CHAPTER 3 PHYTOCHEMICAL ANALYSIS

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1 CHAPTER 3 PHYTOCHEMICAL ANALYSIS

2 CHAPTER 3.1: INTRODUCTION Since ages, the basic requirements of humans like food, shelter, clothing and medicine is dependent on plant diversity. Herbal medicine is the oldest form of healthcare known to human society (Barnes et al., 2007). According to literatures, about 25% of all modern medicines are directly or indirectly derived from higher plants (Duke and Martinez, 1994; WHO, 2005). Plants produce a diverse array of bioactive molecules, making them rich sources of diverse type of medicine. Most of the bioactive molecules have probable evolved as chemical defence against predation or infection (Sofowora, 1986). The term herbal drugs denotes plants or plant parts that have been converted into phytopharmaceuticals by means of simple processes involving harvesting, drying, and storage (EMEA, 1998). Each and every part contains such important phytochemicals. So, it can be said there isn t anything like waste in plant world. This study aims to draw attention to the possible waste-to-wealth utilization of fruit and by-products with the added advantage of providing novel sources of nutraceuticals, phytochemicals, pharmaceuticals and reducing environmental insults (Oikeh et al., 2013). The world health organization has shortlisted certain parameters for standardization and quality control of herbal crude drugs. According to WHO (1992, 1996a and b), standardization and quality control of herbals is the process involved in the physicochemical evaluation of crude drug covering aspects, such as selection and handling of crude material, safety, efficacy and stability assessment of finished product, documentation of safety and risk based on experience, provision of product information to consumer and product promotion. Attention is normally paid to such quality indices such as: 1. Macro and microscopic examination: For Identification of right variety and search of adulterants. 2. Foreign organic matter: This involves removal of matter other than source plant to get the drug in pure form. 3. Ash values: These are criteria to judge the identity and purity of crude drug Total ash, sulphated ash, water soluble ash and acid insoluble ash etc. 42

3 4. Moisture content: Checking moisture content helps reduce errors in the estimation of the actual weight of drug material. Low moisture suggests better stability against degradation of product. 5. Extractive values: These are indicative weights of the extractable chemical constituents of crude drug under different solvents environment. 6. Crude fibre: This helps to determine the woody material component, and it is a criterion for judging purity. 7. Qualitative chemical evaluation: This covers identification and characterization of crude drug with respect to phytochemical constituent. It employs different analytical technique to detect and isolate the active constituents. Phytochemical screening techniques involve botanical identification, extraction with suitable solvents, purification, and characterization of the active constituents of pharmaceutical importance. 8. Chromatographic examination: Include identification of crude drug based on the use of major chemical constituents as markers. 9. Quantitative chemical evaluation: To estimate the amount of the major classes of constituents. 10. Toxicological studies: This helps to determine the pesticide residues, potentially toxic elements, safety studies in animals like LD50 and Microbial assay to establish the absence or presence of potentially harmful microorganisms. Present work was focused on evaluation Ash values, Extractive values, Qualitative chemical evaluation, Chromatographic examination and Quantitative chemical evaluation from fruit and vegetable by-products Ash Value Ash has ancient importance in Indian medicine system. The residue comprising the inorganic mixtures, of metallic salts and silica obtained after controlled incineration of plant drugs is taken to be the ash content (Doshi, 2005). Ash of different plants with Louha Bhasma was used to cure and management of different diseases during vedic period (Pandit et al., 1999). 43

4 Ash contains inorganic radicals like phosphates, carbonates and silicates of sodium, potassium, magnesium, calcium etc. This indicates that ash content of a sample gives an idea about the mineral elements present in the sample (Ansari, 2006; Edeogu et al., 2007). The ash value is a criterion used to judge the purity of a crude drug. Its study gives an idea about the quality and the purity of the drug. Amount and composition of the ash remaining after combustion of plant mineral, varies considerably according to the part of the plant, age of the plant etc. The constituent of ash also varies with time and from organ to organ. Ash usually represents the inorganic part of the plant. It contains inorganic material of the plant because ashing destroys all the organic material present in the sample (Kar et al., 1999). To determine ash content, the plant material is burnt and the residual ash is measured as total, water soluble and acid-insoluble ash. Total ash is the measure of the total amount of material left after burning and includes ash derived from the part of the plant itself. Water soluble ash determines the water soluble minerals. Sometimes, inorganic variables like calcium oxalate, silicate, etc are also present in the ash. Such, variables are removed by treating with acid as these are insoluble in hydrochloric acid and then acid insoluble ash value is determined (AOAC, 2005; Parhi and Mohapatra, 2012) Extractive Value The powdered plant material (crude drug) contains active chemical constituents which are responsible for its biological activity. Extractive value determines the approximate measure of their chemical constituents in a given amount of plant material. These phytochemicals may be soluble in different polar, semi-polar and non-polar solvents. Based upon polarity of required metabolites to be extracted, the solvent selection is depends on the eluotropic series where all the organic solvents are arranged accordingly their polarity from non-polar to highly polar solvents. The polar solvent will extract polar compounds and non-polar solvents will extract non-polar compounds from the sample (Daniel, 1991). The extract yield contains different phytoconstituents. Total soluble quantity of the drug in any particular solvent or mixture is referred to, as its extractive value. Their composition is depending upon the 44

5 nature of plant powder and the used solvent (Tatiya et al., 2012). Generally polar compounds like alkaloids, flavanoids, saponins, tannins etc. are considered as and easily isolated in polar solvents like water, methanol, ethanol, chloroform etc, while non-polar compounds like terpenoids, steroids and sterols are extracted in non-polar solvents like petroleum ether, toluene, hexane, benzene etc (Harborne, 1998). Extractive values of crude drugs are useful for their evaluation, especially when the constituents of a drug cannot be readily estimated by other means. Methanol is known as a super solvent as it is capable of extracting many compounds. It is recommended for extraction of lipids, glycosides, aglycones, phenols and most of the other compounds (Okogun, 2000) Phytochemical Evaluation Phytochemicals (Greek word phyto means plant) are refer to biologically active, naturally formed chemical compounds in plant. Thousands of different phytochemicals have been found from vegetables, fruits, beans, whole grains, nuts and seeds. These chemicals are synthesized in each and every part of plant. These phyto constituents work with nutrients and fibres to form an integrated part of defence system against various diseases and stress conditions. Phytochemicals are basically divided into two groups, i.e. primary and secondary metabolites according to their functions in plant metabolism. Primary metabolites comprise of carbohydrates, amino acids, proteins etc., while secondary constituents consist of alkaloids, phenolic compounds, flavonoids, tannins, glycosides, terpenoids, saponins, and so on (Edeoga et al., 2005; Kumar et al., 2009; Khandare, 2012) Primary Metabolites Plant metabolism is divided in primary and secondary. The substances which are common to living things and essential to cells maintenance are originated from the primary metabolism. Primary metabolites viz, protein, lipid, carbohydrates and so forth are of prime importance and essentially required for metabolic processes such as photosynthesis, respiration and nutrient assimilation. Thus they are directly involved in growth and development of plants. In short they are the building blocks of organisms. Plant synthesizes primary metabolites for the normal growth and development of itself. These compounds are polymeric and usually large molecules. They are universally present in nature and are very essential for all life forms. Many 45

6 primary metabolites lie in their impact as precursors or pharmacologically active metabolites in pharmaceutical compounds and utilized as food by man. They are also used as raw material and food additives (Samuelsson, 1992; Samria and Sarin, 2014) Secondary metabolites The substances originated from several biosynthetic pathways and that are restricted to determined groups of organisms are results of the secondary metabolism. All plants produce an amazing diversity of secondary metabolites. The secondary metabolites such as alkaloids, glycosides, flavonoids, volatile oils etc are biosynthetically derived from primary metabolites as protective mechanisms against predation by many microorganisms, insects and herbivores or any pathogenic attack on plants. They are non-nutritive plant chemicals possessing varying degrees of disease preventive properties. Apart from protection they also help to give plants their characteristic colour, flavour, smell and texture. They are not essentially required for the nourishment of plant but provide extra health benefits against pathogens (Lutterodt et al., 1999; Renu, 2005; Mathew et al., 2012; Chede, 2013). They are sometimes considered as waste or secretary products of plant metabolism and are of pharmaceutical importance. Phytochemical possesses many health protective properties in diverse ways. Secondary metabolites such as polyphenols, carotenoids etc are act as strong antioxidants and protect cells against free radical damage (Devasagayam et al., 2004; Mathew et al., 2012). Alkaloids, phenolic compounds, flavonoids and terpanoids are some important secondary metabolites (a) Phenolic compounds The term phenolic compounds constitute widely distributed and one of the main groups of secondary metabolites. They consist of a wide range of plant substances which are characterized by at least one aromatic ring (C6) bearing one or more hydroxyl groups. They are responsible for the major organoleptic characteristics of plant-derived foods and beverages, particularly colour and taste properties and they also contribute to the nutritional qualities of fruits and vegetables. Plant phenolics are a chemically heterogenous group: Some are soluble only in organic solvents; some are water-soluble carboxylic acids and glycosides. Another group of phenolics are insoluble polymers (Scalbert and Williamson 2000; Tapas et al., 2008; Chirinos et al., 2009). 46

7 Phenolic compounds are mainly synthesized from two metabolic pathways: i) The shikimic acid pathway where, phenylpropanoids are mainly formed and ii) The acetic acid pathway, in which simple phenols are produced (Sánchez-Moreno, 2002). Most plants phenolic compounds are synthesized through the phenylpropanoid pathway (Hollman, 2001). The combination of both pathways leads to the formation of flavonoids, the most plentiful group of phenolic compounds in nature. Plant phenolic compounds are classified as simple phenols or polyphenols based on the number of constitutive carbon atoms in conjunction with the structure of the basic phenolic skeleton. Thus, plant phenolics comprise simple phenols, benzoic acids, phenylopropanoids coumarins, lignins, lignans, condensed and hydrolysable tannins, phenolic acids and flavonoids (Rice-evans et al., 1997; Soto-Vaca et al., 2012). They can also be divided into three classes according to their distribution in nature: i) shortly distributed (as simple phenols, pyrocatechol, hydroquinone, resorcinol, Aldehydes derived from benzoic acids that are components of essential oils, such as vanillin), ii) widely distributed (divided in flavonoids and their derivatives, coumarins and phenolic acids, such as benzoic and cinnamic acid and their derivatives) and iii) polymers (tannin and lignin) (Bravo, 1998). Phenolics have various functions in plants. An enhancement of phenylopropanoid metabolism and the amount of phenolic compounds can be observed under different ecological and physiological stress such as pathogen and insect attack, UV radiation and wounding (Sakihama and yamasaki, 2002; Chung et al., 2003; Diaz et al., 2010; Kennedy and Wightman, 2011). Plant induced the production of Isoflavones and some other flavonoids in the state of infection, injury or temperature and nutrient stress (Takahama and Oniki, 2000; Sakihama and yamasaki, 2002; Ruiz et al., 2003). Most of them have antimicrobial activity. UV-absorbing flavonoids and other phenolic compounds are stored in the vacuoles of epidermal cell to protect deeper tissues from injuries induced by penetration UV-B radiation (Kondo and Kawashima, 2000). Phenolic compounds also have a wide range of pharmaceutical activities such as antiinflammatory, analgesis, antitumour, anti-hiv, antiseptic (Thymol), vasodilatory, 47

8 immunostimulant and antiulcerogenic. Thus they may be used with therapeutic purposes (Lampe, 1999; Beissert and Schwarz, 2002). Phenolic compounds are very strong antioxidants to avoid heart disease (Hoye et al., 2008; Wijngaard et al., 2009; Jin and Mumper, 2010), reduce inflammation (Jin et al., 2006; Zhang et al., 2011; Mohanlal et al., 2012), reduce the frequency of cancers (Slivova et al., 2005; Pieme et al., 2010; Sawadogo et al., 2012) and diabetes (Scalbert et al., 2005; Kusirisin et al., 2009), as well as cut the rates of mutagenesis in human cells (Gomez-Cordoves et al., 2001; Pedreschi and Cisneros-Zevallos,2006; Sawadogo et al., 2012). Flavonoids are some of the most common phenolics. All flavonoids are derived from the aromatic amino acids, phenyalanine and tyrosine. They are low molecular weight compounds, consisting of fifteen carbon atoms. The basic carbon skeleton of a flavonoid composed of two aromatic C6 rings held together by a 3-carbon bridge (Tunen and Mol, 1990; Routray and Orsat, 2012). Flavonoids are classified into different groups based on the scale and pattern of hydroxylation, prenylation, alkalinization and glycosylation reactions that alter the basic molecule structure of three-carbon bridge (Stalikas, 2007). They are mainly divided into two classes: (a) anthocyanins (glycosylated derivative of anthocyanidin, present in colorful flowers and fruits); (b) anthoxanthins (a group of colorless compounds further divided in several categories, including flavones, flavans, flavonols, isoflavones and their glycosides) (Henning et al., 2005; Balasundram et al., 2006; Lotito and Frei, 2006). Flavonoids are often responsible for plant pigmentation (carotenoids and chlorophylls for their blue, purple, green, yellow, orange and red colors), UV-protection, plant defense against pathogens and legume nodulations (Ferreira and Pinho, 2012). Flavonoids are also important constituents of the human diet (Hertog et al., 1992; Jovanovic et al., 1994). Nowadays, flavonoids have attracted attention due to the discovery of their pharmacological activities (Pietta, 2000; Brahmachari and Gorai, 2006). Tannins are phenolic compounds of molecular weight from intermediate to high ( D) in complexes with alkaloids, polysaccharides and proteins, are a group of water-soluble polyphenols. They may be subdivided into two major groups: hydrolysable tannins and nonhydrolysable or condensed tannins. The hydrolysable 48

9 tannins are esters of gallic acid (gallo- and ellagi-tannins), while the condensed tannins (also known as proanthocyanidins) are polymers of polyhydroxyflavan-3-ol monomers. There is a third group of tannins, phlorotannins, which are only found in brown seaweeds and are not commonly consumed by humans (Henning et al., 2005; Balasundram, 2006; Rangkadilok, 2007). Tannins are general toxins, which can reduce the growth of many herbivores when added to their diet. They act as feeding repellents to a great diversity of animals. They have characteristic to bind with proteins. Due to that they cause a sharp, unpleasant, astringent sensation in mouth and can also activate the digestive enzymes (Oates et al., 1980; Clausen et al., 1992). Plant tannins also serve as defences against microorganisms (Schultz et al., 1992) (b) Alkaloids Alkaloids are chemically heterogenous group of low-molecular-weight compounds. They comprise more than 6000 basic nitrogen containing organic compounds found in about 20% of plant species. In most cases, the nitrogen atom is located inside the heterocyclic ring structure (Wink, 1999; Freye, 2009). They are usually colourless, often optically active substances; most are crystalline but a few (e.g. nicotine) are liquids at room temperatures. Chemically, alkaloids are a very heterogenous group, ranging from simple compounds like coniine, the major alkaloid of hemlock, Conium maculatum, to the pentacyclic structure of strychnine, the toxin of the Strychnos bark. Plant amines (e.g. mescaline) and purine and pyrimidine bases (e.g. caffeine) are sometimes loosely included in the general term alkaloid (Harborne, 1980). Alkaloids have significant pharmaceutical activities, such as analgesic (morphine), anticancer (vinblastine), antimalarial, antiarrythmic and antispasmodic. Many of them are also used in the treatment of asthma (ephedrine), coughs and pain, in the treatment of gout and as pupil dilatin. Many alkaloids have high impact on central nervous system. Caffeine provides a mild stimulation but datura plant alkaloids are able to cause severe toxicity or even death (Buss and Waigh, 1995; Freye, 2009; Lee, 2011). In fact, alkaloids are among the most important active components in natural herbs, and some of these compounds have already been successfully developed into chemotherapeutic drugs, such as camptothecin (CPT), a famous topoisomerase I 49

10 (TopI) inhibitor (Huang et al., 2007), and vinblastine, which interacts with tubulin (Li et al., 2007). Phytochemistry is a rapidly expanding area with new techniques for the analysis of organic compounds. Phytochemical compounds are the fundamental source for the establishment of numerous pharmaceutical industries. These elements play an important role in the identification of crude drugs (Savithramma et al. 2011). Phytochemical evaluation is one of the tools for the quality evaluation of plants, which avails us with the prospect to see at a glance about the various phytochemicals present in a plant material (Sofowora, 1986). In order to find out new bioactive compounds, extracts are concurrently evaluated by chemical screening (Wing, 1999) Qualitative analysis and Quantitative analysis Preliminary phytochemical screening is primarily an important aspect for establishing profile of given extract for its chemical compounds produced by plant (Gurumurthy et al., 2008). Quantification of those metabolites will help to extract, purify and identify the bioactive compounds (Geetha and Geetha, 2014). Now a day s new technology have made it possible to identify, screen and isolate these active compounds. The chromatographic and spectral fingerprints play an important role in the quality control of complex herbal medicines (Gong et al., 2005) Thin Layer Chromatographic Analysis Thin layer chromatography (TLC) is the initial and standard technique for separation, detection and monitoring of compounds from the given sample (Panyaphu et al., 2012). Thin layer chromatography was accepted as a method of separation as early as in It is widely adopted for the rapid and positive analysis of drugs and drug preparations. TLC is a simple, quick, and inexpensive procedure that represents a comprehensive qualitative approach for the purpose of species authentication, evaluation of quality and ensuring the consistency and stability given sample. It is also useful in identification of a compound with reference to the Rf value of a known compound. Additional tests involve the spraying of phytochemical screening reagents, which 50

11 cause color changes according to the phytochemicals existing in a plants extract; or by viewing the plate under the UV light. This has also been used for confirmation of purity and identity of isolated compounds. The entire pattern of compounds can then be evaluated to determine not only the presence or absence of desired active constituents but the complete set of rations of all detectable analytes (Chitlange, 2008; Sasidharan et al., 2011; Panyaphu et al., 2012) HPTLC High Performance Thin layer Chromatography (HPTLC) is an advanced, sophisticated and automated form of the thin-layer chromatography (TLC) with better and advanced separation efficiency and detection limits. It is also known as High Pressure Thin layer Chromatography/Planar chromatography. It is a powerful analytical method which provides an image of chromatographic finger print and densitogram to detect the presence of a marker compound in the plant sample equally suitable for qualitative and quantitative analytical tasks (Srivastava, 2011; Sethi, 1996). HPTLC offers better resolution and estimation of active constituents can be done with reasonable accuracy in a shorter time. It can analyze several samples at the same time using a small quantity of mobile phase. This minimizes time and cost of analysis as well as it reduces exposure risks and considerably lessens disposal problems of toxic organic effluents, thereby minimizing possibilities of environment pollution. HPTLC also facilitates repeated detection of chromatogram with same or different parameters (Sethi, 1996). High performance thin layer chromatography (HPTLC) provides a chromatographic fingerprint and is suitable for confirming the identity and purity. (Wagner and Bladt, 2002; Raina, 1993). In the last two decades high performance thin layer chromatography (HPTLC) method has emerged as an important tool for the qualitative and quantitative phytochemical analysis of herbal drugs and formulations. This includes TLC fingerprint profiles and estimation of chemical markers and biomarkers (Ravishankar et al., 2000). 51

12 CHAPTER 3.2: MATERIALS AND METHODS Present work was carried out with the aim of phytochemical analysis of plant waste materials i.e. by-products of fruits and vegetables Selection and extraction of plant waste material Different fruits and vegetable by-products were selected for the study enlisted below. 1. Allium cepa L. (Onion Peel: OP) 2. Allium sativum L. (Garlic Peel: GP) 3. Solanum tuberosum L. (Potato Peel: PTP) 4. Mangifera indica L. (Mango peel: MNP and Mango seed kernel: MNS) 5. Cajanus cajan (L.) Millsp. (Pigeon pea pod shell: TP) 6. Pisum sativum L. (Pea pod shell: PSP) 7. Eugenia jambolana Lam. (Black plum seeds: JS) 8. Annona squamosa L. (Custard apple seeds: SS) 9. Citrus sinensis (L.) Osbeck (Orange peel: ORP) 10. Citrus limetta Risso. (Sweet lime peel: SLP) 11. Citrullus lanatus (Thunb.) Matsum. and Nakai (Watermelon peel: WMP) 12. Arachis hypogaea L. (Peanut pod shell: GNP) 13. Punica granatum L. (Pomegranate peel: PGP) 14. Vigna radiata (L.) R. Wilczek (Split green gram peel: MDP) 15. Capsicum annuum L. (Chilli Pedicel: CA) 16. Artocarpus heterophyllus Lam. (Jackfruit peel: JFP) 17. Cucumis sativus L. (Cucumber peel: CC) 3.2.1(a) Procurement of Plant Material Selected fruits and vegetable by-products were collected from the local markets, restaurants, fruit juice shops as well as author s home at Ahmedabad, Gujarat. After the plants are collected they have been processed for cleaning in order to prevent the deterioration of phytochemicals present in plants. 52

13 3.2.1(b) Cleaning After procurement of plant material, they were cleaned properly. The cleaning process involved the following steps. Very first the decayed or deteriorated plant material was removed. This was followed by washing with tap water and distilled water. The washed plant material was wrapped in blotting paper in order to remove extra water (c) Drying Soon after cleaning, plant material was kept for drying in oven at 45 C temperature. The main purpose of drying is to remove the water content from plants so that the plant material can be stored (d) Powdering The dried plant parts were finely powdered using electric grinder (Maharaja White line), sieved (mesh size 500µ) and packaged in polyethylene bags (size 10 8 cm) until when needed (e) Extraction All plant samples were extracted in four solvents of different polarity viz water, methanol, ethyl acetate and hexane. Powdered plant material (10 gm) was extracted in 150ml methanol using soxhlet apparatus by continuous hot percolation method at 60 C temperature for 24 hours. The resultant content was filtered with watman filter paper no.1 and kept for evaporation of solvent to get the dry concentrated extract. The dried crude concentrated extract was weighed to calculate the extractive yield then transferred to glass vials (6 2 cm) and stored in a refrigerator (4 C), till used for analysis Physicochemical analysis Determination of ash value (Kokate, 1994; Gupta, 2003; Indrayan et al., 2005; Mukherjee, 2008; Manjulika et al., 2014) Residue of the crude drugs after incineration contains mostly inorganic salts known as ash. Ash contains inorganic radicals like phosphates, carbonates and silicates of sodium, potassium, magnesium, calcium etc. sometimes, inorganic variables like calcium oxalate, silica, carbonate content of the crude drug affects. Such variables are 53

14 then removed by treating with acid and then acid insoluble ash value is determined. Types of ash values are total ash, acid insoluble ash, and water soluble ash (a) Determination of total ash For determination of ash content, accurately weighed 3 gm of the powdered plant material was taken in a previously ignited and tarred crucible (of silica) and it was incinerated at a temperature not exceeding 450 C until free from carbon. The sample was cooled in desiccators and weighed. To ensure completion of ashing, it was heated again in the furnace for half an hour, cooled and weighed. This was repeated consequently till the weight became constant (ash became white or greyish white). Weight of ash gave the ash content. The percentage of ash content was calculated with reference to the initial weight of plant material. Total ash value can be calculated by using following formula: % Ash value = Initial weight Final weight Initital weight (b) Determination of water soluble ash To the ash obtained as total ash was boiled with 25ml of distilled water for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water and ignited in a crucible at a temperature not exceeding 450 C to obtain constant weight. The weight of this insoluble matter was subtracted from the weight of total ash, the difference in weight represents the water soluble ash. The percentage of water- soluble ash was calculated with reference to the air dried drug. Water soluble ash content = Weight of total ash Weight of water insoluble ash Weight of crude drug (c) Determination of acid insoluble ash: The total ash obtained was boiled with 25 ml of dilute hydrochloric acid for 5 minutes. The insoluble matter was filter by ash less filter paper and washed with hot acidulated water. Residue was collected on tarred grouch crucible, ignited, cooled and 54

15 weighed. Process was repeated until the constant weight gained. The percentage of acid insoluble ash was calculated with reference to the air dried drug by using following formula. Acid insoluble ash content = Weight acid insoluble ash Weight of crude drug Determination of Extractive Value This method determines the active constituents of plant material with different solvents and help in preliminary phytochemical tests of plant materials. Extractive values were carried out as per standard procedure Indian pharmacopeia (Anonymous, 1996; WHO, 2002) (a) Determination of water soluble extractive value 10gm of coarsely powdered air-dried plant material was placed in a glass-stoppered conical flask and 150ml of distilled water was added in the flask. It was shaken well and allowed standing for 1 hour. A reflux condenser was attached to the flask and the mixture was boiled for 12 hours. Then it was allowed to cool down and filtered with whatman filter paper. Filtrate was collected in empty pre-weighed evaporating dish. The dish was heated until the damp mass was formed. Cooled evaporating dish was then weighted and difference between damp mass and empty evaporating dish was taken to calculate the water soluble extractive value (b) Determination of methanol soluble extractive value 10 gm of plant material was extracted with methanol by soxhlet apparatus for 12 hours at 50 C. Thereafter, it was filtered and the filtrate was evaporated to dryness till constant weight was obtained. The percentage of extractable matter was calculated with reference to the sample taken initially (c) Determination of ethyl acetate soluble extractive value 10 gm of plant material was extracted with ethyl acetate by soxhlet apparatus for 12 hours at 55 C. Thereafter, it was filtered and the filtrate was evaporated to dryness till constant weight was obtained. The percentage of extractable matter was calculated with reference to the sample taken initially. 55

16 (d) Determination of hexane soluble extractive value 10 gm of plant material was extracted with hexane by soxhlet apparatus for 12 hours at 40 C. Thereafter, it was filtered and the filtrate was evaporated to dryness till constant weight was obtained. The percentage of extractable matter was calculated with reference to the sample taken initially. Extractable matter was calculated in mg per gm of air dried plant material and % extractive value was calculated by following formula. % Extractive value = Initial weight Final weight Initial weight Phytochemical Analysis (A) Qualitative phytochemical analysis Qualitative chemical tests were carried out for methanolic extract, to identify different phyto-constituents by using standard methods (Evans, 1996; Harborne, 1980; Dey and Harborne, 1987; Iyengar, 1995; Kokate et al., 1990; Siddiqui and Ali, 1997) (A.1) Alkaloids Mayer s Test: In 1ml of extract 2ml of freshly prepared mayer s reagent was added side by side to test tube. Dull white or creamy precipitates indicate alkaloids are present. Dragendorff s test: 1-2 ml Dragendorff s reagent was added to 1ml extract. Prominent yellow/ orange red precipitate indicated positive test for alkaloids. Hager s Test: 2-3 drops of hager s reagent (saturated aqueous solution of picric acid) was added to 1 ml of extract. A prominent yellow precipitate indicated the test as positive for alkaloid. Wagner s Test: In 1ml of extract, 2ml wagner s reagent was added by the side of the test tube. A reddish brown precipitate confirmed the test as positive. Tannic acid test: Add 2-3 drops of 10% Tannic acid in 1ml of extract. Buff colour indicates presence of alkaloids. 56

17 3.2.3 (A.2) Phenols Ferric chloride test: The extract (50 mg) was dissolved in 5 ml of distilled water or 2 ml ethanol was added to 2ml of the test solution) and few drops of 5% ferric chloride solution and observed for coloration. Test solution gives blue green color with ferric chloride. Lead acetates test: The extract (50 mg) was dissolved in distilled water and 3 ml of 10% lead acetate solution was added. A bulky white precipitate indicated the presence of phenol compounds. Gelatin test: 50 mg of extract dissolved in 5 ml of distilled water and to this; 2 ml of a 1% solution of gelatin containing 10% sodium chloride was added. The appearance of white precipitates indicated the presence of phenolic compounds Potassium Dichromate Test: The extract was added to potassium dichromatic solution, formation of a precipitate shows presence of tannins and phenolics. Alkaline reagent test: Test solution with sodium hydroxide solution gives yellow to red precipitate within short time (A.3) Flavonoids Alkaline Reagent Test: Extracts have to be treated with a few drops of sodium hydroxide solution. Formation of intense yellow color, which becomes colorless on the addition of dilute acid, indicates the presence of falvonoids. Shinoda Test (Magnesium Hydrochloride reduction test): About 0.2 g (4ml) of the extract was dissolved in 2 ml of 50% methanol and heated. A chip of magnesium metal was added to the mixture followed by the addition of a few drops of concentrated HCl. The occurrence of a red or orange colouration was indicative of the flavonoids. Occasionally green to blue color appears after few minutes. Ethyl acetate- Ammonia Test: A portion of the powdered plant samples were separately heated with 10ml of ethyl acetate in a water bath for 3min. The mixtures were filtered and 4ml of each filtrate were shaken with 1ml of dilute ammonia solution. A yellow colour observation indicates the presence of flavonoids. 57

18 Pew s test: Five millilitres (5 ml) of the aqueous solution of the water extract was mixed with 0.1 g of metallic zinc and 8ml of concentrated sulphuric acid. The mixture was observed for red colour as indicative of flavonols (A.4) Tannins Ferric chloride Test: About 0.5 g of the extract was boiled in 10 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride were added, and the solution was observed for brownish green or a blue-black colouration. Blue color was observed for gallic tannins and green black for catecholic tannins. Lead Acetate Test: To the extract added 1ml of lead acetate solution. Formation of white precipitate indicates presence of tannins. Potassium Dichromate Test: The extract was added to potassium dichromatic solution, formation of a precipitate shows presence of tannins and phenolics (A.5) Saponins Frothing test: 0.5 g of extract was added 5 ml of distilled water in a test tube. The solution was shaken vigorously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously after which it was observed for the formation of an emulsion. Sodium bicarbonate test: To the few milligrams of extract few drops of sodium bicarbonate were added and shaken well. Formation of honey comb like frothing indicates positive test for saponins (A.6) Terpenoids Salkowski's test: To 0.5 g of each the extract was added 2 ml of chloroform. Concentrated sulphuric aids (H2SO4), (3 ml) was carefully added to form a layer. A reddish brown coloration of the interface indicates the presence of terpenoids. Liebermann - Burchard s Test: Four milligrams of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated 58

19 solution of sulphuric acid was added slowly and red violet color was observed for terpenoid and green bluish color for steroids. Hesse s reaction: The residue was dissolved in chloroform (4 ml) and an equal quantity of concentrated sulphuric acid was then along the side of the tube. The formation of the pink colored ring, which is on shaking diffused in both the layers, indicating the presence of sterols in the extract (A.7) Glycosides Keller killiani test (test for Deoxy sugars): 0.5 gm of extract was diluted to 5 ml in water, and 2 ml of glacial acetic acid containing one drop of ferric chloride solution was added to it. 1 ml of concentrated sulphuric acid was added to form a layer, and the colour at the interphase was recorded. A brown ring at the interface indicated the presence of a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer; a greenish ring may form just above the brown ring and gradually spread throughout this layer. Borntragers Test: A few ml of dilute sulphuric acid was added to 1ml of the extract. Boiled, filtered, cooled and extract the filtrate with 3ml of chloroform. Chloroform layer was separated and 10%, 1ml ammonia solution was added to it. Pink colour indicated the presence of glycosides or formation of red colour in the ammoniacal layer indicates presence of anthraquinone glycoside. Modified Borntragers Test: A few ml of dilute hydrochloric acid was added to 1ml of the extract. Add few drops ferric chloride solution. Boiled, filtered, cooled and extract the filtrate with benzene. The chloroform layer was treated with 1ml of ammonia. The formation of red colour in the ammoniacal layer indicates presence of anthraquinone glycoside. Legal Test: Fifty mg of the extract was dissolved in pyridine. Sodium nitroprusside solution was added and made alkaline using 10 % sodium hydroxide. Presence of glycoside was indicated by pink colour. Baljet Test: To 1ml of the test extract, 1ml sodium picrate solution was added. The formation of yellow to orange colour indicates presence of glycoside. 59

20 3.2.3 (A.8) Steroids Libermann- Buchard test: Extract is treated with few drops of acetic anhydride, boil and cool, concentrated sulfuric acid is added from the sides of the test tube. Presence of a brown ring at the junction of two layers and the upper layer turns green which shows the presence of steroids and formation of deep red color indicates the presence of triterpenoids. Salkowski test: Treat extract in Chloroform with few drops of concentrated sulfuric acid, shake well and allow standing for some time, red color appears at the lower layer indicates the presence of Steroids and formation of yellow colored lower layer indicates the presence of Triterpenoids. Acetic anhydride-h2so4 Test: To the plant extract add 2 ml of acetic anhydride and add 0.5 gm of extract of each sample with 2 ml of sulphuric acid. Observe for the color change from violet to blue or green in samples indicating the presence of steroids Libermann sterol test: To a solution of glycosides or steroidal aglycones in glacial acetic acid, one drop of concentrated sulphuric acid was added. A play of colours was observed starting from rose, red, violet, blue to green (A.9) Sugar/Carbohydrates The extract (100 mg) was dissolved in 5 ml of water and filtered. The filtrate was subjected to the following tests. Molish s test: To 2 ml of filtrate, two drops of alcoholic solution of a- naphthol (20% in ethyl alcohol) were added, the mixture was shaken well and 1 ml of concentrated sulphuric acid was added slowly along the sides of the test tube and allowed to stand. A reddish violet ring indicated the presence of carbohydrates. Fehling s test: One ml of filtrate was boiled on water bath with 1 ml each of Fehling solutions A and B. Brick red precipitate indicated the presence of sugar. Barfoed s test: 1 ml of filtrate, 1 ml of Barfoed s reagent was added and heated on a boiling water bath for 2 min. Red precipitate indicated presence of sugar. 60

21 Benedict s test: To 0.5 ml of filtrate, 0.5 ml of Benedict s reagent was added. The mixture was heated on a boiling water bath for 2 min. A characteristic colored precipitate indicated the presence so sugar. (Reddish brown precipitate forms if reducing sugars are present. Camnelisation Carbohydrates when treated with strong sulfuric acid, they undergo charring with the dehydration along with burning sugar smell. Iodine Test: To 1ml of the extract added to 3 drops of iodine. Blue colour indicates presence of starch (A.10) Amino Acids and Proteins The extract (100 mg) was dissolved in 10 ml of distilled water and filtered through whatman No.1 filter paper and the filtrate was subjected to tests for proteins and amino acids. Millon s test: To 2 ml of filtrate, few drops of Millon s reagent (2ml) were added. A white precipitate indicated the presence of proteins. Biuret test: An aliquot of 2 ml of filtrate was treated with one drop of 2 % copper sulphate solution. To this, 1 ml of ethanol (95%) was added, followed by excess of potassium hydroxide pellets, pink colour in the ethanolic layer indicated the presence of proteins. Ninhydrin test: Two drops of ninhydrin solution (10 mg of ninhydrin in 200 ml of actone) were added to two ml of aqueous filtrate. A characteristic purple colour indicated the presence of amino acid. Xanthoproteic Test: 1ml of extract was added to 1ml of concentrated nitric acid. A white precipitate is formed; it is then boiled and cooled. Then 20% sodium hydroxide or ammonia is added. Orange colour indicates presence of aromatic amino acid (A.11) Fats and Fixed Oils Spot test: A small quantity of extract was pressed between two filter papers. Oil stains indicates presence of fixed oils. Saponification test: To 1ml of the extract was added to a few drops of 0.5N alcoholic potassium hydroxide along with a drop of phenolphthalein. The 61

22 mixture was heated on a water bath for 2hrs.The formation of soap or partial neutralization indicates the presence of fixed oils (B) Quantitative Phytochemical Analysis (B.1) Total and reducing sugars (Nelson Somogyi, 1944) Principle Monosaccharide readily reduces oxidizing agents such as Ferric cyanide, hydrogen peroxide or cupric ions (Cu ++ ). In such reactions the sugar is oxidized at carbonyl group and the oxidizing agent becomes reduced glucose or other sugars capable of reducing oxidizing agents are called reducing sugars. Thus by measuring the amount of oxidizing agent that is reduced by a sugar solution, it is possible to estimate the sugar concentration. The method involves the reduction of cupric ions (Cu ++ ) to cuprous ions (Cu + ) which in alkaline solution and forms yellow cuprous hydroxide, which in turn is converted by heat of the reaction to insoluble red cuprous oxide (Cu2O). The amount of Cu2O formed can be increased by adding arsenomolybdic acid which in turn is reduced to lower oxides of molybdenum by Cu2O. The coloured complex produced is known as molybdenum blue. The intensity of the colour is related to the concentration of the reducing sugars in the sample and is measured using the spectrometer at 620 nm. The content of non-reducing sugars can be calculated by subtracting the reducing sugars from total sugars. Non reducing sugars = Total sugars - Reducing sugars Procedure 100mg plant material was homogenized with 10ml 80% ethanol and centrifuged at 5,000-10,000 rpm for 10 minutes. Supernatant I was collected and 10ml 80% ethanol was added to residue and again centrifuged at 5,000-10,000 rpm for 10 minutes. Supernatant II was collected and mixed with supernatant I, residue was discarded. Total Sugar: To 1 ml of aliquot, 1ml 1N H2SO4 was added and incubated in water bath at 49ºC for 30 minutes. Add 1-2 ml drop of methyl red indicator followed by 1 N NaOH drop wise for neutrialization (Colour change pink to yellow). After that 1 ml 62

23 Nelson Somogyi reagent was added and incubated for 20 minute in boiling water bath. Then add 1 ml arsenomolybdate Final volume was made up to 20 ml with DW. OD was read at 620 nm Reducing Sugar: To 1 ml of aliquot, 1 ml Nelson somogyi reagent was added and incubated in boiling water at 100ºC for 20 minutes. 1 ml arsenomolybdate was added and final volume was made up to 20 ml with DW. OD was read at 620 nm. Standard curve (Figure 3.2.1) was prepared for sugar and results were derived from regression formula which were represented as mg/gm. Figure 3.2.1: Showing estimation of Sugar concentration using standard curve method and evaluation of the Regression Equation, Correlation Coefficient (r 2 ) (B.2) Starch Content (Chinoy, 1939) Principle The plant material is treated with aqueous sodium hydroxide in the cold to dissolve the starch. This dissolved starch reacts with I2KI and gives a coloured product and the starch content is determined by calculated with the standard curve. Procedure 100mg plant material was weighed and homogenate with 10ml 80% ethanol. It was centrifuged for 10minutes. Supernatant 1 was collected; while 10 ml 80% ethanol was added again to the residue centrifuge it ad supernatant 2 was mixed with supernatant 1 and removed. Residue was used for starch estimation. 63

24 The residue was dissolved in 20ml 0.7% KOH and boiled for gelatinization for 40 minutes. It was centrifuged after cooling and 1ml aliquot (Supernatant), 0.5ml 20% acetic acid; 1ml citrate buffer (0.05M, ph 5.0) and 1ml I2KI were added and incubated at room temperature for 10minutes. O.D. was taken at 600nm. Blank was prepared in the same manner. Standard curve (Figure 3.2.2) was prepared by using same method with known starch concentrations. Starch concentration of sample was determined by using regression formula of standard. The result was expressed as mg/gm plant material. Figure 3.2.2: Showing estimation of Starch concentration using standard curve method and evaluation of the Regression Equation, Correlation Coefficient (r 2 ) (B.3) Determination of Protein content (Bradford, 1976) Principle This is a simple, rapid and sensitive method for estimation of proteins. The procedure is based on interaction of dye, coomassie brilliant blue with proteins. The unbound dye has the absorbance maxima at 465nm. However, on interaction with proteins, the dye turns blue and the absorbance maxima shifts to 595nm. The colour development is virtually complete in 2 minutes and the colour is stable for about 1hour. Procedure Grind 1gm plant material in 10ml, 0.1M phosphate buffer (ph 7.0) using mortar and pestle. Centrifuge the extract at 10,000 rpm for 15 minutes at 4 0 C. Use the supernatant as extract for estimation of total soluble proteins. Take 1ml extract and add 5ml Bradford s reagent and mix well. Read the absorbance of the resultant solution which is a blue coloured complex at 595nm. Standard curve (Figure 3.2.3) was prepared by 64

25 using bovine serum albumin as a standard and results were derived from regression formula which were represented as mg/gm plant material. Figure 3.2.3: Showing estimation of Total Proteins concentration using standard curve method and evaluation of the Regression Equation, Correlation Coefficient (r 2 ) (B.4) Total phenols (Bray et al., 1954) Principle Estimation of phenols using Folin- ciocalteu s reagent is based on the reaction between phenols and an oxidizing agent phosphomolybdate which results in the formation of a blue complex (Bray et al., 1954). Figure 3.2.4: Showing estimation of Phenol concentration using standard curve method and evaluation of the Regression Equation, Correlation Coefficient (r 2 ) Procedure 100mg plant material was weighed and homogenate with 10ml 80% ethanol. It was centrifuged at ,000g for 10minutes. Supernatant 1 was collected; while 10 ml 80% ethanol was added again to the residue, centrifuge it and supernatant 2 was 65

26 mixed with supernatant 1 and used for estimation. Residue was discarded.1ml alcoholic aliquot was mixed with 1ml 20% Na2CO3 and 0.5ml Folin-ciocalteau s reagent. It was boiled for 10minutes at C in water bath. Final volume was made up to 20ml with DW and OD was noted at 660nm. PPT were filtered or centrifuged before reading. Blank was prepared in the same manner. Standard curve (Figure 3.2.4) was prepared by using Tannic acid as a standard and results were derived from regression formula which were represented as mg/gm plant material (B.5) Determination of total Flavonoid content (Park et al., 1997; Ordoñez et al., 2006; Ebrahimzade et al., 2008; Nabavi et al., 2008) Total flavonoid content of the crude extracts was determined by Colorimetric aluminum chloride method. Aliquot of 0.5 ml solution of each plant extracts (contains 0.5 mg of crude extract) in methanol were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminium chloride, 0.1 ml of 1 M potassium acetate, and 2.8 ml of distilled water. This mixture was left at room temperature for 30 minutes. Yellow color indicated the presence of flavonoids. The absorbance of the reaction mixture was measured at 415 nm with a double beam Perkin Elmer UV/Visible spectrophotometer (USA). Total flavonoid contents were calculated as quercetin from a calibration curve. The calibration curve was prepared (Figure 3.2.5) by preparing quercetin solutions at concentrations 0.5 to 5.0 mg ml-1 in methanol. Figure 3.2.5: Showing estimation of Flavonoid concentration using standard curve method and evaluation of the Regression Equation, Correlation Coefficient (r 2 ) 66

27 3.2.3 (B.6) Determination of total Tannin content The total tannin content was determined by using Folin Denis method given by Schanderl (1970). Principle Tannin-like compounds reduce phosphotungstomolybdic acid in alkaline solution to produce a highly coloured blue solution, the intensity of which is proportional to the amount of tannins. The intensity is measured in a spectrophotometer at 700nm (Ram and Mehrotra, 1993; Sane, 2002). Preparation of standard curve Tannic acid was used as standard. To 0.1-1mg/ml standard solution, 0.5ml of Folin- Denis reagent and one ml of 1N sodium carbonate solution was added to each tube. Each tube was made upto 10 ml with distilled water. All the reagents in each tube were mixed well and kept undisturbed for about 30 minutes and read at 760 nm against reagent blank (Schanderl, 1970). The standard Figure was plotted taking optical density on X- axis and concentration of tannin with mg/ml concentration on Y-axis (Figure 3.2.6). The concentration of unknown sample was read from the standard curve. Figure 3.2.6: Showing estimation of Tannin concentration using standard curve method and evaluation of the Regression Equation, Correlation Coefficient (r 2 ) Procedure for sample evaluation To 1 ml of the plant extract or standard was mixed with 0.5 ml Folin-Denis reagent and then added 1 ml of 1N sodium carbonate and total volume made up to 10 ml with 67

28 distilled water. The mixture was allowed to stand for 30 min at room temperature. The blue color produced was read at 760 nm using UV/visible spectrophotometer. The tannin content was calculated by calibration curve of gallic acid and the results were expressed as gallic acid equivalent (mg/g) (C) Thin Layer Chromatography Thin layer chromatography is particularly valuable for the qualitative and quantitative analysis of a number of natural products. It is effective and easy to perform and the equipment required is inexpensive. Different solvent systems were utilized mentioned by Harborne (1998) and the systems gave better results were subjected to HPTLC profile. Preparation of Plates The TLC plates were prepared by applying silica gel GF 254 (E. Merk) slurry (1:2 w/v) in distilled water on 10 X 20cm thin glass plates. The plates were air-dried at room temperature and then activated by heating 100 C for two hours. The spots were applied on these plates equidistantly with the help of micropipette, 2cm above the lower edge of plate. The distance between spots were 1.5cm, the line was drawn at the distance of 10cm from the spots. The spots were dried and then the plates were introduced in saturated sealed chamber containing solvent system. The plates after developing with suitable system were air dried and then observed under short UV (254nm) for quenching of fluorescence and long UV (365nm) for the fluorescence pattern. Spots/area, where quenching and/or fluorescence were observed, were marked and used to calculate the Rf. The separated compounds were visualized by spraying these plates with a specific spray reagent as well as scanning the plates under the long UV radiation. Following solvent system (table 3.2.1) were finalized for HPTLC after TLC screening. Table 3.2.1: Table for solvent systems and spray reagents used for TLC Sr. Components Solvent system Proportion Derivatization No. 1 Alkaloids Chloroform: Methanol 90:10 DDR reagent followed 3 Flavonoids Toluene:Ethylacetate: Formic acid by 10% Ethanolic H 2SO 4 50:40:10 1% AlCl 3 68

29 3.2.3 (D) HPTLC instrumentation Quantitative and qualitative analysis was performed with the help of HPTLC instrument. The HPTLC system (Camag, Muttenz, Switzerland) consists of (1) TLC Scanner connected to a PC running WinCATS software under MS Windows NT; (2) Linomat V Sample applicator, (3) Photo documentation system Camag, Reprostar III. The HPTLC analysis needs sample and solvent preparation. Step 1: Sample preparation All reagents used in this study were of analytical grade. Each extract was re-dissolved at the concentration of 50 mg/ml in respective solvent in which they were extracted in narrow glass vial and used for plate application. Step 2: Plate activation Aluminium sheet back coated with silica gel 60F254 plates were used in the study. The plates were activated in an oven at 50 C (10 min) prior to use. This process helps to remove the moisture and activates the active sites of silica gel for better separation. Step 3: Sample application CamagLinomat V (Camag, Muttenz, Switzerland) was utilized for nitrogen gasassisted and controlled application of samples on to TLC plate. The sample extracts were streaked in form of narrow bands on the pre-coated silica gel 60F254 aluminum TLC plate, at a constant application rate of 250 µl/s and gas flow 10 s/µl employed with help of Camag 100 µl syringe connected to a nitrogen tank; using a CamagLinomat V (Camag, Muttenz, Switzerland). Samples were applied at the height of 10 mm from the base, having specific band width and space between two bands. Step 4: Plate development and chromatographic conditions After sample application, the plates were subjected to linear ascending development, in selected solvent system, up to a distance of about 70 mm. Twin trough glass chamber (with 10 min prior saturation with the solvent system) was used at room temperature. 69

30 Step 5: Scanning of plate Subsequent to the development, the TLC plates were dried in a current of air. Densitometric scanning was carried out using Camag TLC Scanner III (Camag, Muttenz, Switzerland) in the absorbance mode at nm wavelength with a scanning speed of 20 mm/s, data resolution 100 µm/step and a specific slit dimension. The source of radiation utilized was deuterium and tungsten lamp. All remaining measurement parameters were at default settings. The chromatograms were integrated and regression analysis and statistical data were generated using WinCATS evaluation software (Version ). Step 6: Photo documentation of plate After scanning, images were taken at three different wavelengths i.e. 254 nm by UV lamp, 366 nm by mercuric lamp, nm with Photo documentation system Camag, Reprostar III. Step 7: Post chromatographic derivatization of TLC plate Post-chromatographic derivatization of developed TLC plates was also performed wherever necessary. Various spray reagents were used to mark visible spots visible on the plate. This step is optional and used for better resolution and spotting of separated compounds on the plate. 70

31 CHAPTER 3.3 RESULTS AND DISCUSSION Determination of ash value Selected 18 fruit and vegetable by-products were subjected to incineration in order to determine their total ash value. This ash was dissolved in water and acid to find out the content of water soluble and acid insoluble ash. The total ash and acid insoluble ash are vital parameters for detecting the presence of inorganic substances. Results for total ash, water soluble and acid insoluble ash were calculated in percentage with reference to initial weight of plant powder. Figure 3.3.1: Comparison between ash values of selected plant material As shown in figure total ash content was recorded highest in water melon peel (WMP) i.e ±0.1% while custard apple seeds (SS) reported for the minimum ash content i.e. 1.85±0.10%. Maximum content for water soluble ash was found in WMP i.e ±0.05% and minimum in garlic peel (GP) i.e. 0.65±0.25 %. Capsicum annum (CA) pedicle contain the maximum acid insoluble ash i.e. 1.26±0.11% and minimum was reported in Pisum sativum (PSP) pod shell i.e. 0.33±0.06%. All plant material showed higher amount of water soluble ash than acid insoluble ash. Results for onion peel (OP) ash is represented in figure 3.3.1(a). Present study shows that onion peel contains 9.31±1.45% total ash content which falls in the range of 5.93% to 14.12% ( 2015; Sayed et 71

32 al., 2014) reported by other researchers. Studies revealed that onion bulb contains total ash content in the range of 4.26±0.22 % to %. (Nwinuka et al., 2005; Yahaya et al., 2010) and in outer scale it is 8.06±0.01 % (Bello et al., 2013). When all numbers are compared, it can be said that onion peel possesses the highest amount of total ash. Figure 3.3.1(a): OP ash values Figure 3.3.1(b): GP ash values As shown in figure 3.3.1(b), garlic peel (GP) contains 9.46±1.68% ash while studies on garlic bulb shows the range of 1.33±0.04% to 5.00±0.11% (Nwinuka et al., 2005; Odebunmi et al., 2009; Otunola et al., 2010; Okolo et al., 2012; Belemkar et al., 2013; Marina et al., 2014). Several reports on garlic peel indicates the presence of 17.07% ash ( ). Total ash content in potato peel (PTP) was recorded 9.22±0.10% represented in figure 3.3.1(c). The value is closer to other results i.e. 7.65% (Mahmood et al., 1998), 5.46 ± 0.17% ash (Amado et al., 2014), % (Camire et al., 1997) and 5.31% ash (Dhingra et al., 2014). Figure 3.3.1(c): PTP ash values Figure 3.3.1(d): MNP ash values 72

33 Ash content of mango peel (MNP) and seed kernel (MNS) shown in figure 3.3.1(d and e). Total ash content in mango peel and seed kernel was found 4.65±0.05% and 2.88±0.38 % respectively. These values fall within the range i.e ± 0.59% for the peel and 1.46 to 3.2% for Seed kernel as described in literature (Nzikou et al., 2010; Fowomola, 2010; Ashoush and Gadallah, 2011; Ashifat et al., 2012). Water soluble and acid insoluble ash in mango peel and seed kernel was 2.65±0.05, 0.6±0.06 and 1.85±0.08, 0.58±0.06% respectively. Figure 3.3.1(e): MNS ash values Figure 3.3.1(f): TP ash values Pigeon pea pod shell (TP) was found to contain 3.96±0.16 % total ash content (Figure 3.3.1(f)). No reports are available about the proximate study of pea pod shell. But when compared with other parts of pea it has been found that the result of present study resembles with total ash content of seed coat of tur dal i.e. 3.5% reported by Singh et al. (1968) and ash content of seed i.e. 3.17± 0.005% analysed by Balogun (2013).Water soluble ash and acid insoluble ash were recorded 2.52±0.05 and 0.42±0.06 respectively (Figure 3.3.1(f)). Figure 3.3.1(g): PSP ash values Figure 3.3.1(h): JS ash values Pea pod shell (PSP) were observed with 5.57±0.19 % total ash content (Figure 3.3.1(g)) which is close to other findings of ash content for pea peel waste i.e % recorded by Verma et al. (2011), 5.77±0.46% by Rehman et al. (2015) 73

34 and % by Sharoba et al., (2013). Pea pod shell also contained 4.34±0.13% water soluble ash and 0.33±0.06% acid insoluble ash (Figure 3.3.1(g)). As represented in figure 3.3.1(h), the ash content in jamun (black plum) seeds (JS) is 2.45±0.25% total ash, 1.66±0.06% water soluble ash and 0.65±0.05% acid insoluble ash shown in figure 3.3.1(h). When the present results were compared to other studies, a large variation in results was observed. According to other reports, the total ash content of jamun seed ranged within 2.18% to 25%. Current results resembled with the study done by Raza et al. (2015) who recorded seed ash content 2.18±0.06% and Pulp ash 2.04±0.06%. Modi et al. (2010), Chitnis et al. (2012), Agrawal and Argal (2014) noted that the concentration of total ash value, water soluble ash and acid insoluble ash in jamun seed is <10%. Shahnawaz et al. (2009), Ranjan et al. (2011) and Nair et al. (2013) found the total ash value in jamun seeds were %, 21.72% and 25% respectively. Annona squamosa seeds (SS) were found to contain 1.85±0.10 % total ash, 1.04±0.69% water soluble ash and 0.72±0.08% acid insoluble ash represented in figure 3.3.1(i). Results from other studies on total ash i.e. 2.39% (Kulkarni et al., 2013), 2.50 ± 0.20% (Hassan and Muhammad, 2008), 2.2 ± 0.1% (Mariod et al., 2010) and % (Kadarani et al., 2015) were found near to similar with the present observation. Water soluble ash and acid insoluble ash was also recorded in transitional range in compare with previous researches i.e % to 3.79% for water soluble ash and 0.551% to 1.05% for acid-insoluble ash content (Kulkarni et al., 2013; Sharma et al., 2013). Figure 3.3.1(i): SS ash values Figure 3.3.1(j): ORP ash values Present study shows the orange peel (ORP) contains 3.22±0.05 % total ash, 2.41±0.06% water soluble ash and 0.7±0.06% acid insoluble ash (Figure 3.3.1(j)). 74

35 Similar results were reported by mahmood et al. (1998) i.e. 3.78%. Several studies also noted the lesser concentration of ash than the present one i.e. 2.61±0.10% (Nassar et al., 2008), 1.60 ± 0.06% (Oikeh et al., 2013) and 1.50 ± 0.1% (Ververis et al., 2007) while Sadiq et al. (2014), Peter et al. (2013) and Sharoba et al. (2013) found the total ash content in orange peel was 4.8 %, % and 10.03% respectively. As shown in figure 3.3.1(k), ash content of sweet lime peel (SLP) was 4.76±0.12 % (total ash), 3.24±0.10 % (water soluble ash) and 0.38±0.05% (acid insoluble ash). This finding quite resembled with the results obtained by Younis et al. (2015) for sweet lime peel i.e. 3.39%. Figure 3.3.1(k): SLP ash values Figure 3.3.1(l): WMP ash values Total ash, water soluble ash and acid insoluble ash content in watermelon peel (WMP) was recorded 12.73±0.1 %, 11.44±0.05% and 0.88±0.05% respectively as denoted in figure 3.3.1(l). This result was found to be compatible with earlier study done by El-Badry et al. (2014) who documented the ash value 12.93±0.32% for watermelon peel. Several other reports are also available for watermelon peel ash content which shows the much lower ash value than the present one i.e. 0.92±0.04% for unfermented watermelon rinds, 1.61±0.07% for Fermented watermelon rinds (Erukainure et al., 2010) and 3.07±0.00% (Cemaluk and Egbuonu, 2015). Ash content for peanut pod shell (GNP) was found 3.14±0.11 % (total ash), 2.25±0.05 (water soluble ash) and 0.34±0.05% (acid insoluble ash) as presented in figure 3.3.1(m). Other studies also revealed the ash content of peanut hull as 4.23% (Hegazy et al., 1991) and 3.10± 0.22% ash (Fakhriya et al., 2012) which were adjacent to our obtained results. Sim et al. (2012) have analysed two different varieties (Shandong PS and Menglembu PS) of peanut and stated the total ash content 21.7± 0.0% and 13.0± 0.20% respectively which was much higher than the former. 75

36 As shown in figure 3.3.1(m), pomegranate peel (PGP) was found to contain total ash 3.61±0.08 %, water soluble ash 2.81±0.10% and acid insoluble ash 0.71±0.05%. Other studies also reported the similar results for ash content of pomegranate peel i.e. total ash 05 ± 0.14(%) (Naseem et al., 2012), 3.30% (Rowayshed et al., 2013), 3.11% total ash and 0.54% acid insoluble ash (Sangeetha and vijayalakshmi, 2012). Figure 3.3.1(m): GNP ash values Figure 3.3.1(n): PGP ash values Split green gram peel (seed coat) (MDP) was analysed for its ash values which resulted in 2.66±0.17 % for total ash, 1.14±0.08% for water soluble ash and 0.42±0.01% for acid insoluble ash (figure 3.3.1(o)). No reports are available on green gram proximate analysis. But when the present outcomes were compared with the earlier studies done for whole green gram, it was found that there is not much difference in the ash content. Present results are in close agreement (i.e. ±2%) with other findings % (Habibullah et al., 2007), 3.85±0.05% (Paul et al., 2011), 2.91 ± 0.072%, (Shabnum et al., 2012), 3% (Blessing and Gregory, 2010), 2.97% (Pasha et al., 2011), 3.51% to 4.29% (Riaz Ullah et al., 2014) and 3.81±0.03% (Phule et al., 2013). Figure 3.3.1(o): MDP ash values Figure 3.3.1(p): CA ash values Chilli pedicel (CA) was found to contain 12.14±0.39% total ash, 10.28±0.16% water soluble ash and 1.26±0.11% acid insoluble ash as shown in figure 3.3.1(p). Chilli 76

37 pedicel separately is reportedly still untouched for analysis. No reports were found on its proximate composition or phytochemical analysis but several documents containing the information about chilli fruit were studied. Findings on chilli indicated a range of variation in chilli fruit. Sarker et al. (2012), Gupta and Tambe (2003) and Ogunlade et al. (2012) recorded <3% of ash content in different chilli varieties i.e ±0.07%, % and % respectively. Other group of researchers have noted the ash content within the range of 5-10%. These findings are, 5.3 to 7.3% (Esayas et al., 2011), 5.7% (FAO, 2009), 6.27% (Tandon et al., 1964), 8.00% Krishna De (2003), 9.78±0.04% to 10.78±0.04% (Emmanuel et al., 2014) and 9.27 %, (Raimi et al., 2014). A study on different parts of chilli shares the similarity with the present finding. Simonovska et al. (2014) reported ash content in pericarp 16.32±0.29%, seed 3.76±0.41 and placenta 17.99±0.50%. Altogether it can be said that chilli pedicel contains more ash content than the fruit. As shown in Figure 3.3.1(q), jackfruit peel (JFP) holds 5.67±0.15 % total ash, 4.53±0.66% water soluble ash and 1±0.03% acid insoluble ash. Feili et al. (2013) analysed jackfruit peel and found that it contains 5.91±0.22% total ash which is close to the finding of present study. Jackfruit peel is found to be the least explored fruit part for its phytochemical values. Major literatures were confined of jackfruit flesh and seeds. Morton (1987), Eke-Ejiofor (2013) and Koh et al. (2014) studied the jackfruit flesh and stated ash content 2.2 %, 0.43% and 0.99% respectively. Jackfruit seed ash contain was also stated in few reports i.e. 0.15±0.01% (Gupta et al., 2011), 2.70 % (Ocloo et al., 2010) and 5.05±0.07 (Okafor et al., 2015). Relationship of all these studies indicated that jackfruit peel contains more ash content than flesh and seeds. Figure 3.3.1(q): JFP ash values Figure 3.3.1(r): CC ash values 77

38 As presented in Figure 3.3.1(r) cucumber peel (CC) contains 2.91±0.15% total ash, 1.94±0.03% water soluble ash and 0.94±0.05% acid insoluble ash. Findings noted by Ghosia et al. (2014) and Abulude et al. (2007) for cucumber peel shares resemblance with present results i.e. 2.20% and 3.65% respectively. Ash content of cucumber flesh recorded by other studies are 0.4 % (Roe et al., 2013), 1.54% (Gopalakrishnan and Kalaiarasi, 2014) and 9.67% Ash (Okoye Ngozi, 2013) which shared a range of variation in ash content of fruit peel. The total ash is particularly important in the evaluation of purity of drugs, i.e. the presence or absence of foreign matter such as metallic salts or silica. Water soluble ash value is more than acid insoluble ash indicated that the amount of acid-insoluble siliceous matter present was less than that of water soluble ash ( ) Extractive Values Selected plant materials were extracted with four different solvents i.e. water, methanol, ethyl acetate and hexane according to polarity. Extractive values are primarily useful for the determination of exhausted or adulterated drugs and it is an important tool to check the quality and variation in chemical constituents of the drug. The extractive values are indicators of total solvent soluble component. Figure 3.3.2: Comparison between various extractive values of selected plant materials 78

39 Comparative extractive value of all selected fruit and vegetable by-products are shown in figure which indicates that maximum samples have higher extractive value in methanol followed by water (a) Onion peel Table 3.3.2(a): Extractive values of OP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Reddish glittery solid 10.92±0.15 brown 2. Methanol Reddish/ sticky solid 6.93± Ethyl Soxhlet Yellowish solid 1.38±0.17 acetate extraction brown/ sticky 4. Hexane white solid 1.16±0.08 Maximum extractive value for onion peel (OP) was recorded in distilled water followed by methanol, ethyl acetate and hexane as demonstrated in table 3.3.2(a). Extract colour was ranged from reddish brown to white (b) Garlic Peel Table 3.3.2(b): Extractive values of GP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature 1. Water Dark brown solid 4.66± Methanol Soxhlet Dark brown/ Solid 3.71±0.27 extraction sticky 3. Ethyl yellow solid 0.23±0.01 acetate 4. Hexane Whitish/sticky solid 3.78±0.20 Yield (%W/W) Maximum extractive value for garlic peel was recorded in distilled water followed by hexane, methanol and ethyl acetate. Its yield (%), colour and physical nature is shown in table 3.3.2(b) 79

40 3.3.2 (c) Potato Peel Table 3.3.2(c): Extractive values of PTP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Dark brown/ solid 8.78±1.18 less sticky 2. Methanol Brown/ sticky solid 13.53± Ethyl acetate Soxhlet Yellowish solid 0.63±0.03 extraction brown/ sticky 4. Hexane Yellowish/ sticky solid 2.77±0.22 Maximum extractive value for potato peel was recorded in methanol followed by distilled water, hexane and ethyl acetate. Extract colour ranged from dark brown to yellow (d) Mango Peel Table 3.3.2(d): Extractive values of MNP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Brown/ sticky Solid 12.91± Methanol Yellowish dark brown/ sticky semisolid 31.73± Ethyl acetate Soxhlet Dark olive Solid 5.44±0.06 extraction green /sticky 4. Hexane Greenish yellow/ sticky Solid 3.25±0.25 Maximum extractive value for mango peel (MNP) was recorded in Methanol followed by distilled water, ethyl acetate and hexane. Extractive details are mentioned in table 3.3.2(d) (e) Mango Seed Kernel As mentioned in table 3.3.2(e), maximum extractive value for mango seed (MNS) was recorded in Methanol followed by distilled water, hexane and ethyl acetate. Extract colour was ranged from chocolate brown to cream. 80

41 Table 3.3.2(e): Extractive values of MNS with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Chocolate brown solid 16.24± Methanol Chocolate brown solid 20.93± Ethyl Soxhlet extraction /waxy Light yellow semisolid 1.97±0.03 acetate 4. Hexane White/ cream Waxy greasy semisolid 8.06± (f) Pigeon pea pod shell Table 3.3.2(f): Extractive values of TP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Blackish brown solid 7.75± Methanol Soxhlet Reddish brown/sticky mass solid 9.66± Ethyl acetate extraction Dark green/ solid 1.67±0.03 sticky 4. Hexane Brownish green solid 0.97±0.11 Maximum extractive value for pigeon pea pod shell (TP) was recorded in Methanol followed by distilled water, ethyl acetate and hexane. Extractive value details has mentioned in table 3.3.2(f) (g) Pea pod shell Table 3.3.2(g): Extractive values of PSP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Dark brown/ Solid 16.19±0.18 sticky 2. Methanol Soxhlet extraction Greenish brown semisolid 34.59± Ethyl acetate Dark green/ solid 7.36±0.04 sticky 4. Hexane Green/ sticky semisolid 1.40±

42 As shown in table 3.3.2(g), maximum extractive value for pea pod shell (PSP) was recorded in Methanol followed by distilled water, ethyl acetate and hexane. Results were supported by the study done by Hadrich et al. (2014). He found that the extract yield value of pea peel extracts were 3.75±0.3% for ethyl acetate, 33.5±2.3% for methanol and 21.22±1.3% for water was recorded (h) Black plum seeds Table 3.3.2(h): Extractive values of JS with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Dark brown Solid 17.01± Methanol Chocolate semisolid 24.95± Ethyl acetate Soxhlet extraction brown Light yellow semisolid 0.20± Hexane Brown/ sticky solid 0.69±0.08 As represented in table (h), in black plum seeds (JS), methanol was found to have maximum extractive value followed by distilled water, hexane and ethyl acetate. Modi et al. (2010) have recorded 14 %W/W alcohol soluble extractive, 22 %W/W Water soluble extractive, 28 %W/W Petroleum-ether soluble and 36 %W/W Chloroform soluble extractive value which varies at some extend with the present results. Similarly Chitnis et al. (2012) studies three different samples of jamun seed powder and found 45,45, 35% alcohol soluble extractive value, 45, 45, 40% Methanol soluble extractive value, 20, 55, 40% water soluble extractive values. Agrawal and Argal (2014) reported alcohol soluble extractive (14.94%), water soluble extractive values (22.20%) and pet-ether extractive values (27.30%) respectively. Nair et al. (2013) also noted extractive value of jamun seeds in petroleum ether 0.6%, Chloroform 1%, ethyl acetate 1.2%, methanol 0.07% and water 1.16% (i) Custard apple seeds As presented in table 3.3.2(h), custard apple seeds (SS) have maximum extractive value in distilled water followed by hexane, methanol and ethyl acetate. Extract colour ranged from chocolaty brown to yellow. 82

43 Table 3.3.2(i): Extractive values of SS with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Chocolaty brown/ solid 5.51±0.54 crystalline powder 2. Methanol Soxhlet Brown/ waxy solid 2.05± Ethyl acetate extraction Light yellow semisolid 1.81± Hexane Yellow/ oily liquid 3.31± (j) Orange peel Table 3.3.2(j): Extractive values of ORP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Reddish brown Solid 11.65± Methanol Soxhlet Yellowish semisolid 35.86±0.11 extraction brown/ sticky 3. Ethyl acetate Reddish brown/ solid 3.14±0.13 sticky 4. Hexane Yellow/ sticky solid 1.48±0.08 Extractive values of orange peel (ORP) were maximum in methanol followed by distilled water, ethyl acetate and hexane as mentioned in table 3.3.2(j). According to other reports, the extractive value obtained using water as solvent was the highest (17.60 ± 0.75%) while that of chloroform was the lowest (2.83 ± 0.09%). Water extract was followed by methanol extract (12.82 ± 0.76%) then acetone and ethyl acetate were 3.98 ± 0.00% and 3.27 ± 0.13%, respectively (Arawande et al., 2012). Arora and Kaur (2013) also reported extract yield of orange peel in acetone 1.5%, methanol 60.6%, hexane 1.2% and distilled water 12.7% (k) Sweet lime peel Extractive values of sweet lime peel were reported maximum in methanol followed by water, ethyl acetate and hexane as mentioned in Table 3.3.2(k). 83

44 Table 3.3.2(k): Extractive values of SLP with different solvents Sr. No. Solvent Method of Extraction Colour 1. Water Brown glittery/ sticky 2. Methanol Soxhlet Dark yellow/ extraction sticky 3. Ethyl acetate Greenish yellow/ sticky 4. Hexane Brownish green/sticky Physical Yield nature (%W/W) Solid 15.08±0.10 semisolid 25.15±0.21 Solid 1.46±0.10 Solid 0.54± (l) Watermelon peel Table 3.3.2(l): Extractive values of WMP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Reddish brown/ Solid 17.45±0.48 sticky 2. Methanol Soxhlet Dark green semisolid 35.02± Ethyl extraction Cream colour with solid 0.96±0.01 acetate greenish pinch 4. Hexane Dark green/ sticky solid 2.04±0.08 Watermelon peel (WMP) was observed to have maximum extractive value with polar solvents i.e. methanol and water followed by hexane and ethyl acetate. Extract colour was ranged from reddish brown to dark green. Extractive details for WMP have demonstrated in table 3.3.2(l) (m) Peanut pod shell Table 3.3.2(m): Extractive values of GNP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Chocolaty solid 8.52±0.17 brown 2. Methanol Soxhlet Dark brown solid 19.96± Ethyl extraction Yellow/ solid 3.13±0.11 acetate sticky 4. Hexane Cream coloured semisolid 1.69±

45 Extractive value details of peanut pod shell (GNP) have represented in table 3.3.2(m) and it was reported maximum in methanol followed by water, ethyl acetate and hexane (n) Pomegranate peel As presented in table 3.3.2(n), extractive value of pomegranate peel (PGP) was reported maximum in methanol followed by water, ethyl acetate and hexane. Similar findings were recorded by Sangeetha and Vijayalakshmi (2012), they found the water soluble extractive value 42.46% and alcohol soluble extractive 30.53%. Table 3.3.2(n): Extractive values of PGP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Reddish brown Solid 20.46± Methanol Dark reddish solid 35.95±0.21 Soxhlet brown/ sticky 3. Ethyl extraction Cream coloured solid 0.55±0.01 acetate with reddish pinch 4. Hexane White with solid 0.41±0.04 greenish pinch 3.3.2(o) Split green gram peel Table 3.3.2(o): Extractive values of MDP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Chocolate brown/ solid 3.05±0.10 powder 2. Methanol Dark olive green solid 2.17± Ethyl Soxhlet Light green/ sticky solid 0.20±0.01 acetate extraction 4. Hexane Green/ sticky solid 1.80±0.19 Extractive value details of split green gram peel (MDP) have mentioned in table 3.3.2(o). MDP was reported to have maximum extractive value with water, followed by methanol, hexane and ethyl acetate (p) Chilli Pedicel As represented in table 3.3.2(p), extractive value of chilli pedicel (CA) was recorded maximum in methanol followed by water, hexane and ethyl acetate. 85

46 Table 3.3.2(p): Extractive values of CA with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Powdery Brown solid 9.21± Methanol Dark green solid 17.93± Ethyl Soxhlet Dark olive green/ solid 0.70±0.01 acetate extraction sticky 4. Hexane Dark green solid 1.36± (q) Jackfruit peel Table 3.3.2(q): Extractive values of JFP with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water Chocolaty brown solid 6.10± Methanol Brown solid 1.45± Ethyl acetate Soxhlet Yellow/ sticky solid 1.16± Hexane extraction White with yellow pinch/ sticky solid 3.76±0.19 Extractive values for Jackfruit peel (JFP) were recorded maximum with water followed by hexane, methanol and ethyl acetate. Extract colour was ranged from brown to white (r) Cucumber peel Table 3.3.2(r): Extractive values of CC with different solvents Sr. No. Solvent Method of Extraction Colour Physical nature Yield (%W/W) 1. Water brown/ Sticky Solid 8.80± Methanol Yellow/sticky solid 17.86± Ethyl acetate Soxhlet Olive green/ semisolid 1.09±0.04 extraction sticky 4. Hexane Brownish green/ sticky solid 2.14±0.11 Extractive value of cucumber peel (CC) was reported maximum with methanol followed by water, hexane and ethyl acetate. Details of extractive values have mentioned in table 3.3.2(r). 86

47 The water soluble extractive value indicated the presence of sugar, acids and inorganic compounds. The alcohol soluble extractive values indicated the presence of polar constituents like phenols, alkaloids, steroids, glycosides, flavanoids and secondary metabolites present in the plant sample (Sangeetha and vijayalakshmi, 2012). Extractive values give an idea about the chemical constituents present in the drug and indicate the presence of sugar, acids and inorganic compounds; water soluble extractive value found to be more than that of alcohol soluble extractive value suggests that the powdered drug have high water soluble extractive value. ( ) Qualitative Analysis Phytochemical constituents such as tannins, flavonoids, alkaloids or secondary metabolites possess nutritive and pharmacological activities (Britto and Sebastiana, 2011). Thus the preliminary screening test may be useful in the detection of the bioactive principles and subsequently may lead to the drug discovery and development (Doss, 2009) (a) ALKALOIDS Sr. No. Table 3.3.3(a): Results of qualitative screening for alkaloids Plant/Test Mayer s DragendoRff s Hager's Wagner's Test test Test Test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PG MDP CA Tannic acid test 87

48 17. JFP CC Preliminary screening for alkaloids was confirmed by five different tests. It has been observed that OP, GP, PTP, PSP, SS, ORP, SLP, PG and CC extracts gave positive results in all the tests (table 3.3.3(a)). Among all these GP, PTP and SLP were recorded to have maximum alkaloid concentration. The presence of alkaloids is noteworthy as the biological function of alkaloids and their derivatives are very important and are used in analgesic, antispasmodic and bactericidal activities (Stary, 1998) (b) PHENOLS Table 3.3.3(b): Results of qualitative screening for total phenolic compounds Sr. No. Plant/ Test Ferric chloride Lead acetates Gelatin test Potassium Dichromate Alkaline reagent test test test Test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PG MDP CA JFP CC The presence of phenolic compounds was confirmed by five different spot tests. It was observed that all compounds gave positive results for the compounds. TP, PSP, GP, CC, MNP and WMP gave negative results in few tests but rest were indicated the presence of phenolic compounds in all tests (table 3.3.3(b)). Phenolic compounds are an important group of metabolites with strong antioxidant properties (Tang and 88

49 Cronin, 2007). Phenolics are reported also to have antimicrobial activity (Dixon, 2001) (c) FLAVONOIDS Sr. No. Table 3.3.3(c) : Results of qualitative screening for flavonoid compounds Plant/ Alkaline Shinoda Ethyl acetate- Pew test Test reagent Test Ammonia Test test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PG MDP CA JFP CC The presence of flavonoid was confirmed by four different screening tests and noticed that many of the selected fruit and vegetable by-products gave positive results for the tests. Among all selected plants OP, GP, MNP, MNS, JS and PG gave positive results in all tests and rest were found positive in one or two tests while JFP and GNP didn t confirm the presence of flavonoids in any of the tests (table 3.3.3(c)). Flavonoids are found to be distributed throughout the plant kingdom belongs to polyphenolic compounds. They exhibit certain biological effects such as antiinflammatory, anti-hepatotoxic and anti-ulcer actions (Bors et al., 2001) and also reported for antioxidant properties antibacterial and antimicrobial properties (Qian and Nihorimbere, 2004). The presence of this group of polyphenols can be helpful for further exploration of selected plant material for their biological activities. 89

50 3.3.3(d) TANNINS Sr. No. Table 3.3.3(d): Results of qualitative screening for tannins Plant/ Test Ferric chloride Lead Acetate Test Test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PG MDP CA JFP CC Potassium Dichromate Test The presence of tannins in present study was confirmed by three different methods and OP, GP, MNS, JS, SS, ORP, SLP, WMP and PG confirmed presence of tannins in all three tests while rest showed positive results in one or two (table 3.3.2(d)). The extracts of GNP, TP, CA and JFP didn t show any positive results for tannins. Tannins are also polyphenolic compounds. They are very important when it comes to the nutritive value of human diet or phytotherapeutical value of plants (Cobzac et al., 2005) (e) SAPONINS Table 3.3.3(e): Results of qualitative screening for saponins Sr. No. Plant/ Test Frothing test Sodium bicarbonate test 1. OP GP PTP MNP MNS TP

51 7. PSP JS SS ORP SLP WMP GNP PG MDP CA JFP CC -- + Saponins were analysed qualitatively through two selected methods from selected waste materials. All extracts responded positive for the presence of saponins except JFP which showed negative results in both the tests (table 3.3.3(e)). Saponins are important group of secondary metabolites possessing bioactive properties like antimicrobial activity (Lanzotti et al., 2012). Citrus sinensis peels are rich source of saponins with haemolytic activity and cholesterol binding properties (Kumar et al., 2011). Saponins also possess the properties like cholesterol binding, precipitating and coagulating red blood cells and haemolytic activity (Sodipo et al., 2000) (f) TERPENOIDS Table 3.3.3(f) : Results of qualitative screening for terpenoids Sr. No. Plant/ Test Salkowski's Liebermann - Hesse s test Burchard s Test reaction 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PGP MDP

52 16. CA JFP CC Only few waste material showed positive results for the terpenoids i.e. PTP, ORP, PSP, SS, SLP, CA and CC (table 3.3.3(f)). Terpenoids are also an important secondary metabolites with antibacterial activities and also reported for inhibiting human pancreatic adenocarcinoma cells (Sanchez et al., 2010; Madar et al., 1995; Zore et al., 2011) (g) GLYCOSIDES Sr. No. Table 3.3.3(g) : Results of qualitative analysis for glycosides Keller Borntragers Modified Legal killiani Test Borntragers Test test Test Plant/ Test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PGP MDP CA JFP CC Baljet Test Screening tests for glycosides were found to be positive in OP, PTP, MNP, MNS, TP, PSP, JS, SS, ORP, WMP and CA extracts while it was reported absent in JFP, MDP, PGP, SLP, GP and GNP extracts (table 3.3.3(g)). 92

53 3.3.3(h) STEROIDS Sr. No. Table 3.3.3(h): Results of qualitative analysis for steroids Plant/ Libermann- Salkowski Acetic Libermann Test Buchard test test anhydride- sterol test H2so4 Test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PG MDP CA JFP CC Screening tests for steroids were found to be positive for MNS, JS, SS, ORP, PG, CA and SLP while rest of the extracts showed negative results (table 3.3.3(h)). Plant steroids are useful in herbal medicine and cosmetics and also known for their cardio tonic activities (Salah et al., 1995) (i) SUGAR/CARBOHYDRATES Table 3.3.3(i): Results of qualitative analysis for sugar/ carbohydrates Sr. No. Plant/ Test Molish s test Fehling s test Barfoed s test Benedict s test Iodine Test 1. OP GP % + 3. PTP % MNP >2% + 5. MNS % + 6. TP % + 7. PSP % + 8. JS % SS % -- 93

54 10. ORP % SLP % WMP % GNP % PG >2% MDP % CA % JFP % CC % -- Sugar/Carbohydrates are essential primary metabolites which are present in all plants (table 3.3.3(i)). In present study also all the extracts were gave positive results for the confirmative tests (j) AMINO ACIDS AND PROTEINS Sr. No. Table 3.3.3(j): Results of qualitative analysis for Protein Plant/ Test Millon s Biuret Ninhydrin test test test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PG MDP CA JFP CC Xanthoproteic Test Proteins are also an important structural primary metabolites which are present in all plants. All selected waste material extracts showed positive results for screening test of protein. Only one extract i.e. OP was found to have negative results (table 3.3.3(j)). 94

55 3.3.3(k) FATS AND FIXED OILS Table 3.3.3(k): Results of qualitative analysis for fats and fixed oils Sr. No. Plant/ Test Spot test Saponification test 1. OP GP PTP MNP MNS TP PSP JS SS ORP SLP WMP GNP PG MDP CA JFP CC Only a few extracts i.e. MNS, SS, ORP, SLP, PG and JFP confirmed the presence of fats and fixed oils (table 3.3.3(k)) while rest of the extracts expressed negative results for confirmation tests of fats and fixed oils QUANTITATIVE ANALYSIS OF METABOLITES (a) Onion peel As shown in figure 3.3.4(a.1), the concentration of primary metabolites in onion peel was 37.94±0.44 mg/g (total sugar), 35.15±1.27 mg/g (reducing sugar), and 2.79±1.70 mg/g (non-reducing sugar) 3.92±0.26 mg/g (starch), 9.27±2.17 mg/g (Protein). Only few records are available for onion peel which showed higher protein and carbohydrate value than the present one. Bello et al. (2013) noted 2.64±0.01 g/100g (26.4mg/g) crude protein content and Sayed et al. (2014) found 3.06% (30.6 mg/g) protein content and 82.15% (821.5 mg/g) total carbohydrates in onion peel. 95

56 Figure 3.3.4(a.1): Primary metabolites (mg/gm) present in OP Remarkably, the current findings share similarities with the content of onion bulb documented in several reports. These observations are 4.74 ± 0.5 g/100g (47.4mg/g) total sugar content and ± 0.05 g/100g (829.9 mg/g) crude protein content for Bangladeshi onion and 2.32 ± 0.2 g/100g (23.2 mg/g) total sugar content and ± 0.4 g/100g (14.89 mg/g) crude protein content for Indian onion studied by Bhattacharjee et al. (2013). Yahaya et al. (2010) and Nwinuka et al. (2005) noted 6.48±1.23 g/100g (64.8mg/g) and % (104.g mg/gm) crude protein in onion bulb respectively. Figure 3.3.4(a.2): Secondary metabolite (mg/gm) in OP Onion peel was found to contain 71.19±5.21 mg GA/g phenols, 45.28±0.40 mg QE/g flavonoids and 11.40±1.50 mg/g tannin (figure 3.3.4(a.2)). Similar results were recorded by Vanesa et al. (2011) who found that onion peel contains 52.7±0.9 mg GA/g total phenolics and 43.1±1.8 mg QE /g. Other studies on onion peel noted very 96

57 high concentration of phenol and flavonoids. Singh et al. (2009) analysed five extract/fractions of red onion peel and reported ± 5.0 mg GAE/g phenolics and 165.2± 3.2 mg QE/g flavonoids. Comparable high content were observed by Lee et al. (2014) who found mg GAE/g extract phenolic content and mg QE/g extract flavonoid content. Some reports are also available on whole onion which was analysed for total phenolics and flavonoids, Vanesa et al. (2011) observed mg GA/g and 12.9 mg GA/g total phenols; mg QE/g and 119 mg QE/g flavonoid content. This indicates that onion peel contains more phenolics and flavonoids than the whole onion (b) Garlic peel Figure (b.1): Primary metabolites (mg/gm) in GP Garlic peel was found to contain 1.47±0.45 mg/g total sugar, 0.71±0.31 mg/g reducing sugar, 0.76±0.14 mg/g non-reducing sugar, 4.82±0.17 mg/g starch and 6.33±0.66 mg/g protein (figure 3.3.4(b.1)). Ifesan et al. (2014) analysed garlic peel and found 0.57 ± 0.02% (5.7 mg/g) protein content which is close to present finding. But carbohydrate content was notably higher i.e ± 0.04% (932.6 mg/g). When the current findings of peel were compared with the proximate composition of bulb, it was noted that all primary metabolites were higher in bulb than peel. Nwinuka et al. (2005), Odebunmi et al. (2009) and Marina et al.(2014) recorded protein values % (85.8 mg/g), 7.87±0.76% (78.7 mg/g) and 7.87±0.32% (78.7 mg/g) respectively from garlic bulb which are almost ten times more than the values reported for peel. Similarly higher protein (15.33±0.0% and 16.55±0.01%) and 97

58 carbohydrate (73.22±0.0% and 70.31±0.11%) values were documented by Otunola et al. (2010) and Okolo et al. (2012) respectively. Quantified secondary metabolites in garlic peel were 12.23±0.28 mg/g total phenols, 8.72±1.55 mg/g flavonoids and 5.17±1.41 mg/g tannin content. Quantified secondary metabolites in garlic peel were 12.23±0.28 mg/g total phenols, 8.72±1.55 mg/g flavonoids and 5.17±1.41 mg/g tannin content (figure 3.3.4(b.2)). Figure (b.2): Secondary metabolites (mg/gm) in GP Ifesan et al. (2014) also analysed garlic peel and found ± 2.17 (μg/500μg) total phenolic content and ± 2.95 (μg QUE/500μg) total flavonoid content which are much higher than the present findings. Though garlic peel is less explored, many reports are available on garlic buds. According to Otunola et al. (2010) and Fidrianny et al. (2013), garlic contains 7.84 total phenolics, % flavonoids and 0.07% tannins. Othman et al. (2011) and Abdel-Salam (2014) also reported ±2.31 mg GAE/100 g total phenolic content and 7.25 ug/gaemg flavonoid content. This also showed that peel contains higher concentration of secondary metabolites than garlic buds (c) Potato peel Figure 3.3.4(c.1) indicated that potato peel contains 13.50±0.96 mg/g total sugar, 10.75±0.66 mg/g reducing sugar, 2.75±0.31 mg/g non-reducing sugar, 36.01±1.07 mg/g starch and 2.64±1.44 mg/g protein. Mahmood et al. (1998) also reported the similar results for total sugar 1.40% (14.0 mg/g) and reducing sugars 0.91% (9.1 98

59 mg/g) while the results for starch (667.8 mg/g) and protein (147.0 mg/g) were much higher than the present findings. Figure 3.3.4(c.1): Primary metabolites (mg/gm) in PTP Dhingra et al. (2014) also found higher protein content (14.04 %) in potato peel. Amado et al. (2014) also analysed potato peel and found 6.47 ± 0.23% (64.7 mg/g) protein, 0.92 ± 0.17% (9.2 mg/g) soluble sugar and ± 0.43% (869.7 mg/g) for carbohydrates. According to United States Department Agriculture national nutrient database (USDA, 2004), potato tuber contains 2.57% (25.7 mg/g) protein, 12.44% (124.4 mg/g) carbohydrate and 0.78/100G (7.8 mg/g) total sugars. USDA (2004) records are quite close to the present findings while other reports indicated much higher concentration of primary metabolites. Figure 3.3.4(c.2): Secondary metabolites (mg/gm) in PTP 99

60 Figure 3.3.4(c.2) shows secondary metabolites analysed for potato peel were 15.73±0.64 mg/g total phenols, 4.58±0.83 mg/g flavonoids and 8.78±2.57 mg/g tannins. Several reports are available on potato peel analysis and have range of variation in results. Kalpna et al. (2011) determined the phenol and flavonoid content from different extracts of potato peel. She reported that methanolic extract of potato peel possessed ± 0.34mg/g total phenolics and 5.78 ± 0.17 mg/g flavonoids. Likewise other extracts hexane, chloroform and acetone contains 4.45 ± 0.28 mg/g, 9.60 ± 0.51 mg/g and ± 0.71mg/g total phenols respectively and 2.20 ± 0.23 mg/g, 8.93 ± 0.41 mg/g and 5.78 ± 0.17 mg/g flavonoid contents respectively. These results are quite similar to the results obtained in present study. Other studies exhibited lower phenolic content in potato peel when compared with present findings. These reports showed phenolic content 68.7 ± 5.7 mg/100 g and 26.1 ± 0.5 mg/100 g (Farvin et al., 2012), mg/100g (Kanatt et al., 2005), 4.3 ± 0.2mg/g and 2.5 ± mg/g (Kahkonen et al., 1999), 522.1±2.14 ug/g (Azadeh et al., 2012) and 2.91 mg/g (Mohdaly et al., 2010). Peel exhibited higher phenol content as phenolic compounds are mostly distributed between the potato cortex and skin (peel) tissues (Friedman, 1997) and about 50% of the phenolic compounds are located in the potato peel and adjoining tissues, while the rest decrease in concentration from the outside toward the center of potato tubers (Hasegawa et al., 1966) (d) Mango peel Figure 3.3.4(d.1): Primary metabolites (mg/gm) in MNP 100

61 As represented in figure 3.3.4(d.1), mango peel was found to hold 42.28±0.35 mg/g total sugar, 30.6±0.50 mg/g reducing sugar, 11.68±0.59 mg/g non-reducing sugar, 18.84±2.71 mg/g starch and 10.93±2.32 mg/g protein. Mango peel quantification results from other studies were quite higher than the current results. Ashoush and Gadallah (2011) reported 3.6 ± 0.15 % (36 mg/g) protein content and Ashifat et al. (2012) found 4.32 % (43.2 mg/g) protein and 57.92% (579.3 mg/g) carbohydrate. Figure 3.3.4(d.2): Secondary metabolites (mg/gm) in MNP As represented in figure 3.3.4(d.2), mango peel was found to contain 21.19±1.20 mg/g total phenols, 9.68±0.40 mg/g flavonoids and 3.34±0.60 mg/g tannins. When present findings were compared with other literature, it was noticed that total phenolic content (i.e ± 0.30 mg GA/g) noted by Ashoush and Gadallah (2011) shared resemblance with obtained results whereas total flavonoid (F) content shared similarities with results obtained by Kalpna et al. (2011) but she also found the higher total phenolic (TP) content. According to her analysis mango peel contains 1.53 ± 0.16 mg/g (TP), ± 0.82 mg/g (F) for hexane extract, ± 1.17 mg/g (TP) ± 0.42 mg/g (F) for chloroform extract, ± 0.43 mg/g (TP) ± 0.20 mg/g (F) for acetone extract and ± 0.30 mg/g (TP) 4.83 ± 0.08 mg/g (F) for methanol extract (e) Mango seed kernel Mango seed kernel was revealed to contain 43.32±0.06 mg/g total sugar, 33.55±0.37 mg/g reducing sugar, 9.76±0.30 mg/g non-reducing sugar, 21.86±5.45 mg/g starch and 20.62±2.82 mg/g protein (figure 3.3.4(e.1)). Several other studies have also been 101

62 observed on mango seed kernel in comparison with present study. These studies have reported higher concentration of protein and carbohydrates when matched with present findings. Figure 3.3.4(e.1): Primary metabolites (mg/gm) in MNS Nzikou et al. (2010) found 6.36% (63.6 mg/g) crude protein and 32.24% (322.4 mg/g) total carbohydrate. Another study by Fowomola (2010) showed ± 0.12% (100.6 mg/g) crude protein and ± 1.34% (701.2 mg/g) carbohydrate. Similarly Yatnatti et al. (2014) noted 7.53g/100g protein and 69.77g/100g carbohydrate content. Figure 3.3.4(e.2): Secondary metabolites (mg/gm) in MNS As shown in figure 3.3.4(e.2), methanolic extract of mango seed kernel was found to hold 45.57±1.08 mg/g total phenols, 13.5±1.66 mg/g flavonoids and 7.24±1.83 mg/g tannin content. Several studies have been done for the analysis of mango seed kernel and found that present findings showed higher phenol content when compared with 102

63 results of other studies. Ashoush and Gadallah (2011) recorded ± 0.33 (mg GAE/g) phenol content and Deng et al. (2012) found 7.54 mg GAE/g phenols from seed kernel extract. Similarly Eva et al. (2012) noted total flavonoids ranged between 0.057±0.007 to 1.3±0.1 catechin/100g DW and tannins 1.5±0.1 to 11±2 tannic acid/100g DW (f) Pigeon Pea Pod Shell Pigeon pea pod shell contains 30.35±4.73 mg/g total sugar, 23.83±1.37 mg/g reducing sugar, 6.51±3.36 mg/g non-reducing sugar, 13.18±1.37mg/g starch and 24.83±1.22 mg/g protein content (figure 3.3.4(f.1)). Figure 3.3.4(f.1): Primary metabolites (mg/gm) in TP No scientific reports are available on pigeon peapod shell but few studies have been carried out on pigeon pea seed coat, seeds and seed splits. Morton (1976) and Singh et al. (1968) studied seed coat of tur dal and found 5.6% crude protein and 58.7% carbohydrates. Saxena et al. (2010) analysed seed and seed splits and observed 48.4, 57.6% starch; 21.0, 24.6% protein and 5.1, 5.2% soluble sugars respectively. Balogun (2013) and Adamu and Oyetunde (2013) recorded 4.11% and 30.53% protein content and Carbohydrate content. Metabolites content in pigeon pea pod shell is noteworthy even though it is lesser than the seeds, the edible part of plant. Anti-nutritional factors in pigeon pea pod shell as shown in figure 3.3.4(f.2) were recorded as 11.49±1.70 mg/g total phenolics, 3.01±0.23 mg/g flavonoids and 3.86±0.44 mg/g tannins. According to reviewed literature, pigeon pea pod shells are 103

64 still unexplored for its anti-nutritional composition. But, several reports are available on pegion pea seeds. Figure 3.3.4(f.2): Secondary metabolites (mg/gm) in TP It has been mentioned that pegion pea contains 3.0 to 18.3 mg/g total phenols, 0.0 to 0.2 mg/g (Singh, 1988) and 1.05 mg/100g tannins (Balogun, 2013). These findings were lower than the obtained results. Florence et al. (2014) observed 83.0 ± 0.02 mg GA/g total phenols and 46.0 ± 0.01mg pyrocatechol /g flavonoids from pigeon pea (g) Pea pod shell Figure 3.3.4(g.1): Primary metabolites (mg/gm) in PSP Pea pod shell was found to contain 43.25±0.33 mg/g total sugar, 17.47±0.54 mg/g reducing sugar, 25.77±0.88 mg/g non-reducing sugar, 9.52±0.14 mg/g starch and 17.18±3.11 mg/g protein. A few studies have been done for the analysis of pea pod shell which was compared with the present results. Sharoba et al. (2013) reported 104

65 % total protein and Rehman et al. (2015) found 90.33±1.85% total sugar in pea peel which is quite higher than the present results. Mishra et al. (2010) analysed a range of pea varieties and found to 24.67% protein and to 63.53% carbohydrates in pea seeds. Figure (g.2): Secondary metabolites (mg/gm) in PSP Pea pod shell contains 8.79±0.36 mg/g total phenols, 5.99±0.46 mg/g flavonoids and 3.68±1.22 mg/g tannin (figure 3.3.4(g.2)). Pea plant was found less explored as a very few reports are available on pea seed analysis. Pea pod shell is found totally unexplored. Hadrich et al. (2014) analysed different extracts of pea and found phenolic content mg GE/g (ethyl acetate extract), mg GE/g (methanol extract), mg GE/g (water extract) and flavonoid content mg QE/g (ethyl acetate extract), mg QE/g (methanol extract), and mg QE/g (water extract) (h) Black plum seeds As shown in figure 3.3.4(h.1), black plum seeds contains 44.05±0.041mg/g total sugar, 28.32±0.048 mg/g reducing sugar, 15.73±0.02 mg/g non-reducing sugar, 16.79±2.89 mg/g starch and 13.09±2.54mg/g protein content. Present findings were found to resemble with analysis results for jamun seeds recorded by Shahnawaz et al. (2009). He noted that shade dried seed powder contains 1.22 % (12.2 mg/g) reducing sugar, 1.84 % (18.4 mg/g) non reducing sugar and 3.06 % (30.6 mg/g) total Sugar, % (721.7 mg/g) carbohydrates and 5.18 % (51.8 mg/g) protein. Raza et al. (2015) also recorded protein content (19.7±0.59 mg/g) which is close to present results. While previous works have shared resemblance with current work, a study on 105

66 black plum seed has reported values which were higher. Ranjan et al. (2011) documented 6.3 to 8.5% protein and 41% starch in black plum seeds. Figure 3.3.4(h.1): Primary metabolites (mg/gm) in JS Black plum seed extract contains 49.97±1.68 mg/g total phenols, 11.14±0.53 mg/g flavonoids and 7.35±0.27 mg/g tannins. Black plum seeds have an important place in Ayurveda. Hence, they are studied well and also explored for their phytochemical importance. Figure 3.3.4(h.2): Secondary metabolites (mg/gm) in GP Total phenolic content from present finding shares resemblance with the results noted by Shahnawaz et al. (2010). He found that phenolic content in black plum seeds ranged within mg/gm to mg/gm. While Margaret et al. (2015) reported lower phenol content (16.1.mg/g) from methanolic extract but flavonoid content (19.1 mg/g) was found closer to present findings. Ranjan et al. (2011) documented tannin content i.e. 6 to 19% which is much higher than the obtained results. 106

67 3.3.4(i) Custard apple seeds Figure 3.3.4(i.1): Primary metabolites (mg/gm) in SS As represented in figure 3.3.4(i.1), custard apple seeds were found to contain 35.33±1.08 mg/g total sugar, 6.80±0.68 mg/g reducing sugar, 28.52±0.41 mg/g nonreducing sugar, and 17.73±5.76 mg/g starch and 29.29±1.32 mg/g protein. Several other reports on custard apple seeds were also examined and compared with the present findings. The content of crude protein and carbohydrate were reported higher than present findings. Hassan et al. (2008) noted 4.4 ± 0.72% Crude protein and ± 1.80% carbohydrate from seeds which are almost double than the current results. Similarly Mariod et al. found 17.5 ± 0.2a % Protein and 30.0 ± 0.3% carbohydrate which is again almost double than the reports of Hassan et al. (2008). It shows a range of variation in proximate composition of custard apple seeds. Figure 3.3.4(i.2): Secondary metabolites (mg/gm) in SS 107

68 Custard apple seeds contain 14.13±1.20 mg/g total phenols, 10.0±1.36 mg/g flavonoids and 2.65±1.23mg/g tannin content (figure 3.3.4(i.2)). Custard apple seeds are also well explored for their phytochemical importance. Present findings were found similar with the results obtained by Gowdhami et al. (2014). They recorded 11.9 mg/g total phenolics and 6.5mg/gm total flavonoids. Bhardwaj et al. (2014) noted the lower concentration of phenols (250 ug/100 mg) and flavonoids (339 ug/100mg). Whereas Kadarani et al. (2015) and Kothari and Seshadri (2010) documented higher content. Former studied total phenolic content from unripe and ripe seeds and obtained mg GE/gm and 85.3mg GE/gm total phenol content respectively. Later analysed the various extracts of seeds and recorded total phenol and flavonoid content mg GE/g; mg QE/g (Chloroformmethanol extract) and mg GE/g; mg QE/g (Ethanol extract) respectively (Kothari and Seshadri, 2010) (j) Orange peel Figure 3.3.4(j.1): Primary metabolites (mg/gm) in ORP As represented in figure 3.3.4(j.1), orange peel contains 44.15±0.34 mg/g total sugar, 35.39±0.16 mg/g reducing sugar, 8.76±0.50 mg/g non-reducing sugar, 5.95±2.46 mg/g starch and 31.84±2.60 mg/g protein content. Several studies have been reviewed on orange peel analysis. Oikeh et al. (2013) found that the total protein content in orange peel was 3.94 ± 0.25% (39.4 mg/g) which shares resemblance with present result. Peter et al. (2013) also recorded the similar results for protein (4.05±0.25% i.e mg/g) but found higher concentrations for carbohydrate (42.90±1.00%) and fat 108

69 (10.00±0.00%). Another findings by Mahmood et al. (1998) showed alike results for starch content i.e. less than 1.00% (<10.0 mg/g) with current results and also noted the higher concentrations for total soluble sugars 15.0% DW, reducing sugars 9.16% and crude protein 6.53%. Nassar et al. (2008) and Adewole et al. (2014) also recorded the 5.15±0.38% and % protein content and 9.21±0.22% and 40.47±0.37% carbohydrate content respectively which were higher than present study. 8.72±0.36% total protein and 1.57±0.02% total fat content from orange peel was noted by Sharoba et al. (2013). Figure 3.3.4(j.1): Secondary metabolites (mg/gm) in ORP Orange peel contains 10.55±0.52 mg/g total phenols, 3.53±0.63 mg/g flavonoids and 5.86±0.74 mg/g tannins. Several studies have been conducted for phytochemical analysis of orange peel and a range of variation was reported among all results. Park et al. (2014) noticed that more phenolic components were extracted with methanol and acetone solvent and he stated that the phenolic content in orange peel ranged from 1.39 mg GAE/100 g to 1.85 mg GAE/100 g which is lesser than the current obtained results. Bernard et al. (2014) also recorded the lower flavonoid content i.e mg/100g. Current results were found to be closer with the findings of Abd El-aal HA and Halaweish (2010). They stated that total phenolic content of orange peel ranged from ± 1.99 to ± 2.20 mg/100g. Adewole et al. (2014) documented that the orange peel contains 0.07±0.06% phenol, 7.40±0.06% tannin and 0.16±0.01% flavonoid. Total phenol and flavonoid content from other studies were mg/g total phenolic content, µg/g total flavonoid content (Hegazy and Ibrahium, 2012), mg/g total phenolic content, 0.3 mg/g total flavonoid content 109

70 (Singh and Immanuel, 2014) and 158 mg/g total phenolic content (Arora and Kaur, 2013) (k) Sweet lime peel Figure 3.3.4(k.1): Primary metabolites (mg/gm) in SLP Sweet lime peel powder was found to contain 43.58±0.02 mg/g total sugar, 30.94±0.20 mg/g reducing sugar, 12.63±0.18 mg/g non-reducing sugar, 5.58±0.58 mg/g starch and 12.97±1.44 mg/g protein content (figure 3.3.4(k.1)). Only one report is available on sweet lime peel powder which noted higher protein content i.e. 5.39% and 1.58% fat (Younis et al., 2015). Figure 3.3.4(k.2): Secondary metabolites (mg/gm) in SLP 110

71 Sweet lime peel found to contain 11.67±0.32 mg/g total phenols, 4.47±0.46 mg/g flavonoids and 6.53±0.59 mg/g tannin content (figure 3.3.4(k.2)). Similar results were obtained by Muthiah et al. (2012) who found the 7.39 to μg/mg total phenol content and 0.51 to 21.62μg/mg total flavonoid content. Younis et al. (2015) also noted 1.92 ± 0.12% total phenol content in sweet lime peel (l) Watermelon peel Figure 3.3.4(l.1): Primary metabolites (mg/gm) in WMP Watermelon peel found to contain 42.80±0.18 mg/g total sugar, 26.19±0.53mg/g reducing sugar, 16.61±0.35mg/g non-reducing sugar, 5.2±2.15 mg/g starch and 32.35±0.66 mg/g protein (figure 3.3.4(l.1)). Present findings were resembled with the study done by Erukainure et al. (2010). He evaluated fermented and unfermented watermelon rinds and found 1.52±0.05% and 2.80±0.05% protein; 4.68±0.10% and 5.94±0.10% carbohydrate respectively. Fila et al. (2013) have analysed fresh and dry watermelon rind and his observations also shares similarity with current results. According to him fresh and dry watermelon rind holds 0.41±0.02 g/100g and 3.07±0.02 g/100g crude protein; 29.65±0.58 g/100g and 81.62±0.05 g/100g carbohydrates respectively. Similarly, Cemaluk and Egbuonu (2015) have evaluated proximate composition of seeds and peel. In peel they found 7.04±0.00% crude protein, 80.75±0.04% carbohydrate, 0.47±0.01% total sugar and 1.42±0.01% total soluble sugar. Few studies also observed higher proximate composition than the present one. El-Badry et 111

72 al. (2014) and Hanan et al. (2013) reported 8.70±0.28% and 11.17% protein; 76.16±0.52% and 56.02% carbohydrate respectively. Figure 3.3.4(l.2): Secondary metabolites (mg/gm) in WMP Watermelon rind possesses 4.79±0.69 mg/g total phenols, 7.61±0.48mg/g total flavonoids and 2.79±0.73 mg/g tannin content (figure 3.3.4(l.2)). Several analysis have been done on watermelon peel for its phytochemical evaluation and a range of variation have been noticed in all results. Erukainure et al. (2010) analysed fermented and unfermented watermelon peel and stated 12.15±0.09 mg/100g and 13.23±0.42 mg/100g tannin content respectively which are lesser than the obtained results in present study. Similar findings were documented by Johnson et al. (2012) for fresh and dry watermelon rinds. They found that the fresh and dry rind holds 1.05 mg/100g and 1.15 mg/100g (tannin), 0.18 mg/100g and 0.45 mg/100g (phenols) and 8.71 mg/100g and 2.63 mg/100g (flavonoids) respectively. Nessma (2015) also recorded 70.20±0.10 mg/g total phenolic and 45.70±0.10 mg/g tannins which are higher than the current findings and 1.29±0.00 mg/g flavonoids which is lesser than present results (m) Peanut pod shell As presented in figure 3.3.4(m.1), peanut pod shell found to comprise of 24.35±2.90 mg/g total sugar, 5.90±0.22 mg/g reducing sugar, 18.44±3.13 mg/g non-reducing sugar, 6.22±0.31 mg/g starch and 7.61±2.43 mg/g protein. Present results were compare with findings of earlier studies on peanut pod shell and found a range of variation in all results. 112

73 Figure 3.3.4(m.1): Primary metabolites (mg/gm) in GNP Kerr et al. (2006) studied two different varieties of peanut and found protein content ranged within 5.8± 0.0 g/100g to 6.1± 0.5 g/100g. He also noted the carbohydrate content in peanut shell was 2.5% and 37.0% cellulose. Fakhriya et al. (2012), Kerr et al. (1986) and Hegazy et al. (1991) have reported the protein content in peanut pod shell 3.90± 0.12%, 8.2% and 6.90% respectively. Figure 3.3.4(m.2): Secondary metabolites (mg/gm) in GNP Results obtained for peanut pod shell were 16.74±2.20 mg/g total phenols, 2.93±0.13 mg/g total flavonoids and 3.23±0.17 mg/g tannin content (figure 3.3.4(m.2)). Only a few reports are available for peanut pod shell as it is a less explored material. When the present findings were compared with other studies, it was found that Marwin et al. (2011) reported higher phenolic content i.e ±2.82 mg GAE/g and Sim et al. (2012) found lesser phenol content in pod shells of two different varieties i.e. 113

74 Shandong PS (175± 3.9 mg/100g) and Menglembu PS (255± 8.1 mg/100g). Yen and Duh (1995) analysed peanut hulls from various cultivars and stated that phenolic content was available within the range of 4.2mg/g to 10.2mg/g (n) Pomegranate peel Pomegranate peel found to comprise of 44.35±0.20mg/g total Sugar, 20.68±0.04 mg/g reducing Sugar, 23.66±0.16 mg/g non-reducing Sugar, 7.58±1.87 mg/g starch and 9.65±2.10 mg/g protein (figure 3.3.4(n.1)). Present findings were compared with several other studies which analysed pomegranate peel and found to have reduced results than others. Naseem et al. (2012) have observed ± 0.3% total sugar, 30.40± 0.11 %reducing Sugar, 0.98 ± 0.12% non-reducing sugar and 8.719± 0.10 % protein content in pomegranate peel. Figure 3.3.4(n.1): Primary metabolites (mg/gm) in PGP Middha et al. (2013) also documented reducing sugars (16.94%), proteins (4.90%) and fructose (15.622%) from peel. 3.10% Protein and 80.50% Carbohydrate content was recorded by Rowayshed et al. (2013). The results of pomegranate peel analysis showed variation and none of the results are close to each other. Pomegranate peel was found to contain 49.01±6.98 total phenols, 22.98±1.14 total flavonoids and 34.44±5.40 tannin content (figure 3.3.4(n.2)). Pomegranate peel was seems to be highly explored fruit by-product as numerous study reports are available for pomegranate peel with a range of variation. 114

75 Figure 3.3.4(n.2): Secondary metabolites (mg/gm) in PGP Al-Rawahi et al. (2014) documented somewhat similar results with present findings. He noted 64.2 mg GAE/g total phenolic content and 1.4 mg CE/g dry total flavonoid content. Similarly Middha et al. (2013) documented higher phenolic content and similar flavonoid content for pomegranate peel. He stated methanolic and water extract of pomegranate peel possesses 185 ± 2.45 to ± 4.86 mg GA/ g total phenols and ± 1.54 to 49.8 ± 2.14 mg/g total flavonoids. Elfalleh et al. (2012) also stated the concentration of total phenols, flavonoids and tannins as ± 4.87 mg GAE/ g, ± 8.14 mg/g and ± 4.25 mg/g respectively. Nuamsetti et al. (2012) and Shiban et al. (2012) reported total phenolic content mg GAE/100g and 91.2 mg GAE/g respectively. While Rajan et al. (2011) and Yehia et al. (2011) documented higher concentration of phenol, flavonoids and tannins in comparison with current findings. Former studied ethanol and aqueous extracts of pomegranate rind and showed ±8.08 mg/g and 81.33±6.1 mg/g flavonoid content, 81.66±3.51 mg/g and ±12.16 mg/g tannins and ±6.42 mg/g and ±5.29 mg/g total phenols. Later found ±3.21 mg GAE/g total phenols and 47.27±1.54 mg QE/g flavonoid content. Similarly Negi and Jayaprakasha (2003) and Kulkarni et al. (2004) analysed pomegranate peel from Kashmir district and reported mg GAE/g phenolic content and mg CE/g flavonoid content. 115

76 3.3.4(o) Split green gram peel Figure 3.3.4(o.1): Primary metabolites (mg/gm) in MDP Split green gram peel (moong dal) contains 1.73±0.06 mg/g total sugar, 2.54±0.14 mg/g reducing sugar, 0.80±0.08 mg/g non-reducing sugar, 18.47±2.43 mg/g starch and 46.13±1.01 mg/g protein content (figure 3.3.4(o.1)). No relevant studies have found for split green gram peels but few reports are available on green gram and green gram splits. Habibullah et al. (2007), Paul et al. (2011) and Riaz et al. (2014) have analysed few varieties of vigna radiata and found protein content %, carbohydrates % and crude fat %. Similarly Phule et al. (2013) studied green gram splits and reported 1.71±0.002% Fat, 22.16±1.1% protein, 0.30±0.001% Fiber and 59.12±1.5% carbohydrates. All above reviewed results were higher than the current findings of green gram peel. Figure 3.3.4(o.2): Secondary metabolites (mg/gm) in MDP 116

77 Split green gram commonly known as mung dal. Its peel was analysed for its phytochemical composition and results were found as 8.34±0.60 mg/g total phenols, 3.28±0.13 mg/g total flavonoids and 4.21±0.80 mg/g tannin content (figure 3.3.4(o.2)). No research reports are available on non-nutritional components of green gram or green gram splits and its peels (p) Chilli Pedicel Chilli pedicel found to contain 13.04±0.58 mg/g total sugar, 8.51±0.04 mg/g reducing sugar, 4.52±0.62 mg/g non-reducing sugar, 10.97±2.33 mg/g starch and 18.71±2.71 mg/g protein (figure 3.3.4(p.1)). No related studies have been reported for chilli pedicel but a number of reports are available on chilli and its different varieties with a series of variation in findings. Figure 3.3.4(p.1): Primary metabolites (mg/gm) in CA Several results on chilli cultivars were found to close with the present findings. Twelve cultivars of chilli have been evaluated by Gupta and Tambe (2003) and they reported that large differences for the content of protein (1.44 to 2.1%), carbohydrate (6.05 to 11.11%) and fat (0.92 to1.56%). Similar observation was noted by Ogunlade et al. (2012). He found the proximate composition values (%) were: crude protein (2.64 to 3.51%), fat (1.52 to 2.87%), crude fibre (2.37 to 4.71%) and carbohydrates (4.62 to 6.71%). Various other studies were also matched and noticed higher content of primary metabolites in chilli fruit. Zaki et al. (2013) analysed chemical composition of chilli at different harvest time and findings were ranged as ± 4.8 to ± 3.3 g/100 g carbohydrate, ± 2.6 to ± 4.5 g/100 g protein and 6.88 ± 0.47 to ± 0.11 g/100 g total sugar. Similarly Sarker et al. (2012) and 117

78 Emmanuel et al. (2014) reported 5.42±0.43% and 11.67±0.03% protein content; 7.41±0.87% and 66.98±0.05% carbohydrate content respectively. Simonovska et al. (2014) studied different parts of red hot pepper i.e. pericarp, seed and placenta and noted 14.13±0.36, 20.88±0.75 and 25.19±0.35 Proteins (%) and 19.73±0.82, 2.95±0.38 and 20.42±0.73 reductive sugar (%) respectively. As shown in figure 3.3.4(p.2), chilli pedicel was found to consist of 10.23±0.94 mg/g total phenols, 2.10±0.34 total flavonoids and 9.12±0.80 tannin content. Chilli pedicel was found to be unexplored for its phytochemical characterization but chilli fruit has been evaluated in numerous studies with diverse range of findings. When the findings of other studies for chilli fruit were compared with current obtained results for chilli pedicel, it was noticed that pedicel contains more concentration of non-nutritive compounds Figure 3.3.4(p.2): Secondary metabolites (mg/gm) in CA Medina-Juárez et al. (2012) analysed five different varieties of capsicum annuum and recorded total phenol within the range of 59.34mg/100g to mg/100g and total flavonoid from mg/100g to 60.36mg/100g. Similarly Hernández-Ortega et al. (2012) studied different colored two varieties of C. annuum and noted the total phenol content ranged from ±16.43 μg /g ±33.33 μg /g. Shaha et al. (2013) also stated that total phenolic content in chilli were in range of g/100 g for crude extract and g/ 100 g for dry powder. Kevers et al. (2007) reported total phenol content for different colored chilli viz. red, yellow and green, as 296,

79 and 215mg/100g, respectively. Sun et al. (2007) stated that flavonoids in c. annuum are ranged between not detectable to 80mg QE/100mg. Shaha et al. (2013) have observed that the maximum amount of total phenolic content (4135 µg GAE/g) was recorded at full ripening stage while for flavonoid (2.59 µmol Q/g) it was in intermediate ripening stage. Esayas et al. (2011) analysed different varieties of chilli and tannin content was reported within the range of to mg/100g. Highest concentration of metabolites were recorded by Emmanuel et al. (2014) who stated 1.44±0.22% Tannins, 4.91±0.31% flavonoid, 1.55±0.11% alkaloids, 3.00±0.12% phenolics (q) Jackfruit peel Jackfruit rind have found to contain 36.21±1.07mg/g total sugar, 18.00±0.51 mg/g reducing sugar,18.20±1.40 mg/g non-reducing sugar, 8.27±3.33 mg/g starch and 15.01±2.76 mg/g protein content (figure 3.3.4(q.1)). Figure 3.3.4(q.1): Primary metabolites (mg/gm) in JFP Feili et al. (2013) and Koh et al. (2014) analysed jackfruit rind and found protein content 4.52% and 1.54% respectively. The concentration reported by Koh et al. (2014) was found closer to present data. They also reported 79.32% carbohydrates and 1.71% crude lipid. Jackfruit peel is been explored less than the seeds and flesh. Eke-Ejiofor (2013) and Morton (1987) noted 1.12 and 1.8% protein content; and 25.4% carbohydrate content respectively. Protein content reported in these studies share resemblance with the current findings. Protein and carbohydrate content in 119

80 jackfruit seed was reported %, 11.85%, 15.88% and %, 26.20%, 29.52% by Ocloo et al. (2010), Gupta et al. (2011) and Okafor et al. (2015) respectively. Figure 3.3.4(q.2): Secondary metabolites (mg/gm) in JFP Jackfruit peel possesses 7.94±1.22 mg/g total phenols, 1.29±0.71 mg/g total flavonoids and 3.54±0.80 mg/g tannin content (figure 3.3.4(q.2)). As per the reviewed literature, jackfruit peel seems least analysed for its phytochemical values. Several reports were available on jackfruit seeds and reported for less phenolic and flavonoid content when compared with current obtained results. Gupta et al. (2011) analysed jackfruit seeds and obtained the values 1.45±0.007 (µg GAE/ mg) for total phenolic content and 290.6±3.414 (µg RE/ mg) for total flavonoid content (r) Cucumber peel Figure 3.3.4(r.1): Primary metabolites (mg/gm) in CC 120

81 As shown in figure 3.3.4(r.1), cucumber peel contain 28.01±1.10 mg/g total sugar, 18.6±0.15 mg/g reducing sugar, 8.37±3.66 mg/g starch, 9.41±1.25 mg/g reducing sugar, and 4.42±0.66 mg/g protein. Similar study was commenced by Abulude et al. (2007) and Ghosia et al. (2014) and they found that cucumber peel contains 3.84% protein, % fat, 7.53% carbohydrates and 0.5 % Brix (Sugar content). Roe et al. (2013) analysed raw cucumber skin and flesh and reported starch content <0.1% and total sugar was 1.2% which is resembled with the present findings. He also mentioned about protein (1.0%), fat (0.6%) and sugars like Glucose (0.5%), Fructose (0.7%), Sucrose (<0.1%), Maltose (<0.1%) and Lactose (<0.1 %). Okoye Ngozi (2013) studied cucumber fruit and noted the findings as protein 17.25±0.16% and 63.06±0.20% Carbohydrate. Figure 3.3.4(r.2): Secondary metabolites (mg/gm) in CC Cucumber peel was found to contain 5.21±0.30 mg/g total phenols, 2.19±0.27 total flavonoids and 1.75±0.24 mg/g tannin content. Currently obtained results were compared with the other findings in reviewed literature. Agarwal et al. (2012) reported that cucumber peel contains 7.4 mg GA/g total phenolic content which shares resemblance with present finding and 17.1 mg QE/g total flavonoid content which is higher than the present one. Other studies showed the analysis results of cucumber fruit and were noticed lesser than the present findings. Budhiraja et al. (2014) found 6.23 µg / mg total phenolics and 1.82 µg / mg total flavonoids. Similarly Gopalakrishnan and Kalaiarasi (2014) 121

82 noted 2.63 mg/kg total flavonoids, 0.12 mg/kg tannins and 0.18mg/kg phenols. This shows higher secondary metabolites content in peel than the pulp HPTLC based Chemical Fingerprinting of Selected Plants The fruit and vegetable waste materials were investigated for their phytochemical profile and also subjected to the HPTLC analysis. TLC (Thin layer Chromatography) is a technique to separate the compounds from a mixture according to their polarity. Selected by-products were found positive for several phytochemicals in qualitative and quantitative phytochemical screening. Various phyto-chemicals present in the crude extracts have specific λmax and can be seen in visible light and/or in the UV range. Spray reagents are used to facilitate the calculation for Rf value the separated compounds on TLC plate. The reagents react with the components and spots can be easily seen in the visible light. Khan and Nasreen (2010), used various derivatization reagents such as dregendroff's reagent, mayer's reagent, wagner's reagent, iodine vapours, ammonium vapours etc. In the current study, HPTLC profiles of selected fruit and vegetable by-products were developed. Three extract of all plants were used to develop the profile. Solvent system (mobile phase) used in the study were optimized (shown in table 3.3.5) and the systems with better results were subjected for their HPTLC chemical profiling. The chromatogram represents the profile of plants used in the experiment. Thus these profiles can be utilized for the future quality control of formulation/product. Four solvent systems were finalized are as follows. Table 3.3.5: Selected solvent systems for HPTLC analysis Sr. Solvent system Proportion Derivatization Components No. (v/v) 1 Chloroform: 90:10 DDR reagent followed by Alkaloids Methanol 10% Ethanolic H2SO4 2 Toluene:Ethylacetate: Formic acid 50:40:10 1% AlCl3 Flavonoids 122

83 3.3.5 (A) Results for Solvent System I (Suitable for alkaloid detection) Total 18 samples each with three different extract were analysed. On a single plate, 9 samples were spotted and allowed to run with solvent system. The instrument details are as follows. Solvent system: Chloroform: Methanol (90:10) v/v Spray reagent: DDR reagent followed by 10% Ethanolic H2SO4 Spotting of sample: For each solvent system two plates were prepared comprising of 9 samples on each plate. Sequence of the samples shown in table 3.3.5(A). Table 3.3.5(A): Sequence of samples loaded on TLC plate Sample Sample Name Sample Sample Name No. No. 1. OP - Onion Peel 10. ORP - Orange peel 2. GP - Garlic peel 11. SLP - sweet lime peel 3. PTP - Potato peel 12. WMP - Watermelon peel 4. MNP - mango peel 13. GNP - peanut pod shell 5. MNS - mango seed kernel 14. PGP - pomegranate peel 6. TP - pigeon pea pod shell 15. MDP- Split green gram peel 7. PSP - pea pod shell 16. CA - chilli Pedicel 8. JS- black plum seeds 17. JFP - Jackfruit peel 9. SS - custard apple seeds 18. CC- cucumber peel Band width: 8.0 mm TLC plate size: 20.0 x 10.0 cm Distance between two bands: 21.2 mm Sample concentration: 2mg/ml Sample volume: 10.0 μl 123

84 Figure 3.3.5(A-1): TLC chromatogram for Methanol extracts visualized in various lights representing separated compounds Figure 3.3.5(A-2): TLC chromatogram for Ethyl acetate extracts visualized in various lights representing separated compounds Figure 3.3.5(A-3): TLC chromatogram for Hexane extracts visualized in various lights representing separated compounds 124

85 Figure 3.3.5(A-4): 3-D graphical display of absorbance peaks for Methanol extracts Figure 3.3.5(A-5): 3-D graphical display of absorbance peaks for Ethyl acetate extracts Figure 3.3.5(A-6): 3-D graphical display of absorbance peaks for Hexane extracts 125

86 Sample 1: OP Figure 3.3.5(A-7): Densitometric chromatogram showing peak display of onion peel extracts (Density (AU) vs Rf ) As shown in figure 3.3.5(A-7), scanning at different wavelengths exhibited that maximum bands i.e. 20 were observed at 254 nm for methanolic extract of onion peel. This was followed by 366 nm and 400 nm with 16 and 15 bands respectively. Rf value for the compounds were recorded in-between 0.02 to Ethyl acetate extract of onion peel showed 20 bands with Rf value range at 254 nm. 9 Numbered peak was found to cover maximum area of the chart i.e % with Rf and 14 bands were recorded for 366 nm and 400nm respectively with the Rf value range of 0.06 to th peak and 7 th peak with maximum chart area of 41.89% and 53.33% were reported for 366 and 400 nm respectively. Hexane extract represented 20, 9 and 6 bands at 254, 366 and 400 nm respectively. Minimum Rf value was 0.01 and maximum was Maximum chart area was covered by 7 th band with 77.97% at 366nm. Sample 2: GP As represented in figure 3.3.5(A-8), methanolic extract of garlic peel showed maximum 21 bands at 254 nm followed by 17 at 366 nm and 14 at 400 nm. Rf value was ranged between 0.05 to Compound no. 11 th at 254 nm was found to cover maximum 23.51% area at 0.77 Rf. Ethyl acetate extract showed 26 and 17 separated compounds when scanned at 254 and 366, 400 nm. Recorded Rf value ranged between 0.05 to At all three wavelength, the maximum chart area was covered 126

87 by the same compound having 0.72 Rf value. It covered 18.03%, 34.42% and 31.48% respectively for 254, 366 and 400 nm. Figure 3.3.5(A-8): Densitometric chromatogram showing peak display of Garlic peel extracts (Density (AU) vs Rf ) For hexane extract, 20, 5 and 4 bands were recorded when scanned at 254, 366 and 400 nm. Rf value was ranged between 0.01 to Maximum chart area was covered by 19 th compound with 11.78% at 254 nm. While at 366 and 400, 4 th compound was reported to cover maximum density with 34.56% and 35.52% respectively Sample 3: PTPEA Figure 3.3.5(A-9): Densitometric chromatogram showing peak display of Potato peel extracts (Density (AU) vs Rf ) Potato peel methanolic extract exhibited 21bands when scanned at 254 nm with 0.03 minimum and 0.98 Rf values. Maximum area i.e % covered by peak no. 11 with 127

88 Rf value It showed 19 and 13 separated compounds at 366 and 400 nm respectively. Rf value ranged between 0.07 to 0.98 and maximum chart area was covered by 1 st compound with 33% at 400 nm. Potato peel EA extract was separated in 20 bands with 0.06 minimum and 0.94 maximum Rf values when analysed under 254 nm wavelength. 10 th Numbered peak was found to cover maximum area of the chart i.e % with Rf and 12 bands were found at 366 and 400 nm respectively. Maximum chart area was covered by 9 th compound with 31.50% at 366 nm. 7 h peak was found to cover maximum area of the chart i.e % at 400 nm. Hexane extract was separated in 16 and 8 bands when scanned under 366 and 400 nm. Rf value was ranged between 0.02 to Maximum chart area was covered by 2 nd compound with 57.63% at Rf 0.02 for 366 nm (Figure 3.3.5(A-9)). Sample 4: MNP As shown in figure 3.3.5(A-10), mango peel methanol extract was found to be separated in 22 bands when scanned at 254 nm with the Rf value ranged within 0.24 and It showed 19 bands when scanned under 366 nm with minimum 0.03 and maximum 0.95 Rf values. Maximum area i.e % was covered by 10 th peak with Rf separated compounds were observed when scanned at 400 nm with minimum 0.05 Rf and maximum 0.95 Rf values. Maximum area i.e % was covered by peak no. 8. Figure 3.3.5(A-10): Densitometric chromatogram showing peak display of mango peel extracts (Density (AU) vs Rf ) 128

89 Similarly mango peel ethyl acetate extract showed 23 separated compounds with 0.06 minimum and 0.99 maximum Rf values when analysed under 254 nm wavelength. 1st peak was found to cover maximum area of the chart i.e % with Rf It showed 18 bands with 0.05 minimum and 0.99 maximum Rf values when analysed under 366 nm wavelength. 1st peak was found to cover maximum area of the chart i.e % with Rf Hexane extract showed to be separated in 22 bands when scanned at 254 nm with 0.02 minimum and 0.94 maximum Rf values. 7th peak was found to cover maximum area of the chart i.e. 8.52% with Rf bands with 0.04 minimum and 0.93 maximum Rf values were recorded when analysed under 366 nm wavelength. 3rd peak was found to cover maximum area of the chart i.e % with Rf 0.26 at 400 nm it showed 9 bands with 0.05 minimum and 0.82 maximum Rf values. 3rd peak was found to cover maximum area of the chart i.e % with Rf SAMPLE 5: MNS As represented in figure 3.3.5(A-11), mango seed kernel methanolic extract exhibited 22 bands when scanned at 254 nm, 22 bands when analysed under 366 nm wavelength and 17 different bands at 400 nm. Mango seed kernel EA extract was showed 24 bands when analysed under 254 nm, 18 bands with 0.02 minimum and 0.88 maximum Rf values when analysed under 366 nm wavelength and 12 bands with 0.02 minimum and 0.99 maximum Rf values when analysed under 400 nm wavelength. Figure 3.3.5(A-11): Densitometric chromatogram showing peak display of mango seed kernel extracts (Density (AU) vs Rf ) 129

90 Mango seed kernel HE extract was found to be separated in 18 bands when analysed under 254 nm wavelength, 8 bands with 0.04 minimum and 0.98 maximum Rf values when analysed under 366 nm wavelength and 7 bands with 0.03 minimum and 0.94 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 6: TP Figure 3.3.5(A-12): Densitometric chromatogram showing peak display of TP extracts (Density (AU) vs Rf ) Pigeon pea pod shell methanolic extract exhibited 17 bands when scanned at 254 nm with 0.08 minimum and 0.91 Rf values, 18 bands when analysed under 366 nm wavelength with 0.06 minimum and 0.97 maximum Rf values and 17 bands at 400 nm with maximum 0.96 Rf and minimum 0.10 Rf. Ethyl acetate extract was separated in 23 bands with 0.07 minimum and 0.94 maximum Rf values when analysed under 254 nm wavelength, 11 bands with 0.06 minimum and 0.97 maximum Rf values when analysed under 366 nm and 12 bands with 0.04 minimum and 0.97 maximum Rf values when analysed under 400 nm wavelength. Hexane extract was separated in 20 bands when analysed under 254 nm wavelength, 10 bands with 0.03 minimum and 0.96 maximum Rf values when analysed under 366 nm wavelength and 9 bands with 0.04 minimum and 0.96 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(A-12)). 130

91 SAMPLE 7: PSP As shown in figure 3.3.5(A-13), pea pod shell methanol extract exhibited 28 bands when scanned at 254 nm with 0.09 minimum and 0.98 Rf values, 11 bands when analysed under 366 nm wavelength with 0.06 minimum and 0.98 maximum Rf values and 11 bands at 400 nm wavelength. Figure 3.3.5(A-13) - Densitometric chromatogram showing peak display of PSP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 23 bands with 0.03 minimum and 0.97 maximum Rf values when analysed under 254 nm wavelength, 18 bands with 0.04 minimum and 0.99 maximum Rf values when analysed under 366 nm wavelength and 19 bands with 0.04 minimum and 0.98 maximum Rf values when analysed under 400 nm wavelength. While hexane extract was separated in 18 bands with 0.01 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 7 bands with 0.04 minimum and 0.80 maximum Rf values when analysed under 366 nm wavelength and 5 bands with 0.05 minimum and 0.87 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 8: JS As shown in figure 3.3.5(A-14), black plum seeds methanolic extract exhibited 24 bands when scanned at 254 nm with 0.04 minimum and 0.94 Rf values, 17 bands when analysed under 366 nm wavelength with minimum 0.02 and 0.97 maximum Rf values and 11 bands at 400 nm which indicates 11 different compounds. Where minimum Rf value was 0.03 (band 2) and maximum Rf value was Ethyl acetate extract was separated in 20 bands with 0.03 minimum and 0.93 maximum Rf values 131

92 when analysed under 254 nm wavelength, 16 bands with 0.02 minimum and 0.97 maximum Rf values when analysed under 366 nm wavelength and 16 bands with 0.02 minimum and 0.97 maximum Rf values when analysed under 400 nm wavelength. Hexane extract was separated in 16 bands with 0.09 minimum and 0.94 maximum Rf values when analysed under 254 nm wavelength, 8 bands with 0.07 minimum and 0.99 maximum Rf values when analysed under 366 nm wavelength and 7 bands with 0.05 minimum and 0.99 maximum Rf values when analysed under 400 nm wavelength. Figure 3.3.5(A-14): Densitometric chromatogram showing peak display of JS extracts (Density(AU) vs Rf ) SAMPLE 9: SS Figure 3.3.5(A-15): Densitometric chromatogram showing peak display of SS extracts (Density (AU) vs Rf ) 132

93 As shown in figure 3.3.5(A-15), custard apple seeds methanolic extract exhibited 26 bands when scanned at 254 nm with 0.03 minimum and 0.95 Rf values, 16 bands when analysed under 366 nm wavelength with minimum 0.02 and 0.97 maximum Rf values and 11 bands at 400 nm with the highest recorded Rf value 0.97 and lowest Ethyl acetate extract was separated in 28 bands with 0.03 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 21 bands with 0.03 minimum and 0.99 maximum Rf values when analysed under 366 nm wavelength and 21 bands with 0.03 minimum and 0.98 maximum Rf values when analysed under 400 nm wavelength. HE extract was separated in 22 bands with 0.01 minimum and 0.95 maximum Rf values when analysed under 254 nm wavelength, 13 bands with 0.01 minimum and 0.92 maximum Rf values when analysed under 366 nm wavelength and 10 bands with 0.01 minimum and 0.99 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 10: ORP Figure 3.3.5(A-16): Densitometric chromatogram showing peak display of ORP extracts (Density(AU) vs Rf ) Orange peel methanolic extract exhibited 21 bands when scanned at 254 nm with 0.01 minimum and 0.90 Rf values, 21 bands when analysed under 366 nm wavelength with 0.01 minimum and 0.97 maximum Rf values and 15 bands (i.e. 15 compounds) when scanned at 400 nm. Maximum Rf value was recorded 0.90 and minimum was Ethyl acetate extract was separated in 26 bands with 0.02 minimum and 0.97 maximum Rf values when analysed under 254 nm wavelength, 18 bands with 0.02 minimum and 0.96 maximum Rf values when analysed under 366 nm wavelength,

94 bands with 0.02 minimum and 0.93 maximum Rf values when analysed under 400 nm wavelength. Hexane extract was separated in 18 bands with 0.01 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 8 bands with 0.01 minimum and 0.91 maximum Rf values when analysed under 366 nm wavelength and 6 bands with 0.04 minimum and 0.75 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(A-16)). SAMPLE 11: SLPMH Figure 3.3.5(A-17): Densitometric chromatogram showing peak display of SLP extracts (Density(AU) vs Rf ) As shown in figure 3.3.5(A-17), sweet lime peel methanol extract exhibited 26 bands when scanned at 254 nm with 0.06 minimum and 0.97 Rf values, 26 bands when analysed under 366 nm wavelength with 0.02 minimum and 0.98 maximum Rf values and 25 bands when scanned under 400 nm. Minimum Rf value was recorded 0.09 and maximum was Ethyl acetate extract was separated in 24 bands with 0.05 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 16 bands with 0.06 minimum and 0.94 maximum Rf values when analysed under 366 nm wavelength and 17 bands with 0.06 minimum and 0.98 maximum Rf values when analysed under 400 nm wavelength. Hexane extract was separated in 17 bands with 0.01 minimum and 0.97 maximum Rf values when analysed under 254 nm wavelength, 12 bands with 0.01 minimum and 0.99 maximum Rf values when analysed under 366 nm wavelength and 10 bands with 0.01 minimum and 0.99 maximum Rf values when analysed under 400 nm wavelength. 134

95 SAMPLE 12: WMPMH Figure 3.3.5(A-18): Densitometric chromatogram showing peak display of WMP extracts (Density(AU) vs Rf ) As represented in figure 3.3.5(A-18), watermelon peel methanolic extract exhibited 25 bands when scanned at 254 nm with 0.02 minimum and 0.90 Rf values, 25 bands when analysed under 366 nm wavelength with 0.02 minimum and 0.94 maximum Rf values and 22 bands when analysed at 400 nm. Ethyl acetate extract was separated in 15 bands with 0.02 minimum and 0.95 maximum Rf values when analysed under 254 nm wavelength, 17 bands with 0.02 minimum and 0.78 maximum Rf values when analysed under 366 nm wavelength and 11 bands with 0.02 minimum and 0.93 maximum Rf values when analysed under 400 nm wavelength. Watermelon rind hexane extract was separated in 16 bands with 0.03 minimum and 0.93 maximum Rf values when analysed under 254 nm wavelength, 10 bands with 0.04 minimum and 0.78 maximum Rf values when analysed under 366 nm wavelength and 11 bands with 0.04 minimum and 0.98 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 13: GNPMH Peanut pod shell methanolic extract exhibited 29 bands when scanned at 254 nm with 0.07 minimum and 0.99 Rf values, 15 bands when analysed under 366 nm wavelength with 0.03 minimum and 0.97 maximum Rf values and 18 bands with maximum 0.95 Rf and minimum 0.09 Rf at 400 nm. Ethyl acetate extract was separated in 25 bands with 0.04 minimum and 0.98 maximum Rf values when analysed under 254 nm 135

96 wavelength, 21 bands with 0.04 minimum and 0.99 maximum Rf values when analysed under 366 nm wavelength and 15 bands with 0.04 minimum and 0.99 maximum Rf values when analysed under 400 nm wavelength. Figure 3.3.5(A-19): Densitometric chromatogram showing peak display of GNP extracts (Density(AU) vs Rf ) Peanut pod shell hexane extract was separated in 20 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 14 bands with 0.02 minimum and 0.93 maximum Rf values when analysed under 366 nm wavelength and 16 bands with 0.01 minimum and 0.93 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(A-19)). SAMPLE 14: PGPMH Figure 3.3.5(A-20): Densitometric chromatogram showing peak display of PGP extracts (Density (AU) vs Rf ) 136

97 As shown in figure 3.3.5(A-20), pomegranate peel methanolic extract exhibited 30 bands when scanned at 254 nm with 0.05 minimum and 0.95 Rf values, 28 bands when analysed under 366 nm wavelength with 0.03 minimum and 0.99 maximum Rf values and 28 bands at 400 nm with maximum and minimum Rf values 0.95 and 0.06 respectively. Ethyl acetate extract was separated in 25 bands with 0.04 minimum and 0.97 maximum Rf values when analysed under 254 nm wavelength, 25 bands with 0.04 minimum and 0.95 maximum Rf values when analysed under 366 nm wavelength and 16 bands with 0.04 minimum and 0.95 maximum Rf values when analysed under 400 nm wavelength. Pomegranate peel hexane extract was separated in 12 bands with 0.30 minimum and 0.90 maximum Rf values when analysed under 254 nm wavelength, 9 bands with 0.04 minimum and 0.75 maximum Rf values when analysed under 366 nm wavelength and 10 bands with 0.02 minimum and 0.97 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 15: MDP Green gram split peels methanolic extract exhibited 29 bands when scanned at 254nm with 0.02 minimum and 0.95 Rf values, 27 bands when analysed under 366nm wavelength with 0.02 minimum and 0.97 maximum Rf values and 13 bands with minimum 0.06 Rf and maximum 0.90 Rf. Figure 3.3.5(A-21): Densitometric chromatogram showing peak display of MDP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 26 bands with 0.01 minimum and 0.99 maximum Rf values when analysed under 254 nm wavelength, 16 bands with 0.03 minimum and 0.96 maximum Rf values when analysed under 366 nm wavelength and 137

98 14 bands with 0.04 minimum and 0.93 maximum Rf values when analysed under 400 nm. Green gram splits peel hexane extract was separated in 14 bands with 0.37 minimum and 0.96 maximum Rf values when analysed under 254 nm wavelength, 12 bands with 0.03 minimum and 0.97 maximum Rf values when analysed under 366 nm wavelength and 9 bands with 0.03 minimum and 0.80 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(A-21)). SAMPLE 16: CAMH Chili pedicel methanolic extract exhibited 29 bands when scanned at 254 nm with 0.02 minimum and 0.94 Rf values, 26 bands when analysed under 366 nm wavelength with 0.02 minimum and 0.96 maximum Rf values and11 bands with minimum 0.14 Rf and maximum 0.92 Rf. Ethyl acetate extract was separated in 25 bands with 0.05 minimum and 0.97 maximum Rf values when analysed under 254 nm wavelength, 15 bands with 0.02 minimum and 0.96 maximum Rf values when analysed under 366 nm wavelength and 14 bands with 0.02 minimum and 0.97 maximum Rf values when analysed under 400 nm. Figure 3.3.5(A-22): Densitometric chromatogram showing peak display of CA extracts (Density(AU) vs Rf ) Chili pedicel hexane extract was separated in 9 bands with 0.31 minimum and 0.88 maximum Rf values when analysed under 254 nm wavelength, 12 bands with 0.04 minimum and 0.96 maximum Rf values when analysed under 366 nm wavelength and 8 bands with 0.04 minimum and 0.95 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(A-22)). 138

99 SAMPLE 17: JFPMH Figure 3.3.5(A-23): Densitometric chromatogram showing peak display of JFP extracts (Density(AU) vs Rf ) As shown in figure 3.3.5(A-23), jackfruit peel methanolic extract exhibited 17 bands when scanned at 254 nm with 0.03 minimum and 0.92 Rf values, 27 bands when analysed under 366 nm wavelength with 0.03 minimum and 0.94 maximum Rf values and 24 bands at 400 nm with minimum 0.04 Rf and maximum 0.89 Rf. Ethyl acetate extract was separated in 24 bands with 0.05 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 12 bands with 0.06 minimum and 0.92 maximum Rf values when analysed under 366 nm wavelength and 10 bands with 0.07 minimum and 0.95 maximum Rf values when analysed under 400 nm wavelength. Jackfruit rind hexane extract was separated in 10 bands with 0.35 minimum and 0.88 maximum Rf values when analysed under 254 nm wavelength, 8 bands with 0.02 minimum and 0.97 maximum Rf values when analysed under 366 nm wavelength and 8 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 18: CCMH As shown in figure 3.3.5(A-24), cucumber peel methanolic extract exhibited 17 bands when scanned at 254 nm with 0.05 minimum and 0.98 Rf values, 27 bands when analysed under 366 nm wavelength with 0.06 minimum and 0.98 maximum Rf values and 13 bands with 0.08 minimum Rf and 0.96 maximum Rf. at 400 nm. 139

100 Figure 3.3.5(A-24): Densitometric chromatogram showing peak display of CC extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 26 bands with 0.04 minimum and 0.92 maximum Rf values when analysed under 254 nm wavelength, 19 bands with 0.07 minimum and 0.98 maximum Rf values when analysed under 366 nm wavelength, 15 bands with 0.07 minimum and 0.99 maximum Rf values when analysed under 400 nm wavelength. No remarkable results were reported in hexane extract of cucumber peel (B) Results for Solvent System II (Suitable for flavonoids detection) Total 18 samples each with three different extract were analysed. On a single plate, 9 samples were spotted and allowed to run with solvent system. The instrument details are as follows. Solvent system: Toluene: Ethyl acetate: Formic acid (50:40:10) v/v Spray reagent: 1% AlCl3 Spotting of sample: 10 samples were plotted on each plate. First spotted sample was quercetin referred as standard in case of this solvent system which is generally used for flavonoids detection. Quercetin was considered as sample zero and sequence for rest of the samples were the same as mentioned in previous solvent system. Band width: 8.0 mm TLC plate size: 20.0 x 10.0 cm Distance between two bands: 21.2 mm Sample concentration: 2mg/ml Sample volume: 10.0 μl 140

101 Figure 3.3.5(B-1): TLC chromatogram for Methanol extracts before derivatization visualized in various lights representing separated compounds Figure 3.3.5(B-2): TLC chromatogram for Methanol extracts after derivatization visualized in various lights representing separated compounds Figure 3.3.5(B-3): TLC chromatogram for Ethyl acetate extracts before derivatization visualized in various lights representing separated compounds 141

102 Figure 3.3.5(B-4) : TLC chromatogram for Ethyl acetate extracts after derivatization visualized in various lights representing separated compounds Figure 3.3.5(B-5): 3-D graphical display of absorbance peaks for Methanol extracts Figure 3.3.5(B-6): 3-D graphical display of absorbance peaks for Ethyl acetate extracts 142

103 Chromatograms of all samples for methanol, ethyl acetate and hexane extracts were scan under 254, 366 and 400 nm wavelengths. Among all, chromatograms for hexane extract didn t show any remarkable results. Standard compound: Quercetin Figure 3.3.5(B-7): Densitometric chromatogram showing peak display of Quercetin Quercetin was used as standard compound for flavonoid detection and spotted first on the plate. Scanning at 254nm detected 8 bands. The very first sharp peak represented quercetin at 0.05 Rf value with 7.75% total area covered on chart. At 366nm 4 peaks were noted and the first sharp peak was of quercetin with 0.05 Rf value and 6.91% covered chart area. 3 peaks were observed at 400nm and quercetin was detected with 0.05 Rf value with 5.37% covered chart area. Sample 1: OP Figure 3.3.5(B-8): Densitometric chromatogram showing peak display of onion peel extracts (Density (AU) vs Rf ) As shown in figure 3.3.5(B-8), onion peel methanolic extract exhibited 18 bands when scanned at 254 nm with 0.03 minimum and 0.92 Rf values, 8 bands when analysed under 366 nm wavelength with 0.03 minimum and 0.92 maximum Rf values and 7 bands when scanned under 400 nm. Minimum Rf value was recorded 0.03 and maximum was First separated compound represented the standard quercetin with 0.03 Rf and confirms its presence in onion peel. Ethyl acetate extract was separated in 10 bands with 0.01 minimum and 1.00 maximum Rf values when analysed under 254 nm wavelength, 8 bands with 0.01 minimum and 0.95 maximum Rf values when 143

104 analysed under 366 nm wavelength and 7 bands with 0.06 minimum and 0.95 maximum Rf values when analysed under 400 nm wavelength. Sample 2: GP Figure 3.3.5(B-9): Densitometric chromatogram showing peak display of Garlic peel extracts (Density (AU) vs Rf) As shown in figure 3.3.5(B-9), garlic peel methanolic extract exhibited 25 bands when scanned at 254 nm with 0.11 minimum and 0.99 Rf values, 9 bands when analysed under 366 nm wavelength with 0.11 minimum and 0.80 maximum Rf values and 7 bands when scanned under 400 nm. Minimum Rf value was recorded 0.10 and maximum was Ethyl acetate extract was separated in 19 bands with 0.10 minimum and 0.99 maximum Rf values when analysed under 254 nm wavelength, 08 bands with 0.12 minimum and 0.89 maximum Rf values when analysed under 366 nm wavelength and 8 bands with 0.05 minimum and 0.90 maximum Rf values when analysed under 400 nm wavelength. Sample 3: PTPEA Figure 3.3.5(B-10): Densitometric chromatogram showing peak display of Potato peel extracts (Density (AU) vs Rf ) 144

105 Potato peel methanolic extract exhibited 13 bands when scanned at 254 nm with 0.01 minimum and 0.94 Rf values, 11 bands when analysed under 366 nm wavelength with 0.05 minimum and 0.93 maximum Rf values and 09 bands when scanned under 400nm. Minimum Rf value was recorded 0.01 and maximum was First separated compound represented the standard quercetin and confirms its presence in potato peel. Ethyl acetate extract was separated in 14 bands with 0.01 minimum and 0.95 maximum Rf values when analysed under 254nm wavelength, 09 bands with 0.01 minimum and 0.99 maximum Rf values when analysed under 366 nm wavelength and 11 bands with 0.06 minimum and 0.99 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(B-10)) Sample 4: MNP As shown in figure 3.3.5(B-11), mango peel methanolic extract exhibited 18 bands when scanned at 254 nm with 0.01 minimum and 0.97 Rf values, 15 bands when analysed under 366 nm wavelength with 0.01 minimum and 0.94 maximum Rf values and 13 bands when scanned under 400 nm. Figure 3.3.5(B-11): Densitometric chromatogram showing peak display of mango peel extracts (Density(AU) vs Rf ) Minimum Rf value was recorded 0.01 and maximum was Second separated compound represented the standard quercetin and confirms its presence in mango peel. Ethyl acetate extract was separated in 9 bands with 0.32 minimum and 0.95 maximum Rf values when analysed under 254 nm wavelength, 7 bands with 0.34 minimum and 0.95 maximum Rf values when analysed under 366 nm wavelength and 12 bands with 0.07 minimum and 0.96 maximum Rf values when analysed under 400 nm wavelength. 145

106 SAMPLE 5: MNS Figure 3.3.5(B-12): Densitometric chromatogram showing peak display of mango seed kernel extracts (Density (AU) vs Rf ) As shown in figure 3.3.5(B-12), mango seed kernel methanolic extract exhibited 15 bands when scanned at 254 nm with 0.04 minimum and 0.87 Rf values, 11 bands when analysed under 366 nm wavelength with 0.01 minimum and 0.99 maximum Rf values and 5 bands when scanned under 400 nm. Minimum Rf value was recorded 0.01 and maximum was Ethyl acetate extract was separated in 20 bands with 0.02 minimum and 0.96 maximum Rf values when analysed under 254 nm wavelength, 15 bands with 0.01 minimum and 0.96 maximum Rf values when analysed under 366 nm wavelength and 15 bands with 0.03 minimum and 0.96 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 6: TP Figure 3.3.5(B-13): Densitometric chromatogram showing peak display of TP extracts (Density (AU) vs Rf ) As shown in figure 3.3.5(B-13), pigeon pea pod shell methanolic extract exhibited 23 bands when scanned at 254 nm with 0.07 minimum and 0.97 Rf values, 13 bands 146

107 when analysed under 366 nm wavelength with 0.07 minimum and 0.98 maximum Rf values and 11 bands at 400 nm with maximum 0.96 Rf and minimum 0.07 Rf. Ethyl acetate extract was separated in 17 bands with 0.06 minimum and 0.99 maximum Rf values when analysed under 254 nm wavelength, 10 bands with 0.06 minimum and 0.99 maximum Rf values when analysed under 366 nm and 9 bands with 0.06 minimum and 0.40 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 7: PSP As shown in figure 3.3.5(B-14), pea pod shell methanolic extract exhibited 25 bands when scanned at 254 nm with 0.07 minimum and 0.95 Rf values, 10 bands when analysed under 366 nm wavelength with 0.09 minimum and 0.94 maximum Rf values and 09 bands at 400 nm wavelength. Quercetin was found to be present in the sample when compared with the standard compound characteristics. Figure 3.3.5(B-14): Densitometric chromatogram showing peak display of PSP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 20 bands with 0.07 minimum and 0.98 maximum Rf values when analysed under 254nm wavelength, 12 bands with 0.18 minimum and 0.92 maximum Rf values when analysed under 366nm wavelength and 12 bands with 0.12 minimum and 0.95 maximum Rf values when analysed under 400nm wavelength. SAMPLE 8: JS As shown in figure 3.3.5(B-15), black plum seeds methanolic extract exhibited 16 bands when scanned at 254 nm with 0.10 minimum and 0.94 Rf values, 10 bands when analysed under 366 nm wavelength with minimum 0.11 and 0.99 maximum Rf 147

108 values and 11 bands at 400 nm which indicates 11 different compounds. Where minimum Rf value was 0.11 and maximum Rf value was Figure 3.3.5(B-15): Densitometric chromatogram showing peak display of JS extracts (Density (AU) vs Rf ) Ethyl acetate extract was separated in 26 bands with 0.06 minimum and 0.99 maximum Rf values when analysed under 254nm wavelength, 12 bands with 0.06 minimum and 0.98 maximum Rf values when analysed under 366nm wavelength and 12 bands with 0.03 minimum and 0.98 maximum Rf values when analysed under 400nm wavelength. SAMPLE 9: SS Figure 3.3.5(B-16): Densitometric chromatogram showing peak display of SS extracts (Density (AU) vs Rf ) Custard apple seeds methanolic extract exhibited 13 bands when scanned at 254nm with 0.09 minimum and 0.74 Rf values, 11 bands when analysed under 366nm wavelength with minimum 0.01 and 0.94 maximum Rf values and 15 bands at 400 nm with the highest recorded Rf value 0.92 and lowest Ethyl acetate extract was separated in 16 bands with 0.06 minimum and 0.68 maximum Rf values when 148

109 analysed under 254nm wavelength, 11 bands with 0.05 minimum and 0.98 maximum Rf values when analysed under 366nm wavelength and 10 bands with 0.05 minimum and 0.98 maximum Rf values when analysed under 400nm wavelength (Figure 3.3.5(B-16)). SAMPLE 10: ORP As shown in figure 3.3.5(B-17), orange peel methanolic extract exhibited 13 bands when scanned at 254nm with 0.06 minimum and 0.99 Rf values, Quercetin was detected at 0.06 Rf value with highest area covered on the graph i.e %. 8 bands when analysed under 366nm wavelength with 0.06 minimum and 0.84 maximum Rf values. Quercetin was reported at first peak with 0.06 Rf value and 21.16% area covered on chart. 10 bands (i.e. 15 compounds) when scanned at 400 nm. Maximum Rf value was recorded 0.99 and minimum was Quercetin was detected at 0.05 Rf value with highest area covered on the graph i.e %. Figure 3.3.5(B-17): Densitometric chromatogram showing peak display of ORP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 14 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 254nm wavelength, 10 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 366nm wavelength, 12 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 400nm wavelength. SAMPLE 11: SLPMH As shown in figure 3.3.5(B-18), sweet lime peel methanolic extract exhibited 18 bands when scanned at 254 nm with 0.04 minimum and 0.95 Rf values, 10 bands 149

110 when analysed under 366 nm wavelength with 0.05 minimum and 1.00 maximum Rf values and 07 bands when scanned under 400 nm. Minimum Rf value was recorded 0.04 and maximum was Figure 3.3.5(B-18): Densitometric chromatogram showing peak display of SLP extracts (Density (AU) vs Rf ) Ethyl acetate extract was separated in 14 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 13 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 366 nm wavelength and 12 bands with 0.02 minimum and 0.98 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 12: WMPMH Figure 3.3.5(B-19): Densitometric chromatogram showing peak display of WMP extracts (Density(AU) vs Rf ) As represented in figure 3.3.5(B-19), watermelon peel methanolic extract exhibited 29 bands when scanned at 254 nm with 0.04 minimum and 1.00 Rf values, 11 bands when analysed under 366 nm wavelength with 0.04 minimum and 0.85 maximum Rf 150

111 values and 12 bands when analysed at 400 nm with minimum 0.04 and maximum 0.90 Rf values. Ethyl acetate extract was separated in 29 bands with 0.01 minimum and 0.96 maximum Rf values when analysed under 254 nm wavelength, 11 bands with 0.01 minimum and 0.93 maximum Rf values when analysed under 366 nm wavelength and 10 bands with 0.01 minimum and 0.76 maximum Rf values when analysed under 400 nm wavelength. SAMPLE 13: GNPMH Peanut pod shell methanolic extract exhibited 19 bands when scanned at 254 nm with 0.04 minimum and 0.99 Rf values, 12 bands when analysed under 366 nm wavelength with 0.03 minimum and 0.97 maximum Rf values and 13 bands with maximum 0.97 Rf and minimum 0.03 Rf at 400 nm. Figure 3.3.5(B-20): Densitometric chromatogram showing peak display of GNP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 21 bands with 0.01minimum and 0.95 maximum Rf values when analysed under 254 nm wavelength, 13 bands with 0.01 minimum and 0.95 maximum Rf values when analysed under 366 nm wavelength and 14 bands with 0.01 minimum and 0.95 maximum Rf values when analysed under 400 nm wavelength (figure 3.3.5(B-20)). SAMPLE 14: PGPMH Pomegranate peel methanolic extract exhibited 19 bands when scanned at 254 nm with 0.03 minimum and 1.00 Rf values, 10 bands when analysed under 366 nm wavelength with 0.03 minimum and 0.99 maximum Rf values and 10 bands at 400 nm with maximum and minimum Rf values 0.84 and 0.03 respectively. 151

112 Figure 3.3.5(B-21): Densitometric chromatogram showing peak display of PGP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 20 bands with 0.01 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 13 bands with 0.01 minimum and 0.97 maximum Rf values when analysed under 366 nm wavelength and 15 bands with 0.01 minimum and 0.97 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(B-21)). SAMPLE 15: MDP As shown in figure 3.3.5(B-22), green gram split peels methanolic extract exhibited 29 bands when scanned at 254 nm with 0.02 minimum and 0.94 Rf values, 15 bands when analysed under 366 nm wavelength with 0.02 minimum and 0.99 maximum Rf values and 12 bands with minimum 0.02 Rf and maximum 0.99 Rf at 400 nm. Figure 3.3.5(B-22): Densitometric chromatogram showing peak display of MDP extracts (Density (AU) vs Rf ) 152

113 Ethyl acetate extract was separated in 17 bands with 0.01 minimum and 0.96 maximum Rf values when analysed under 254 nm wavelength, 10 bands with 0.01 minimum and 0.96 maximum Rf values when analysed under 366 nm wavelength and 09 bands with 0.04 minimum and 0.78 maximum Rf values when analysed under 400 nm. SAMPLE 16: CAMH Chili pedicel methanolic extract exhibited 27 bands when scanned at 254 nm with 0.01 minimum and 0.97 Rf values, 13 bands when analysed under 366 nm wavelength with 0.02 minimum and 1.00 maximum Rf values and13 bands with minimum 0.01 Rf and maximum 0.96 Rf at 400 nm. Figure 3.3.5(B-23): Densitometric chromatogram showing peak display of CA extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 21 bands with 0.01 minimum and 0.99 maximum Rf values when analysed under 254 nm wavelength, 1 bands with 0.01 minimum and 0.66 maximum Rf values when analysed under 366 nm wavelength and 11 bands with 0.01 minimum and 0.70 maximum Rf values when analysed under 400 nm (Figure 3.3.5(B-23)). SAMPLE 17: JFP Jackfruit peel methanolic extract exhibited 29 bands when scanned at 254 nm with 0.03 minimum and 1.00 Rf values, 13 bands when analysed under 366 nm wavelength with 0.03 minimum and 0.82 maximum Rf values and 11 bands at 400 nm with minimum 0.05 Rf and maximum 0.82 Rf. 153

114 Figure 3.3.5(B-24): Densitometric chromatogram showing peak display of JFP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 20 bands with 0.05 minimum and 0.96 maximum Rf values when analysed under 254 nm wavelength, 12 bands with 0.06 minimum and 0.98 maximum Rf values when analysed under 366 nm wavelength and 13 bands with 0.06 minimum and 0.76 maximum Rf values when analysed under 400 nm wavelength (Figure 3.3.5(B-24)). SAMPLE 18: CCMH As shown in figure (3.3.5(B-25)), cucumber peel methanolic extract exhibited 24 bands when scanned at 254 nm with 0.12 minimum and 0.97 Rf values, 06 bands when analysed under 366 nm wavelength with 0.12 minimum and 0.85 maximum Rf values and 07 bands with 0.10 minimum Rf and 0.81 maximum Rf. at 400 nm. Figure 3.3.5(B-25)- Densitometric chromatogram showing peak display of JFP extracts (Density(AU) vs Rf ) Ethyl acetate extract was separated in 18 bands with 0.07 minimum and 0.98 maximum Rf values when analysed under 254 nm wavelength, 10 bands with 0.10 minimum and 0.73 maximum Rf values when analysed under 366 nm wavelength,

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