to my parents, family members and relatives for their support, encouragement and inspiration throughout the whole academic year.

Abbreviations

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Abstract

Quercetin, a flavonol was isolated from the ethyl acetate soluble portion of the methanol extract of outer scale of onion (Allium cepa Linnaeus) by repeated sephadex LH-20 column chromatography. The isolated quercetin was characterized by comparing melting point, Rf values, UV and IR spectra with authentic quercetin. Recently, attention has focused on phytochemicals as new sources of natural antioxidants. Therefore, the methanol extracts, 50% aqueous methanol extracts and 70% aqueous acetone extracts of different parts of four medicinal plants Ficus benghalensis, Elaeocarpus sphaericus, Ipomea carnea and Azeratum conyzoides from Kirtipur, Kathmandu, Nepal were screened for total phenolic content, total flavonoid content and free radical scavenging activity. Total phenolic content was measured spectrophotometrically by using Folin-Ciocalteu reagent and total flavonoid content by using aluminum chloride colorimetric method. Gallic acid was used as the standard for the calibration of phenolics and quercetin for flavonoids. Free radical scavenging activity was evaluated using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. All investigated medicinal plant extracts contain high amount of phenolic but the highest amount of phenolic was detected in 70% aqueous acetone extract of Elaeocarpus sphericus (298.769±9.034 mg GAE/g) and lowest amount in 50% aqueous methanol extract of Ficus benghalensis (6.730±0.737 mg GAE/g). The highest amount of flavonoid was found in methanol extract (78.188±2.719 mg quercetin/g) of Ficus benghalens and the lowest amount was detected in 50% aqueous methanol extract (2.125±0.250 mg quercetin/g) of Ficus benghalenss. DPPH assay was carried out only for 70% acetone extracts of medicinal plants, IC50 value was calculated and correlated with total phenolic contents. A strong linear correlation between total phenolic content and antioxidant activity was found (correlation coefficient, R2 = 0.931), indicating that the major antioxidant compounds are phenolic.

CHAPTER ONE

1.1. Introduction

1.1.1. Introduction and Rationale of study

Natural products are chemical compounds or substances produced by living organisms found in nature that usually has a pharmacological or biological activity.1 They have restricted distribution being found mostly in plants and micro-organisms. However, animals have also been a source of some interesting compounds.2

Plants provide a large bank of rich, complex and highly varied structures which are unlikely to be synthesized in laboratories. Further, these potent compounds are synthesized in plants partly as a response to ecological and physiological pressures such as pathogen and insect attack, UV radiation and wounding.3

Even today, the number of plants that have been extensively studied is relatively very few and the vast majorities have not been studied at all. Major classes of natural products found in plants include terpenoids, phytosterols, alkaloids, natural phenols and polyphenols.4

The term phenolic compound embraces a wide range of plant substances which posses in common an aromatic ring bearing one or more hydroxyl substituents.5 It encompasses approximately 8000 naturally occurring compounds, all of which posses one common structural feature, a phenol (an aromatic ring bearing at least one hydroxyl substituents). Thus, plant phenolics are secondary metabolites with diverse chemical nature and include simple phenols, phenolic acids, flavonoids, tannins, coumarins, lignans, xanthones and stilbenes. Current classification divides the broad category of phenolics into polyphenols and simple phenols, based solely on the number of phenol subunits present. Polyphenols possessing at least two phenol subunits include the flavonoids, and those compounds possessing three or more phenol subunits are referred to as the tannins (hydrolysable and non hydrolysable).6

Phenolic acids are one of the main phenolic classes within the Plant Kingdom and occur in the form of esters, glycosides or amides, but rarely in free form. Phenolic acids have two parent structures: hydroxycinnamic and hydroxybenzoic acid. Hydroxycinnamic acid derivatives include ferulic, caffeic, p -coumaric and sinapic acids, while hydroxybenzoic acid derivatives consist of gallic, vanillic, syringic and protocatechuic acids.7

Flavonoids form the broad major group and are characterized by the presence of a C6-C3-C6 carbon skeleton consisting of the two aromatic rings linked by an aliphatic three carbon chain. This skeleton is made up of two biogenetically distinct fragments: the C6-C3 fragment forming the B-ring and the C6 fragment forming the A ring.8

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Figure1. Flavonoid Structure

Flavonoids are a group of about 4,000 naturally occurring polyphenolic compounds found universally in foods of plant origin. These are the compounds that are ubiquitous in nature and are categorized according to chemical structure into flavonols (quercetin, kaempferol, myrcetin, etc.), flavones (apigenin, luteolin, etc.), flavanones (hesperidin, hesperitin etc.), isoflavones (daidzein, genistein etc.), and anthocyanidine (pelargonidin, cyanidin, etc). Flavonoids are isolated from a wide variety of plants, and are responsible for much of the coloring found in vascular plants. A single plant may contain dozens of different flavonoids.9

Phenolics are molecules that act as antioxidants to prevent heart disease, reduce inflammation, lower the incidence of cancers and diabetes, as well as reduce rates of mutagenesis in human cells. Reactive oxygen species such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), and free radicals such as the hydroxyl radical (˙OH) and superoxide anion (O2˙ˉ) are produced as the normal products of cellular metabolism. Rapid production of free radicals can lead to oxidative damage to biomolecules and may cause disorders, such as cancer, diabetes, inflammatory disease, asthma, cardiovascular diseases, neurodegenerative diseases, and premature aging.10

Phenolics behave as antioxidants, due to the reactivity of the phenol moiety (hydroxyl substituent on the aromatic ring). Although there are several mechanisms, the predominant mode of antioxidant activity is believed to be radical scavenging via hydrogen atom donation. Other established antioxidant, radical quenching mechanisms are through electron donation and singlet oxygen quenching. Substituents on the aromatic ring affect the stabilization and therefore affect the radical-quenching ability of the phenolics. Different phenolics therefore have different antioxidant activity.11

Many medicinal plants contain large amounts of antioxidants, such as phenolics, terpenoids, vitamin C, Vitamin E, selenium, β-carotene, lycopene, lutein, and other carotenoids, which play important roles in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides.12

Data from various studies indicate that medicinal plants contain a wide variety of natural antioxidants, such as phenolics, which posses more potent antioxidant activity than common dietary plants. Compounds responsible for such antioxidant activity should be isolated and used for prevention and treatment of free redical related disorders.13

Recently much attention has paid to find naturally occurring antioxidants to use in food or pharmaceutical to replace synthetic antioxidants.10 However, due to the high price and limited source of natural antioxidants they are not widely used and they are replaced by synthetic antioxidants such as butylated hydroxyl toluene (BHT) and butylated hydroxy anisole (BHA) which have hemorrhaging and carcinogenic effects. Therefore, synthetic antioxidants need to be replaced by natural antioxidants and it is of great importance to find new sources of safe and inexpensive natural antioxidants to use them in food and pharmaceutical preparations.14

Nepal is natural store house of medicinal plants. Approximately, 70 to 80% of the population of Nepal depends on traditional medicines. Indigenous people residing in different belts depend on local plant and plant products to meet their daily requirements for food, fodder, medicines, etc. Local herbs and other plant resources found in rural area are the principle source of medicine for treating disease since time immemorial. Despite widespread use of wild plants as medicines in Nepal, little is known about the antioxidant potential and chemical composition of these plants.15

The aim of the present study is to evaluate and compare 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and the phenolic and flavonoid content of traditional medicinal species of Nepal. In addition, the study sought to determine the relationship between the DPPH radical scavenging activity and total phenolic content of some medicinal plant extract that might be promising sources of natural antioxidants.

1.2. Objectives of study

The objectives of the present study are:

1. Isolation and identification of quercetin from Allium Cepa which is used as a standard for the construction of calibration curve for the determination of Total Flavonoid Content (TFC) in different extracts.

2. Determination of Total Phenolic Content (TFC) and Total Flavonoid Content (TFC) of methanolic, 50% methanolic and 70% acetone extracts of Ficus benghalensis, Elaeocarpus sphaericus, Ipomea carnea and Ageratum conyzoides.

3. Determination of antioxidant activity of selected extrats using DPPH free radical scavenging assay.

4. Correlation between Total Phenolic Content and their antioxidant activity.

1.3. Literature Survey

1.3.1. Allium cepa Linnaeus

The onion (Allium cepa), which belongs to alliaceae family, also known as the bulb onion or common onion, is the most widely cultivated species of the genus Allium. In Nepali, it is called as pyaaj. It was first officially described by Carlous Linnaeus in his 1753 work Species Planturam. A. cepa is one of the edible species of a large genus Allium, consisting of more than 700 species. It is now cultivated throughout the world. Although the origin remains debatable, the middle Asiatic countries in the region of Iran and Pakistan are considered the primary centre of origin of onion. The near east Asiatic and Mediterranean regions are considered to be the secondary centres of origin. Although, temperate in origin, it has been bred to adapt to the tropics. It is distributed throughout Nepal to about 3000 m. They are not found in New Zealand and Australia.16

The onion plant (A. cepa) is unknown in the wild but has been grown and selectively bred in cultivation for at least 7,000 years. It is a biennial plant but is usually grown as an annual. Modern varieties typically grow to a height of 15 to 45 cm (6 to 18 in). The leaves are bluish-green and grow alternately in a flattened, fan-shaped swathe. They are fleshy, hollow and cylindrical, with one flattened side. They are at their broadest about a quarter of the way up beyond which they taper towards a blunt tip. The base of each leaf is a flattened, usually white sheath that grows out of a basal disc. From the underside of the disc, a bundle of fibrous roots extends for a short way into the soil. As the onion matures, food reserves begin to accumulate in the leaf bases and the bulb of the onion swells. In the autumn the leaves die back and the outer scales of the bulb become dry and brittle, and this is the time at which the crop is normally harvested. If left in the soil over winter, the growing point in the middle of the bulb begins to develop in the spring. New leaves appear and a long, stout, hollow stem expands, topped by a bract protecting a developing inflorescence. The flower-head takes the form of a globular umbel of white flowers with parts in sixes. The seeds are glossy black and triangular in cross section.17

Onion bulb contains moisture, protein, fat, carbohydrates, fiber, minerals (calcium, phosphorus, iron), thiamine, riboflavin, niacin, and vitamin C. Onion root contains caffeic acid, ferulic acid, gibberellin A-4, para-hydroxybenzoic acid, tuliposide A, tuliposide B. Leaf of onion contains ascorbic acid, caffeic acid, citric acid, ferulic acid, fructose, glucose, malic acid, methanol, oxalic acid, para-coumaric acid, para-hydroxybenzoic acid, propionaldehyde, protocatechuic acid, raffinose, sinapic acid, succinic acid, sucrose. Presence of quercetin, sterol glycosides, gibberllin is also reported. Onion stalks contains moisture, protein, fat, fibre, carbohrdrates, and minerals (calcium, phosphorus, iron), riboflavin, niacin, and vitamin C. Dried onion skins are the best natural source of quercetin. It contains stigmasterol, cholesterol, β-sitosterol, kaempferol, quercetin, and quercetin-3-glucoside. The phenolic acids reported to be present are p -hydroxy-benzoic acid, protocatechuic acid and vanillic acid. There are seven major flavonoid compounds in onions. They are quercetin aglycone, i.e. with no sugar molecule attached, quercetin monoglucoside, quercetin diglucoside, isorhamnetin (a methyl ether of quercetin), isorhamnetin monoglucoside, rutin and kaempferol. Quercetin aglycone, quercetin-3,4'- O -diglucoside and quercetin-4'- O -glucoside are three predominant forms of quercetin in onion.18-19

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Figure 3. Kaempferol

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Figure 4. Quercetin-4’-O-glucoside

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Figure 5. Quercetin-3,4’-O-diglucoside

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Figure 6. Quercetin-3-O-glucoside

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Figure 7. Isorhamnetin (Quercetin-3’-methyl ether)

Figure 8. Isorhamnetin monoglucoside (Isorhamnetin -3-O-glucoside)

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Figure 9. Rutin (Quercetin-3-rutinoside)

A. cepa has high medicinal values. The fresh bulb is taken orally for the treatment of tuberculosis, gastrointestinal infections and menstrual and uterine pain. The dried bulb is used orally as a contraceptive, used externally as a liniment and as an emmenagogue in the form of Unani medicine. Hot water extract of the onion bulb is taken orally to treat hypertension, inflammations, dropsy, urinary problems, renal and biliary calculi, bronchitis, scurvy, body heat, epilepsy, hysterical fits, nosebleed, jaundice, unclear vision, spleen enlargement, rheumatic pain, strangury and as an anti-diabetic. It is used externally for the treatment of wounds, ulcers, bruices, sores, skin diseases, irritations and eruptions, erysipelas and burns. Fresh bulb juice is used externally as an anti- inflammatory agent on insect bites and for bronchitis. Fresh bulb essential oil, administered by inhalation, is used for the treatment of colds.20

1.3.2. Ficus benghalensis Linnaeus

Ficus bengalensis, which belongs to moraceae family, is commonly known as Banyan tree or Vata or Vada tree in Ayurveda. There are more than 8000 species and 2000 varieties of Ficus species, most of which are native to the old world tropics. It is endemic to Bangladesh, India and Sri Lanka. It is distributed east to west of Nepal, in tropical to sub- tropical regions and known as Bar.21

F. benghalensis is a very large tree upto 30 m in height with widely spreading branches bearing many aerial roots functioning as prop roots, bark greenish white, leaves simple, alternate, often in clusters at ends of branches, stipulate,10 to 20 cm long and 5 to 12.5 cm broad, broadly elliptic to ovate, entire, strongly 3 to 7 ribbed from the base; the fruit recacles are axillary, sessile, in pairs, globose, brick red when ripe, enclosing male, female and gall flowers; fruits small, crustaceous achenes, enclosed in the common fleshy receptacles.22

Phytochemical investigation of F. bengalensis led to the exploration of a wide variety of constituents. Leaves contain flavonols (quercetin-3-galactoside and rutin) and terpenoids (friedelin, 3-friedelanol, β-sitosterol , 20-traxasten-3-ol, lupeol or betulinic acid and β-amyrin). Stem bark contains bengalenosides that is, glycosides of flavonoids, 5,7-dimethyl ether of leucopelargonidin-3-O-α-L- rhamnoside and 5,3-dimethyl ether of leucocyanidin-3-O-β-D-galactosyl cellobioside, and 5, 7, 3-trimethoxyleucodelphinidin-3-O-α-L-rhamnoside. The bark also contains the esters like keto-n-cosanyl stearate, hydroxypentacosanyl palmitate and phenyl tetradecanyl oleiate. The tiglic acid ester of ψ-traxasterol has been also isolated from the heart wood of F. benghalensis. 23

According to Ayurveda, Ficus benghalensis is useful in treatment of biliousness, ulcers, erysipelas, vomiting, vaginal complains, fever, inflammations and leprosy. According to Unani system of medicine, its latex is aphrodisiac, tonic, vulnerary, maturant, lessens inflammations; useful in piles, nose-diseases, gonorrhea, etc. The aerial root is styptic, useful in syphilis, biliousness, dysentery, inflammation of liver, etc. Milky juice is used for pains, rheumatism, lumbago and bruises. Seeds are cooling and tonic in nature. Its leaf buds are astringent, leaves infusion is given in diarrhea and dysentery, poultice of hot leaves is applied on abscesses. The bark is astringent and tonic and used in diabetes and leucorrhoea, lumbago, sores, ulcers pains and bruises.24

1.3.3. Elaeocarpus sphaericus Gaertn. K. Schum

Elaeocarpus sphaericus (syn. Elaeocarpus ganitrus Roxb.), which belongs to Elaeocarpaceae family, is locally known as Rudraksha and commonly known as Bead tree.25 The trees are found on the areas starting from Manila, Philippines and fleeting through Myanmar to whole North-East India, Bangladesh, Nepal and Bhutan.26 In Nepal, it is distributed between 700-1700 m from east to central part.27

E. sphaericus is a large, evergreen, drought-tolerant, perennial broad- leaved tree with a large spreading crown. The tree attains a height of about 50-200 feet. The stem is cylindrical with a dirty white and coarse textured bark, leaves are 10-15 cm long, simple, alternate, oblong-lanceolate, acute or acuminate, obscurely and irregularly crenate- serrate or sub entire. They are shining green on the upper side with dull-fibrous on dorsal side. As the tree matures, the leafy crown obtains a pyramidal shape in nature and the roots rise up narrowly near the trunk and radiate out along the ground- surface. Flowering occurs in the month of April- May. The flowers are small, white with fringed petals (about 8 mm long), half way down and ciliolate. Fruiting is delayed, taking about 7-8 years to fruit. The fruits are borne in the month of June and gradually ripen over a period of time extending from August to October.28-29

E. sphaericus has been reported to possess alkaloids, glycosides, steroids, flavonoids (quercetin), tannins (gallic and linoleic acids), carbohydrates, proteins and ash. Alkaloids (rudrakine, (±)-elaeocarpine and (±)-iso-elaeocarpine, phenolics (quercetin, gallic acid and ellagic acid) are reported from the leaves of E. sphaericus.30-31

The parts of E. sphaericus have been extensively used in the treatment of stress, anxiety, depression, palpitation, nerve pain, epilepsy, migraine, asthma, hypertention, arthritis and liver diseases. In Ayurveda, the fruits are used for psycho-somatic diseases, epilepsy, asthma, hypertention, arthritis and liver diseases.32-33

1.3.4. Ipomea carnea Jacq

Ipomea carnea, family convulvulaceae, is commonly known as “Morning glory” in English. It is widely distributed throughout the American tropics, Argentina, Brazil and Bolivia. This plant species has also been reported from India, West-Pakistan and Sri Lanka. It is also distributed in Nepal from east to west and also known as Beshram and Behaya.34

I. carnea which is glory species grows to a height of 6 m on terrestrial land, but shorter in the aquatic habitats. The stem is thick and develops into a solid trunk over several years with many branches from base. The stem is erect, woody, hairy, more or less cylindrical in shape and greenish in colour, and bearing alternate leaves. It attains 1.25-2.75 m long and 0.5-0.8 cm diameter. The leaf is simple, alternate, exstipulate and petiolate. Petiole is cylindrical, attains 4-7.5 cm length and 2.5-3.0 mm diameter. The leaf blade is cordate with symmetric base, measures 13- 23 cm in length and 5.5-9.5 cm in width, with entire margin and reticulate pinnate venation, slightly hairy on both surfaces, the upper surface is dull green and the lower one is paler. Flowers of this plant are axial, solitary or arranged in monochasium scropioid cymose inflorescence.35

It is found that the plant possess various bioactive compounds such as glycosides, alkaloids, reducing sugars, flavones, fatty acid, esters, alcohols, flavonoids and tannins. The leaves of this plant showed the presence of thirteen compounds which include hexadecanoic acid, steric acid, 1, 2 diethyl phthalate, n-octadecanol, octacosane, hexatriacontane, tetraacontane, 3-diethylamino-1- propanol. The leaves, stems and flowers of I. carnea contain significant amount of phenols and flavonoids. The flowers contain the maximum and the stem contains the minimum amount of phenolic compounds. The leaves of I. carnea contain 1-3 flavonol glycosides and ergine (D-Lysergic acid amide). Polyhydroxylated alkaloids were isolated from the leaves, flowers and seeds. Leaf extract also resulted in the isolation of swainsonine, 2-epilentiginosine, calystegines B (1), B (2), B (3) and C (1) and N- methyl-trans-4-hydroxy-l-proline and β-sitosterol.36-37

The ash and milky juice of I. carnea is used for the treatment of skin diseases and leucoderma respectively. It is also used for the treatment of polluted tanks. The plant had immense potential in cardiovascular activity, immunemodulatory, anti-diabetic, antioxidant, anti-inflammatory, antimicrobial activities and wound healing properties.38

1.3.5. Ageratum conyzoides Linnaeus

Ageratum conyzoides (family Asteraceae) is an annual herb with a long history of traditional medicinal use in the tropical and sub-tropical region of the world, commonly known as Billy goat weeds. In Nepal, it is called Ganmanghaans or Ganaauneghaans or Bokeghaans or Raawanne and distributed between 200-2000 m from east to west.39

It is an annual branching herb which grows to approximately 1 m in height. The stems and leaves are covered with fine white hairs, the leaves are ovate and up to 7.5 cm long. The flowers are purple to white, less than 6 mm diameter and arranged in close terminal inflorescences. The fruits are achene and are easily dispersed while the seeds are photoblastic and often lost within 12 months.40

The chemical constituents of A. conyzoides include flavonoids, alkaloids, cumarins, essential oils, and tannins. The most common component of the essential oil of A. conyzoides is 7-methoxy-2,2-dimethylchromene. Other related compounds obtained from the oil include encecalin, 6-vinyl-7-methoxy-2,2-dimethylchromene, dihydroencecalin, dihydrodemethoxyencecalin, demethoxyencecalin, demethylencecalin and 2-(1-oxo-2-methylpropyl.)-2- methyl-6,7-dimethoxychromene. A. conyzoides is rich in polyoxygenated flavonoids and 21 of them have been reported in the whole plant. Among them there are 14 polymethoxylated flavones. The polyhydroxyflavones include quercetin, kaempferol and their glycosides, that are quercetin-3- rhamnopiranoside, kaempferol -3- rhamnopiranoside and kaempferol 3,7- diglucopiranoside. The two major common sterols sitosterol and stigmasterol together with minor sterol were isolated together with the triterpene friedelin. Other common substances are sesamine, fumaric acid, caffeic acid, phytol, and long chain hydrocarbons.41-42

A. conyzoides has been known since ancient times for its curative properties and has been utilized for the treatment of various ailments, such as burns and wounds, headaches pneumonia, analgesic, inflammation, asthma, spasmodic and haemostatic effects, stomach ailments, gynaecological diseases, leprosy and other skin diseases.43

CHAPTER TWO

2. Experimental Section

2.1 Materials

2.1.1. Plant Materials

Plant materials were collected from Kirtipur, Kathmandu, Nepal on February 2013. They were authenticated by Prof. R. P. Chaudhary, Executive Director of Research Center for Applied Science and Technology (RECAST), Tribhuvan University, Kirtipur, Kathmandu, Nepal. Name of plants, local names and collected parts of plants are shown in table 1.

Table 1. Name of plants, local names and collected parts of plants

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2.1.2. Instruments

Rotavapour Buchi Rotavapour R-200

Buchi Heating Bath B-49

Buchi Vacuum controller V 800

Buchi Vacuum Pump- V500

Buchi Labourtechnic AG, Flawil, Swiss

UV Lamp HP-UVIS, SARSREDT-GRUPPE, DESAGA GmbH, Wiesloch, Germany

Ultrasound Bath Barnstead International ISO 9001

UV Spectrophotometer WPA Linton Cambridge UK, Type S104, No. 385

IR Spectrophotometer IRPrestige-21, FTIR Spectrometer (SHIMADZU)

2.1.3. Solvents, Chemicals and Chromatographic Materials

Solvents like methanol, ethyl acetate, hexane, toluene (Qualigens Fine Chemicals, Mumbai), acetone (Thermo Fisher Scientific India Pvt. Ltd., Mumbai) and chloroform, n-butanol, acetic acid, formic acid (Merck Limited, Mumbai) were of analytical grade and purchased from local vender. Distilled and double distilled water was prepared in the lab by self assembled distillation plant. Similarly, chemicals like aluminium chloride (Sd. Fine Chemical Limited, Mumbai) and sodium hydroxide, sodium nitrite, sodium carbonate (Qualigens Fine Chemicals, Mumbai) were also of analytical grade and purchased from local vender. Standard Gallic acid was purchased from Merck Limited, Germany. Authentic quercetin was provided by Prof. Dr. Sarbajna Man Tuladhar, RECAST, T.U. DPPH was purchased from Sigma Chemical Company, USA. The chromatographic materials such as TLC foils (precoated) Silica gel 60 GF254, 0.2 mm, and TLC foils (precoated) Cellulose F, 0.2mm, were purchased from Merck, Darmstadt, Germany. Sephadex LH-20 was purchased from Pharmacia Biotech, Uppasala, Sweden.

2.2 Methods

2.2.1. Extraction of Allium cepa

Powdered 200 g of onion scale was extracted in a soxhlet extractor with 1 liter methanol for twelve hours. The methanol extract was concentrated under reduced pressure in a rotavapour to get viscous liquid.

2.2.2. Test for Phenolic and Flavonoid Compounds

The presence of phenolic and flavonoid compounds in methanolic extract were identified by reactions with specific reagents.44

2.2.2.1. Test for Phenolic Compounds

The methanol extract (1 mg) was diluted with about 1 ml water or methanol. This solution was treated with FeCl3 solution (0.5 ml).

2.2.2.2. Test for Flavonoid Compounds

2.2.2.2.1. Shinoda’s Test

The methanol extract (1 mg) was diluted with about 1 ml methanol in a test tube. Then, the solution was treated with Mg powder in presence of 5-6 drops of conc. HCl.

2.2.2.2.2. Shibata’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.

2.2.3. Liquid- liquid Extraction

The methanol extract (51.4 g) was suspended in 100 ml distilled water and then extracted each with 100 ml of hexane eight times in a separatory funnel. The aqueous layer was again extracted ten times each with 100 ml ethyl acetate. The lower ethyl acetate layer was collected and evaporated in a rotavapour under reduced pressure.

2.2.4. Thin Layer Chromatography

The ethyl acetate extract was examined by TLC in different solvents and the spots were visualized in day light as well as under UV light, both at 254 and 366 nm. The chromatogram was furthered visualized by spraying with ammonia. The solvent systems used for the development of the chromatogram are:

Ethyl acetate - Methanol - Water 100 : 10 : 5

n-Butanol - Acetic acid - Water 4 : 1 : 5

Toluene - Ethyl acetate - Formic acid 10 : 8 : 1

Chloroform - Acetic acid - Water 10 : 9 : 1

Methanol - Water - Formic acid 55 : 42 : 3

2.2.5. Isolation of Quercetin

2.2.5.1. Sephadex LH- 20 Column Chromatography of Ethyl Acetate Extract of Allium cepa

The ethyl acetate extract (6 g) was chromatographed on a sephadex LH- 20 column (32 cm x 2.1 cm I.D.) filled with 97g sephadex, equilibrated and eluted with methanol. Fractions were collected consisting of 10-12 ml in test tubes. Each fraction was monitored by TLC (silica gel GF254 ) using solvent system ethyl acetate - methanol - water (100 : 10 : 5). The sub-fractions were pooled into four major fractions on the basis of TLC character- fraction F1, F2, F3, and F4.

2.2.5.2. Sephadex LH-20 Column Chromatography of Fraction F4

The fraction F4 (2.11 g) was chromatographed on a sephadex LH- 20 column (32 cm x 2.1 cm I.D.) filled with 97 g sephadex and equilibrated and eluted with 75% aqueous methanol. Fractions were collected consisting of 10-12 ml in test tubes. Each fraction was monitored by TLC (silica gel GF254 ) using solvent system ethyl acetate - methanol - water (100:10:5). The sub-fractions were pooled into five major fractions on the basis of TLC character- fraction F4A, F4B, F4C, F4D and F4E.

2.2.5.3. Flow Chart of Column Chromatography

Abbildung in dieser Leseprobe nicht enthalten

2.2.6. Measurement of Spectra

UV spectrum was measured in methanol in UV-VIS Recording Spectrophotometer. IR spectrum was measured against KBr pellet.

2.2.7. Preparation of Extracts for the Estimation of Total Phenolic and Flavonoid Content

2.2.7.1. Preparation of Methanol Extracts (Soxhlet Extraction)

Each 20 g of the dried and powdered plant material was extracted with methanol (250 ml) in a soxhlet extraction apparatus for 12 hours. The solvent was evaporated in rotavapour under reduced pressure to get the respective crude extract.

2.2.7.2. Preparation of 50% aqueous Methanol Extracts (Reflux method)

The plant residue after extraction with methanol was dried and subjected to extraction with 250 ml of 50% aqueous methanol under reflux for four hours. The solvent was evaporated in a rotavapour under reduced pressure to get the 50% methanolic extract.

2.2.7.3. Preparation of 70% aqueous Acetone Extracts (Percolation)

Each 20 g of dried and powdered sample was percolated for 24 hours at room temperature and subjected to ultrasound – assisted extraction for another 30 min. The extract was filtered and the solvent was evaporated in a rotavapour under reduced pressure to get the 70% acetone extract.

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