Natural Products Extraction, Essays (high school) of Biochemistry

Download Natural Products Extraction and more Essays (high school) Biochemistry in PDF only on Docsity! Republic of the Philippines Batangas State University College of Arts and Sciences Batangas City CHEM 347 - CHEMISTRY ELECTIVE 2 Phytochemical Screening of Melothria pendula (Pipinong Gubat) Submitted to Angelica A. Macalalad, MChem, LPT Submitted by Babao, Scarlett Angelli M. Bendaña, Shaira Mae G. Hernandez, Princess Kayla D. INTRODUCTION The therapeutic application of herbs is rooted in plant chemistry. The potential medicinal value of plants can be deeply understood by knowing their chemical composition. The extensive and diverse pharmacological effects of medicinal plants are based on their phytochemical components. These components are typically classified into two categories: primary and secondary metabolites. They are categorized based on their involvement in fundamental metabolic processes. The term "secondary metabolite" was initially introduced by Albrecht Kossel, who received the Nobel Prize in Physiology or Medicine in 1910. Around thirty years later, Czapek characterized them as final products. He stated that these substances originate from nitrogen metabolism through what he termed 'secondary modifications,' like deamination. In the mid-twentieth century, advancements in analytical methods, such as chromatography, facilitated the identification of an increasing number of these compounds. This progress laid the groundwork for the development of the field of phytochemistry (Hussein & El-Anssary, 2018). Unlike primary metabolites which are essential in growth and development, secondary metabolites are not directly involved in these processes. Secondary metabolites are products derived through biosynthetic modifications of primary metabolites such as methylation, glycosylation, and hydroxylation. The complexity of the structural composition and side chains of secondary metabolites is greater than that of the primary metabolites (Twaij & Hasan, 2022). Secondary metabolites exhibit a range of biological effects, forming the scientific foundation for herbs' traditional medicinal use in ancient communities. They are known for their antibiotic, antifungal, and antiviral properties, which help safeguard plants against pathogens. Additionally, they serve as crucial UV-absorbing compounds, preventing significant leaf damage from light exposure (Hussein & El-Anssary, 2018). Confirmatory qualitative phytochemical screening of plants refers to the process of determining and identifying secondary metabolites present in plant extracts using standard protocols (Dubale et al., 2023). In this experiment, phytochemical screening will be conducted utilizing Melothria pendula, also locally known as “pipinong gubat”. M. pendula is a plant species belonging to the genus Melothria and the Cucurbitaceae family. This plant is an annual * Causes damage to organs (liver, kidneys) through prolonged or repeated exposure (inhalation, oral) Concentrated H2SO4 - Appearance: Clear, colorless to brown liquid - Odor: Odorless - Melting point: 10°C - Boiling range: 290-338°C - Signal word: Danger - IRRITANT * May cause respiratory irritation - CORROSIVE * May be corrosive to metals * Causes severe skin burns and eye damage Keep containers tightly closed in a dry, cool, and well-ventilated place. Keep away from moist air and incompatible materials such as organic materials, strong acids, strong bases, metals, alcohols, cyanides, and sulfides. Dispose of waste material according to local and/or national regulations. 1% Gelatin - Appearance: Off-white solid - Odor: Odorless - pH: 6.0 Not classified as hazardous Keep containers tightly closed in a dry, cool, and well-ventilated place. Dispose of waste material according to local and/or national regulations. Sodium chloride (NaCl) - Appearance: White solid - Odor: Odorless - Melting point: 801°C - Freezing point: 1461°C Not classified as hazardous Keep containers tightly closed in a dry, cool, and well-ventilated place. Protect from moisture and incompatible materials such as strong oxidizing agents, metals, and strong acids. Dispose of waste material according to local and/or national regulations. Copper acetate solution - Appearance: Blue green liquid - Odor: Odorless - Solubility: Moderately soluble - TOXIC * Slightly toxic by ingestion Store in a cool, dry, and well-ventilated place. Keep away from incompatible materials. Dispose of waste material according to local and/or national regulations. Sodium hydroxide - Appearance: Clear, colorless liquid - Odor: Odorless -Melting/Freezin g point: Approx. 0°C - Boiling point: Approx. 100°C - Signal word: Danger - CORROSIVE *May be corrosive to metals * Causes severe skin burns and eye damage * Causes serious eye damage Store in a cool, dry, conditions in well-sealed containers. Keep away from extreme heat, ignition sources, or open flame. Dispose of waste material according to local and/or national regulations. B. Laboratory Glassware and Apparatus Used ● Beaker ● Erlenmeyer flask ● Analytical balance ● Mechanical blender ● Spatula ● Graduated cylinder ● Funnel ● Filter paper ● Wash bottle ● Test tube ● Stirring rod ● Pipette ● Aspirator ● Dropper ● Test tube rack ● Iron stand ● Iron clamp ● Thermometer ● Hot plate C. Methods Followed I. Extraction a. Air-dry the plant sample for a few weeks. Dry until the weight is constant. b. Upon drying, cut the plant sample into smaller pieces. Then, powderize it using a mechanical blender. c. Sieve the powderized sample to obtain only the smaller particles. d. Obtain 20 grams of the powdered sample and transfer it to a 250 mL beaker. e. Dissolve the powdered sample with 100 mL of ethyl alcohol. f. Cover the beaker with foil to avoid light exposure. Leave the sample for 24 hours. g. After 24 hours, filter the sample using a filtration setup. Dispose of the residue and obtain the filtrate for analysis. II. Phytochemical Screening a. Wagner’s Test: Test for Alkaloids 1. Prepare 5 mL of sample in two separate test tubes. 2. Dissolve the sample with 1 mL of dilute hydrochloric acid. 3. Filter the sample. Upon filtration, add 3 drops of the Wagner’s reagent. 4. Observe the formation of reddish brown color on the solution. This indicates a positive test for alkaloids. b. Test for Saponins 1. Prepare 5 mL of sample in two separate test tubes. 2. Add 5 mL of distilled water into the sample. Figure 1.3. Obtaining 20 grams of M. pendula Sample After 24 hours, the sample was taken and observed to settle at the bottom of the beaker. A filtration setup was prepared in which the M. pendula sample was filtered, discarding the precipitate and obtaining the filtrate. An approximately 50 mL of filtrate was obtained from the filtration, which was not an enough amount for all the tests to be conducted. Due to this, an additional 10 mL of ethyl alcohol was added to the filtrate in order to compensate for the amount of sample needed for all tests. The appearance of the filtrate is as seen on Figure 1.4, which was initially observed to have a dark green color. Figure 1.4. Filtrate of M. pendula Obtained from Extraction B. Phytochemical Screening Two trials were conducted for the first three tests performed, namely the tests for diterpenes, flavonoids, and saponins. However, due to the unforeseen scarcity of sample, only one trial was performed for the remaining three tests, namely the tests for alkaloids, phytosterols (Salkowski’s test), and tannins. For each trial, five mL of the sample was prepared in each test tube. The results of all tests are as reflected in Table 1. Table 1. Results of Phytochemical Tests Performed Tests Result Illustration Implication Test for Alkaloids Showed a reddish-brown color upon addition of Wagner’s reagent + Test for Saponins Upon shaking, little foam formed + Test for Phytosterols (Salkowski’s Test) Sample turned brown and did not form any layers. _ Test for Tannins Formed white precipitate upon addition of gelatin + Test for Diterpenes (Copper Acetate Test) No color change was observed Cannot be determined Figure 1.8. Appearance of M. pendula Sample Upon Subjecting to Salkowski’s Test IV. Test for Tannins In the test for tannins, only one trial was performed utilizing 5 mL of the M. pendula sample. Initially, a 1% gelatin solution was prepared containing sodium chloride (NaCl), which was maintained to be in liquid form by continuous heating at a moderate temperature. Five mL of the gelatin solution was obtained and added to the sample. Based on observations made, little amount of white precipitate was seen on the bottom of the test tube, as seen on Figure 1.9. This indicates that the M. pendula sample has the presence of tannins. Figure 1.9. Appearance of M. pendula Sample Upon Subjecting to Test for Tannins V. Test for Diterpenes (Copper Acetate Test) Two trials were conducted for the test for diterpenes, in which 5 mL of the M. pendula sample was prepared in two separate test tubes. Each set of samples was subjected to addition of two drops of copper acetate solution. By default, a positive test for this test is shown with a color change of the sample to emerald green. However, in the case of this experiment, the result for this test was difficult to distinguish due to the dark green color of the M. pendula sample, as shown in Figure 1.10. Figure 1.10. Appearance of M. pendula Sample Upon Subjecting to Test for Diterpenes VI. Test for Flavonoids (Alkaline Reagent Test) In the test for flavonoids, which was specifically performed through the alkaline reagent test, two trials were also conducted. Five mL of sodium hydroxide (NaOH) were added to two separate test tubes each containing 5 mL of the M. pendula sample. Upon addition, it was observed that the sample in both trials exhibited a color change to yellowish-green, as shown on Figure 1.11. This observation indicates a positive result for the presence of flavonoids. Figure 1.11. Appearance of M. pendula Sample Upon Subjecting to Test for Flavonoids DISCUSSION In this experiment, qualitative phytochemical screening of Melothria pendula (pipinong gubat) was conducted. Several studies have noted the use of M. pendula as a medicinal plant, especially in the country Mexico. In fact, as mentioned in a study conducted by Guerrero-Torres et al. (2022), M. pendula is used for various skin conditions including skin rashes, sores, and skin burns, as well as other conditions like animal bites, heart pain, urinary diseases, anemia, gastrointestinal pains, gonorrhea, and hemorrhoids. However, even with its diverse uses as a traditional medicine, not much is known about its phytochemical constituents. The first part of this experiment involves the extraction of the M. pendula sample, specifically the leaves and stems. The extraction was conducted by means of maceration, which, according to an article written by Abubakar and Haque (2020), is the process by which a coarsely powdered sample is subjected to addition of solvent, storage for at least 24 hours, and filtration. In the experiment, the samples were initially obtained and air-dried three weeks prior to extraction. Air-drying the samples within a sufficient amount of time allows the complete evaporation of the water and moisture content in order to ensure a higher accuracy of the results. The air-dried samples were taken and powderized using a mechanical blender, followed by the Figure 1.13. Reaction Mechanism of Saponins with Water (Parbuntari et al., 2018) The third test conducted in this experiment was the Salkowski’s test, which involves the analysis for the presence of phytosterols. In this test, one trial was conducted, utilizing 5 mL of the M. pendula extract. This test involved the initial addition of chloroform, followed by filtration and the subsequent addition of concentrated sulfuric acid (H2SO4). By definition, phytosterols are steroids commonly derived from plants which represent the majority of the unsaponifiables in plant lipids. These compounds are composed of a steroid skeleton with a saturated bond between the carbons 5 and 6, a hydroxyl group on the carbon-3 position, and an aliphatic side chain bound to the carbon-17 atom. In addition, phytosterols exhibit a variety of bioactivities indicating its pharmacological benefits including antidiabetic, antiatherosclerotic, anti-inflammatory, chemopreventive, antioxidant, and cardioprotective activities (Salehi et al., 2020). Qualitatively testing the presence of phytosterols in plant samples is commonly conducted by means of Salkowski’s test. By principle, a positive test for phytosterols through the Salkowski’s test involves the formation of two layers, one of which is the chloroform layer with a bluish red to violet color and the other is the sulfuric acid layer with a yellowish green color. The formation of layers may be attributed to the lipid nature of phytosterols. Furthermore, the addition of concentrated sulfuric acid allows the formation of a yellowish green layer, which is produced through the dehydration caused by such acid to phytosterol, as shown in the reaction mechanism illustrated in Figure 1.14 (IGNOU: The People’s University, n.d.). Reaction with chloroform also causes the formation of another layer, with bluish red to violet color, although the mechanism remains unclear. In the case of the M. pendula sample, changes noted only entailed a color change to brown, with no additional layers formed. This indicates that the M. pendula sample doesn’t contain phytosterols. Figure 1.14. Reaction Mechanism Involved in the Addition of Concentrated Sulfuric Acid (IGNOU: The People’s University, n.d.) The fourth pythochemical test conducted, is the test for the presence of the compound tannins. In this test, only one trial was performed, utilizing only 5 mL of the M. pendula sample. This test involves the addition of a 1% gelatin solution, which was prepared containing sodium chloride (NaCl), and continuously heated at moderate temperature to prevent it from solidifying. Tannins are a diverse group of naturally occurring polyphenolic compounds found in various plant materials and widely distributed in nature. They are known for their ability to interact with proteins and other organic molecules, as well as for their astringent taste and ability to precipitate proteins. Tannins also have various biological activities which are known to have antioxidant, antimicrobial, and anti-inflammatory properties. There are several methods that can be employed to qualitatively verify the presence of tannins in a sample, in this specific experiment we carried out a gelatin test as aforementioned. By principle, a positive test result in a gelatin test for tannins is described by turbidity or the formation of a white precipitate. To further explain, based on composition and structure, gelatin is a protein derived from collagen and is soluble in hot water. Tannins, on the other hand, have an affinity for proteins and can form complexes with them. So, in this test, if tannins are present in the solution, they will interact with the gelatin molecules, causing them to aggregate and precipitate out of solution. In the case of our M. pendula sample, after the addition of the gelatin solution, small amount of white precipitate was observed on the bottom, indicating a positive result and confirming the presence of tannins in the tested solution. The next and fifth phytochemical test is the test for the compound diterpenes. Diterpenes are a class of organic compounds composed of four isoprene units, making them structurally similar to terpenes. They can be found in various plants, fungi, and some marine organisms, often serving as natural products with diverse biological activities. Some of their most notable biological activities are their antimicrobial properties, anti-inflammatory, anticancer potential, antioxidant, neuroprotective and cardioprotective effect. Going on, in this test two trials were performed; 5 mL of the M. pendula sample was prepared in two separate test tubes, then followed by the addition of about 2-3 drops of copper acetate solution to the sample. Qualitative tests for diterpenes are typically used to detect the presence of unsaturation in organic compounds which involve chemical reactions that produce characteristic color changes or precipitates. The positive result can then be specifically described by a visible color change to emerald green or formation of a greenish or blue-green precipitate. When diterpenes react with copper acetate solution, they formed a colored complex. The color change indicates the presence of double bonds or other unsaturated functional groups in the diterpene molecule. Not alone, the intensity of the color change can also provide us information about the concentration or structure of the diterpenes present. In the case of our M. pendula sample, due to the physical nature of the extract which happens to be already dark green in color, it was hard for us to distinguish an accurate result. With this test alone we cannot fully deduce a valid assumption, as the change of color can be barely noticeable. Therefore, we cannot determine whether our sample (Pipinong gubat) has diterpenes or not. The last phytochemical test conducted in this experiment, is the test for flavonoids. In this test two trials were performed; on two separate test tubes, 5 mL of sodium hydroxide (NaOH) were added to 5 mL of the M. pendula sample. Flavonoids are a diverse group of natural compounds found in plants, particularly fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine. They are characterized by their chemical structure, which consists of two benzene rings linked by a linear three-carbon chain, forming a C6-C3-C6 skeleton. There are several qualitative tests that can be used to detect the presence of flavonoids in a sample, in this we have carried out an alkaline reagent test or shinoda test, which the procedure has already been mentioned. A positive test result can be described by a change of color to a yellowish solution, REFERENCES Abubakar, A. R., & Haque, M. (2020). Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes. Journal of Pharmacy and Bioallied Sciences, 12(1), 1. https://doi.org/10.4103/jpbs.jpbs_175_19 Dianito, N. A., Dagalea, F. M. S., Vicencio, M. C. G., & Lim, K. M. C. (2022). Physicochemical Properties and Antibacterial Activity of Biosynthesized Silver Nanoparticles from Melothria pendula Linn. (Pipinong-Gubat) Leaf Extract. 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