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1. TO ANALYZE EXTRACTION OF THE ESSENTIAL OIL AND PECTIN FROM LEMON ( Citrus limon ) PEELS A PROJECT WORK SUBMITTED TO DEPARTMENT OF CHEMISTRY DHWALAGIRI MULTIPUS CAMPUS INSTITUTE OF SCIENCE AND TECHNOLOGY TRIBHUVAN UNIVERSITY NEPAL FOR THE AWARD OF BACHELOR OF SCIENCE (B.Sc.) IN CHEMISTRY BY NABIN PAUDEL ROLL No: 500530016 T.U. REGISTRATION No: 5-2-37-2484-2017 ASHADA 2079 i

2. Abstract Key words

3. sodhsar

4. 1. Introduction 1.1 Lemon ( Citrus limon ) Small tree with evergreen leaves and yellow edible fruits Belongs to citrus fruit family Rustaceae . Important source of Vtamin C. Chemical Compounds: Phenolic compounds, mainly flavonoids ( eg : diosmin , hesperidin , limocitrin ) and Phenolic acids ( eg : ferulic , synaptic, p- hydroxybenzoic acids). ( Agniezka et al., 2020) Has valuable paharmaceutical , cosmetic, flavorings and cleaning properties.

5. 1.2. Description of plant Lemon, Citrus limon , is a small evergreen tree in the family Rutaceae grown for its edible fruit which, among other things, are used in a variety of foods and drinks. The tree has a spreading, upright growth habit, few large branches and stiff thorns. Fruit : Berries globose or ovoid, bright-yellow, usually with a nipple-shaped extremity, rind thick, pulp acid. Seeds ellipsoid, white, glabrous. Flowering and Fruiting Time : Throughout the year. Significance :                                 It is a rich source of vitamin C. It is used in the preparation of lemonade, squash and home made sherbet

6. classificatuion of plant Kingdom: Plantae Order: Sapindales family: Rutaceae Genus: Citrus Species: C . limon (Bharat raj Ojha , Department of Botany, IOST, DMC, T.U.)

7. 1.3 Lemon Peels Chemical Compounds: pectin, polyphenols and flavonoids and is rich in volatile ingredients like terpenes , esters and alcohols. ( Chengyu et al., 2021) Environmental issues: Due to improper disposal of lemon peels, it generates large amount of microbial flora around the waste which causes communicable disease and generates bad odour . Lemon peels can be utilized for the extraction of essential oil. Lemon oil extraction methods: Acetone distillation, and ethanol distillation. (Mohamed et al., 2016) Uses of lemon oils: Anti-cancer, Anti-oxidant, Anti-inflammatory, Anti-bacterial, Anti-fungal, Anti-viral, Anti-allergic, Anti-obesity and hepato -regenerative activities. (Bora et al., 2020)

8. 1.4 Essential oils Essential oils are concentrated volatile aromatic compounds produced by plants- they easily evaporated essences that gives plants their wonderful scents. ( Rao et al., 2007) Has flavor, therapeutic, cosmetics, cleaning properties. Different methods of essential oil extraction: solvent extraction ( Acetone and Alcohol )

9. 1.4.1 Antifungal activities destroying fungi or inhibiting their growth

10. 1.5 Pectin Extraction Pectin (derived from Greek meaning ―congealed and curdled‖) is a structural heteropolysaccharide contained in the primary cell walls of terrestrial plants. . Pectin is a naturally occurring biopolymer that is finding increasing applications in the pharmaceutical and biotechnology industry.

11. 1.6. Rationale of the study To increase the commercial value of lemon and improve economic condition of small subsistence farmers. To reduce possible environmental impacts due to improper disposal of lemon peels. Pharmacological and phytochemical analysis of lemon extract.

12. 1.7. Obectives of the study 1.7.1 General objectives To extract and analysis optimum amount of Citrus limon essential oil and pectin from lemon peels by solvent methods. 1.7.2 Specific objectives To analyze pectin from Citrus limon peels To investigate antifungal activity of essential oil form Citrus limon peels extracts

13. 2. LITERATURE REVIEWS Fabio et al.,2014 described essential oils as the natural products, derived from aromatic plants, traditionally used all over the world for disinfection, as anti-inflammatory relaxing and stimulating substances and with potential and modern exploitation in clinical medicine. In 2016, Himed Louiza , Merniz Salah and Barkat Malika did the study which evaluated the chemical composition, antioxidant and antimicrobial effects properties of essential oil of Citrus limon . The composition of this oil was limonene (61.3%), β- pinene (9.7%), α- citral (4.2%) and α- terpinene (3.8%). Mohamed Amine Ferhatet et al., 2016 did a comparative study about cold pressing (CP), hydro-distillation (HD) and microwave assisted dry distillation (MAD) of Citrus Essential oil from Algeria. Their study showed MAD offers significant advantages over traditional alternatives, namely, shorter distillation times (30 min., against 3 hr for HD and 1hr for CP). improved yields, environmental impact (energy cost is fairly superior to perform HD and for mechanical motor (CP) than that required for rapid MAD extraction), cleaner features (as no residue generation and no water or solvent used) and provides a more valuable essential oil (with elevated quantities of oxygenated compounds). Hence, it also provides the ability for enhanced reproduction of natural fragrance of the essential oil from citrus fruit. Marta klimek Szczykutowicz et al., 2020 studied important botanical, chemical and pharmacological characteristics of Citrus Limon - a species with valuable biological activity of Citrus limon is determined by its high content of phenolic compounds, mainly flavonoids ( diosmin , hesperidin , limocitrin ) and phenolic acids ( eg : ferulic , synaptic, p- hydroxybenzoic acids). The essential oil is rich in bioactive monoterpenoids such as D-limonene, β- pinene , γ- terpinene . Their review presented the use of limon in food industry and cosmetology.

14. 3 . MATERIAL AND METHODS

15. 3 .1 Collection and Preparation of materials: The fresh unaffected citrus fruits; Lemon ( Citrus limon ) will be collected. Lemons will be washed and peeled. The fresh lemon peels will be grated and readied for the experiment.

16. 3 .2 Methods of Essential Oil Extraction Fresh lemons were purchased from a local market in Baglung and washed thoroughly under tap water. The peels were removed using a sterile knife and shade dried for 4-5 days at room temperature. The dried peels were pulverized using an electric blender and stored in airtight containers for further use. Two different solvents namely methanol and acetone were used for extraction. 10grams of lemon peel powder was soaked in 100 mL of the respective solvents for 72 hours by maceration technique. The supernatant was filtered using Whatmann filter paper 1 and Buchner funnel and concentrated using rotary evaporator and dry residue was preserved at 5°C until further use.

17. 3.2.1 Phytochemical screening According to standard method Mace, Wager, Evans and Kokate studied phytochemical screening to analyze the Alkaloids, Glycosides, Saponins,t erpenoids , Steriods , Carbohydrate and protein. Ramakrishnan etal describe the presence of carbohydrates and proteins were also tested according to standard method given. General reactions in this analysis revealed the presence or absence of these compounds. The qualitative analysis of the essential oil (hot ethanol extract, hot acetone extracts) was assessed to confirm the phyto -chemical properties.

18. 3.2.2 Flavonoids Test: 3.2.2.1Alkaline reagent Test Two mL of essential oil was added to few drops of sodium hydroxide. The change in the colour solution from intense yellow color to colorless on addition of dilute acid proves the presence of flavonoids. 3.2.2.2 Lead Acetate Test 2 ml of the essential oil was added to few drops of the lead acetate. The yellow colour ppt was obtained, proves the presence of falvonoids .

19. 3.2.3 Test for flavonoids 0.5 ml of sample solution was mixed with 2 ml of distilled water and subsequently with 0.15 ml of a 5% NaNO2 solution. After 6 min, 0.15 ml of a 10% AlCl3 solution was added and allowed to stand for 6 min, then 2 ml of 4% NaOH solution was added to the mixture. Water was added immediately to bring the final volume to 5 ml, then the mixture was thoroughly mixed and allowed to stand for another 15 min. An appearance of pink colour indicates the presence of flavonoids16. 3.2.4 Phenol Test: Two mL of essential oil extract was added to 2 mL of 5% ferric chloride solution (FeCl3), the formation of a deep bluish green solution confirms the presence of phenols.  

20. 3.2.5 Saponins Test: Two ml of the filtered extract sample was mixed with 1ml of distilled water in a test tube and it was rocked vigorously to obtain a stable persistent forth. The frothing was mixed with 3 drops of olive oil and mixed vigorously. The foam appearance indicates the presence of saponins. 3.2.6 Alkaloids Test: Two grams of extract was added to 10 mL solution of methanol and 1% HCl on water bath and then filtered. A precipitated orange colour infers the presence of alkaloid.

21. 3.2.7 Mayer’s test: 1 ml of the filterate was treated with few drops of Mayer’s reagent. Turbidity or precipitation indicated the presence of alkaloids9. 3.2.8 Test for proteins 0.5 ml of aqueous extract was treated with equal volume of 1% sodium hydroxide, to which a few drops of copper sulphate solution was gently added. The solution turning to purple colour, indicated the presence of proteins.  

22. 3.3 Antifungal Activity PDA   media was ready in the botanical laboratory using the power of the given PDA( 1 liter water, 200gm potato, 20gm dextrose and 20 gm agar) then take 45 min in cooker, finally the media was ready. Then our work started, sprit was use for disinfection the surrounding area. Then the plate smoothly filled with media, 3 plates are ready and 2inside the two plate freely putted the fungi founded in laboratory probably Fusarium but in one separated in two equal size then make a hole by the put and pressed by needle and add a 2 drops of the essentional oil. They are left for 3 weeks.

23. 3.4 Pectin extraction The 100gm dried peels were separately transferred into a beaker containing 500ml of water 2.5 ml hydrochloric acid was added to give a pH of 2.2. Each of the fruits was then boiled for 45min separately. Thereafter, the peels were removed from the extracts by filtering through a filter paper filter study. The cake was washed with 250 ml boiled water and the combine filter allowed to cool to 25°c to minimize heat degradation of the pectin. The exerted pectin was precipitated by adding 200ml 95% ethanol to 100ml of the extracted pectin with through stirring, left for 30min to allow the pectin float on the surface. The gelatinous substances flocculants was then skimmed off. The extracted pectin was purified by washed in 200ml ethanol and then pressed on a nylon cloth to remove the residual HCl and universal salt. The resulting pectin was weighed and shredded into small pieces and weighed and was air dried. Finally, the dried pectin was further reduced into smaller pieces using a pestle and mortar and weighed using a digital weighing balance. Percentage yield of pectin from initial wet peels was then determined on both wet and dry weight basis.

24. Analysis of equivalent weight of pectin

25. 4. Result And Discussion 4.1 Phytochemical Screening The qualitative and quantitative analysis from this study revealed the presence of phyto -chemical properties in the analysed essential oil form samples 1 (Acetone), 2 (hot ethanol). The absence of flavonoids in these essential oil samples from Citrus limon peel is contrary to what was reported by Sun. Flavonoids, among others are known to be produced by plants in response to microbial infection. In this present study saponin was present in all essential oil samples; this is in accordance to the work of (Kumar et al.,2007) who reported that essential oil obtained from Citrus limon peel using ethanol had high quantity of saponin .

26. Phyto-chemicals Inference of Extracts     1 2 Flavonoids ­- + Phenols + + Terpenoids +   Saponins + + Alkaloids +   Keys: Extract 1: Acetone hot ; Extract 2: Hot Ethanol. +Present, - Absent

27. 4.2 Pectin Colour add equivalent weight of pectin Dried pectin samples were observed visually and the colours of samples were noted down. Solubility in hot and cold water (dry pectin) S.No. Parameter Pectin 1 Colour White 2 Solubility in cold water Insoluble, inubides smells after vigorous shaking and forms suspension. 3 Solubility in hot water(85-90°c) for 15 min The mixture Dissolve 4 Solubility of pectin suspension in cold alkali The pectin suspension forms yellow precipitated 5 Solubility of pectin suspension in hot alkali The pectin suspension dissolve and turned milky .

28. 4.3Antifungal Activities After the 3 weeks we can seen that the in 2 plates in which we freely added the fungus they are grow and spreaded but in 1 plate in which essential oil added, inhibiting their growth .

29. 5. Conclusion The study reveals that lemon peel is a rich source of active compounds with various medicinal and pharmacological properties. The equivalent weight of pectin was analyzed from lemon peels and shows inhabitation the growth of Fuserium . The Phytochemicals were found in essential oil extract of lemon peel. Phenols, Terpenoids, Saponins, and Alkaloids were found in Acetone extract of lemon peels and Flavonoids’, Phenols and Saponins were found in ethanol extract of lemon peels. add equivalent weight of pectin

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31. acknowledgement

32. annex Add representative photos with attraction for your research

33. thanks

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37. 7. ACKNOWLEDGEMENT The time has come lastly for which I was waiting to express my sincere gratitude and gratefulness to my supervisor Mr. Narendra Pratap Singh Budhathoki for this Dissertation. I would like to express my sincere appreciation to chief of B.Sc. program, Mr. Prakash Prasad Sapkota, Dhaulagiri Multiple Campus, Baglung for permission to undertake this dissertation work. My sincere gratitude goes to respectable Campus Chief of DMC Professor Dr. Ram Prasad Upadhaya , for commitment to provide every facility required to laboratory work. My sincere gratitude goes to Mr. Bharat Raj Ojha for collaboration in antifungal analysis. Special thanks to Mr. Bishnu Kalfe and Mr. Lakhan Thapa, for their cooperation. Lastly, I am thankful to all my teachers and to all the staffs of DMC, Baglung and also for my family for their sweet cooperation.