PHYTOCHEMICAL ANALYSIS AND ISOLATION OF FLAVONOID COMPOUND FROM METHANOL EXTRACT OF THE LEAVES OF SCURRULA PARASITICA L.

PHYTOCHEMICAL ANALYSIS AND ISOLATION OF FLAVONOID COMPOUND FROM METHANOL EXTRACT OF THE LEAVES OF SCURRULA PARASITICA L.

Laldingngheta Jerry *, Lalnundanga 1 and Vabeiryureilai M

Department of Forestry, Mizoram University, Aizawl, Mizoram, India.

ABSTRACT: Scurrula parasitica L. is a parasitic plant, traditionally used by the people of Mizoram for the treatment of various diseases, and locally called Vatesawi. The bioassay-guided purification of the methanol extract of the leaves of Scurrula parasitica L. led to the isolation of 2-(3, 4-dihydroxy phenyl)-3,5,7-trihydroxy-4H-Chromen-4-one. Quercetin is a flavonol that belongs to one of the subclasses of flavonoids. For separation and purification of compounds, thin layer chromatography and column chromatography were performed using the plant extract. Then identification was done by high-performance liquid chromatography (HPLC) with mass spectral analysis, and confirmation of the isolated compound was performed with thin layer chromatography. The results showed that the solvent system of ethyl acetate 7: chloroform 3 had accurate separation, and the fraction no-200 from column chromatography was processed for identification of the compound. Structures were established with the mass spectrum and confirmation of the compound was done by comparing with standard quercetin, and both appeared at the same wavelength of 254nm (RT 2.63).

Keywords: Scurrula parasitica L., Column chromatography, Thin layer chromatography, HPLC, Quercetin, mass spectral analysis

INTRODUCTION: Medicinal plants possess therapeutic properties against certain diseases or serve as the origin of useful drugs ^1-2. The true efficacies of the substances in medicinal plants have not been recognized until the 19^th century. Plants possess active compounds that have proven to be useful in numerous medical fields for centuries ³. These phytochemicals are the major principles in curing human ailments. Different plants contain certain active principles in them; these active ingredients may differ even in different organs like root, bark, leaves, seeds, twigs and fruits of the same plant.

The substances derived from plants are used for the manufacture of synthetic drugs ⁴. Medicinal plants are rich in secondary metabolites; thus modern pharmaceutical industries have tried to isolate these potential bioactive compounds for the treatment of various diseases.

Scurrula parasitica L. is a parasitic shrub found growing on Dendrophthoe falcate, Myrica esculenta, and Mangifera indica. It belongs to the family Loranthaceae. It is found in Sri Lanka, Nepal, Bhutan, Bangladesh, Myanmar, Thailand, Taiwan, Vietnam, Indonesia, Philippines, Moluccas, and Timor. In India, it is distributed all over the Western Ghats regions of Maharashtra and parts of Uttar Karnataka and Tamil Nadu.  The plant is used as a traditional medicinal herb as an antioxidant and against microbial infections, hypertension, and cancer in Asia ⁵. In Java and Indonesia, the infusion of Scurrula parasitica L. is used for the treatment of cancer ⁶.

In southern China, the leaves and stem of Scurrula parasitica L. are used as antineoplastic, cardio-tonic, as an antioxidant and for treatment of Schizophrenia ^7-8. The plant shows anti-diabetic, cytotoxic, anticancer, anti-hepatoxic and immuno-modulatory activity ⁹. Recently it was observed that the leaves of Scurrula parastica L. were frequently used by the tribal inhabitants of Mizoram for the treatment of diabetes. 21 days anti-hyperglycemic experiment using streptozotocin-induced diabetic rats was performed with two doses of the plant extract, and the results show a significant lowering of blood glucose, cholesterol, LDL, triglyceride, SGOT, ALP, and SGPT, along with an increase in HDL and body weights in rats. 5 anti-diabetic compounds were also detected, which might be the reason for its anti-hyperglycemic properties ¹⁰. Important phytochemicals responsible for numerous biological activities like terpenoids, flavonoids, alkaloids, viscotoxins, amines, lectins have been tested to be present on Scurrula parastica L. ^11-12 This paper focus on the ethnomedicinal claims and isolation of active compound, extracted from the leaves of Scurrula parasitica L. Isolation and identification of active compound is done through standard techniques like chromatography and spectral methods ¹³.

MATERIALS AND METHODS:

Plant Material and Extraction: The leaves of Scurrula parasitica L. were collected from Kolasib, Mizoram. Taxonomic identification was done at the Botanical Survey of India, Shillong (No: BSI/ ERC/ Tech/ 2017/ 43), and authentication was done at the department of environmental science, Mizoram University, with a voucher number of MZU 742. Fresh leaves of Scurrula parasitica L. were air-dried at room temperature and prepared into a coarse powder, and stored in a beaker. Approximately 200gm of the plant powder was weighed and subjected to continuous hot extraction using the Soxhlet apparatus at 60°C. The extraction was carried out successively using petroleum ether, chloroform, and methanol. Subsequently, the extracts were evaporated under pressure using a rotary evaporator until all the solvents have evaporated to give pure crude extracts. The methanol extract was used for the experiment.

Phytochemical Screening of the Crude Extract: Preliminary phytochemical screening was performed to detect the presence of important secondary metabolites in the crude extract of Scurrula parasitica L. The alterations in color and formation of precipitates in the plant extracts performed in separate test tubes denote positive results. Observations were recorded.

Thin Layer Chromatography: Thin-layer chromatography (TLC) is a method of separation in which differential migration of solvents occurs along with a fine powder spread on a glass plate. Several compounds are spotted on a thin layer of solid adsorbent, then the compounds along with the mobile phase travel to distances varying on the particle coefficient of each molecule. Readymade aluminum-coated silica gel plates (Merck and Co. Ltd.) were used for the research. The plate is cut into smaller pieces into a 4 × 10 cm dimension. The plates are kept in an oven for 120°C for half an hour before use ¹⁴. With the help of capillary tubes, 2.5µl of plant extracts are spotted carefully on the silica gel plate. Spotting was done 1 cm above the bottom of the plate and kept in a development chamber. The lid of the development chamber was closed to avoid tailing, and the solvent front was then allowed to travel upwards up to a distance of 3/4^th from the baseline, and then the lid of the chamber is opened, and the plate is removed and allowed to dry in the air.  The plate is then kept in an iodine chamber for a few minutes. The solvent system is based on elusive power because the rate of migration of compounds on a given adsorbent depends on the solvents being used ^15-16.

Column Chromatography: The column chromatographic technique is most commonly used for the separation of compounds into several fractions according to the affinity or solvating capacity of the compounds to the solvent used. The study involves fractionation and isolation of compounds from pharmacologically active ethanol extract. The structure of the compound was tried to establish by spectroscopic methods. A Glass column of a 20 mm dimension was taken and held firmly with the help of a stand and fitted with a stopcock. Solvent system of Ethyl acetate: chloroform (7:3, 8; 2 and 9:1) was used. The stationary phase was made with Silica gel (60-120 mesh size), which was heated at a maximum temperature of 120 °C and placed in the column. Cotton is inserted into the column and the bottom portion is plugged and the silica gel (60-120 mesh size) is packed into the column which is done by the wet package method. Then the stationary phase is allowed to settle down without entrapment of air bubbles ¹⁷. The column is allowed to stand uniformly without disturbances for two hours. Fractionation of extract was carried out using the above solvent system to obtain the bioactive compound. Fractions were collected, and TLC was performed and similar fractions were pool together.

Preparative Thin Layer Chromatography: IPPC or Preparative planar (thin-layer) chromatography is used for the isolation/ separation of compounds. The main purpose is the production of 10 to 1000mg of the separated compounds for structural identification like mass spectrum, Infrared/ Ultraviolet, and for analysis of other activities. Glass plates were used, in which the glasses were cut into 6x4 inch dimensions. A total of 10 slides were made for the experiment.

The glasses were washed properly with water and dried in the oven. Then, Silica gel 'G' as 30 gm of silica gel was weighed and made to a homogenous suspension with 60 ml distilled water for two minutes; this suspension was layered over a glass plate which was air-dried until the transparency of the layer disappeared. The plates were dried in a hot air oven at 110 °C for 30 min and then stored in a dry atmosphere and used whenever required.

Mass Spectral Analysis: The mass spectrum of the isolated bioactive compound was analyzed using LCMS Accucore (C18, 150 × 2.1, 2.6um), SAIF, CSIR-CDRI, Lucknow, India. Then the molecular weight of the peak compound given by the spectrum was integrated with the NIST database online library for confirmation of the specific compound.

High-Performance Liquid Chromatography: HPLC was performed with Quaternary LC Pump Model 200Q/410 with series 200 Autosampler. The mobile phase was determined based on an isocratic system was using appropriate solvents to detect the wavelength, and then it was incorporated with a standard compound. Both the standard and isolated compounds were eluted together. The peak and retention time were observed. The final result obtained was then overlapped.

Thin Layer Chromatography Analysis for Isolated Compound: Further, the isolated compound was subjected to TLC analysis for compound confirmation. Both the standard and isolated compounds were eluted, and movement was observed up to a certain point. It was then visualized under Iodine dye in a closed chamber.

RESULTS:

Phytochemical Screening of the Crude Extract: The preliminary phytochemical screening of the leaves of Scurrula parasitica L. revealed the presence of important bioactive components alkaloid, flavonoid, phenols, saponin, tannin, terpenoids, reducing sugar and phytosterols in the methanolic extract and absence of glycosides.

Thin Layer Chromatography: The results of thin-layer chromatography of methanol extract of Scurrula parasitica L. are presented in Table 3. A definite spot showing yellowish-red was detected in the solvent system of ethyl acetate and chloroform (7: 3) and Ethyl acetate: Chloroform: Methanol (7:2:1) after keeping it inside an iodine chamber. But ethyl acetate and chloroform (7:3) display the best result. Thus, it was selected to be used as a solvent system.

TABLE 1: THE SOLVENT SYSTEM USED IN THIN-LAYER CHROMATOGRAPHY

FIG. 1: ETHYL ACETATE: CHLOROFORM (7:3)

Column Chromatography: Around 200 to 250 fractions were collected and recorded, in which fractions of the elutes namely F 1 – 37, F 38 – 76, F 77 – 108,  F 109 – 146, F 147 – 168, F 169 – 204 all shows single spots and are combined and transferred to a small beaker evaporated to dryness. The pooled fractions were named A, B, C, D, E, and F, respectively. The elutes were then kept in a refrigerator for 12 hours to enhance purification.

According to fractions collected from column chromatography, Fraction No. F-200 with TLC profile of ethyl acetate: chloroform (7:3) obtaining a single spot with an R_f value of 0.44 mm showed the best results and was chosen for further analysis.

Preparative Thin Layer Chromatography: From the results of column chromatography, the solvent system of ethyl acetate 7: chloroform 3 was prepared and quantification was done on the TLC plates. The process was continued until a substantial amount of the compounds were available for further analysis. Then the compound was selected for Mass spectral analysis for identification. Both HPLC and TLC analyses were done for isolated compound confirmation.

Identification of the Isolated Bioactive Compound: Identification of the isolated compound was done by Mass spectral analysis, HPLC, and TLC along with the NIST database.

Mass Spectral Analysis: The ESI-Mass spectrum of the sample (full positive scan mode) indicates the molecular peak at m/z 302.23 [M+H] ⁺. Further, the molecular weight of the isolated compound was incorporated with the NIST database and found to be 302.23, which is Quercetin. The compound was further confirmed by matching with NIST Database online library.

FIG. 2: LC-ESI-MS/MS SPECTRUM OF THE ISOLATED COMPOUND FROM SCURRULA PARASITICA L.

HPLC for Confirmation of Isolated Bioactive Compound: HPLC was accomplished with an Isocratic system (A) 60% Acetonitrile and (B) 40% Methanol (60: 40). The detection wavelength was 254nm. The isolated compound was incorporated with standard Quercetin obtained from HiMedia (RM6191-25g). The compound was confirmed by HPLC with standard Quercetin at a wavelength of 254nm (RT 2.63). This was followed by TLC using a similar solvent system.

FIG. 3: HPLC ANALYSIS OF ISOLATED COMPOUND FROM SCURRULA PARASITICA L. AND QUERCETIN

Thin Layer Chromatography Analysis for Isolated Compound: Finally, Quercetin and the isolated compound were eluted by the TLC method, and the movement was observed up to a certain point. Both appeared at the same level.

FIG. 4: ANALYSIS OF ISOLATED COMPOUND FROM SCURRULA PARASITICA L. S- Spotted area, D- detection level, Q- quercetin, I- isolated compound.

DISCUSSION:

FIG. 5: CHEMICAL STRUCTURE OF QUERCETIN

Quercetin is characterized as a flavonol that belongs to one of the subclasses of flavonoid compounds. IUPAC Name is 2-(3, 4-dihydroxy phenyl)-3,5,7-trihydroxy-4H-Chromen-4-one.

The molecular formula of quercetin is C₁₅H₁₀O₇ and has a molecular weight of 302.23 g/mole. Quercetin is aglycone lacking an attached sugar. It is yellowish, hardly soluble in hot water, insoluble in cold water and quite soluble in alcohol solvents. This bioflavonoid has shown significant results in lowering blood pressure, endothelial functions and inflammations ^18-20. Quercetin also displays beneficial effects in protein oxidation-reduction and LDL-C ^21-22. Numerous investigations of quercetin in guinea pigs through oral administration or inhalation have shown anti-asthmatic activity ^23-25. Intake of non-tea quercetin reduces the risk of developing colon, however not rectal cancer ²⁶. Supplementation of quercetin reduces the development of cardiac hypertrophy in rats ²⁷. Quercetin induces a progressive and sustained reduction in blood pressure in rat models of hypertension and metabolic syndrome ²⁸. In-vitro antibacterial activity of quercetin proved to be effective against microorganisms associated with the start and progression of periodontal diseases like Actinomyces naeslundii wvl, Actinomyces viscosus, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis ^29-30. Quercetin demonstrates in-vitro antiviral activity against parainfluenza virus type 3, poliovirus type 1, HIV transcriptase, hepatitis C and respiratory syncytial virus^31-32.

CONCLUSION: Thin layer chromatography of the methanol extract of the leaves of Scurrula parasitica L. results indicates that the solvent system of Ethyl acetate 7: Chloroform 3 proved to be the most accurate. The fraction (Fraction no 200) from column chromatography was further processed for identification. Then, confirmation was done in which the isolated compound was compared to standard quercetin HiMedia (RM6191-25g), and they both appeared at the same wavelength of 254nm (RT 2.63). The isolation of quercetin from the methanol extract of the leaves of Scurrula parasitica L. will prove to be useful in the manufacture of biopharmaceutical products in the future.

ACKNOWLEDGEMENT: The authors are grateful to Robert Singh, Head, Department of Biotechnology, Mizoram University, for granting permission to use their laboratory and equipment for the success of this research.

CONFLICTS OF INTEREST: There is no conflict of any kind with anybody or amongst the authors

REFERENCES:

1. Ebadi M: Pharmacodynamic basis of herbal medicine, 2^nd CRC Press 2006.
2. Lewis RA: Scientific terminology in Ethnopharmacology. Journal of Ethnopharmacology 1981; 4: 121.
3. Cordell GA: Introduction to the alkaloids. Biogenetic Approach. John Wiley and Sons, New York 1981.
4. Morsy N: Phytochemical analysis of biologically active constituents of medicinal plants. Main Group Chemistry 2014; 13: 7-21.
5. Lim YC, Rajabalaya R, Shirley HFL, Tennakoon KU, Quang-Vuong L, Idris A, Zulkipli IN, Keasberry N and David SR: Mistletoes of the genera Scurrula and Viscum: from bench to bedside. Molecules 2016; 21: E1048.
6. Puneetha GK and Amruthesh KN: Phytochemical screening and in-vitro evaluation of the antioxidant activity of various extracts of Scurrula parasitica. Int J of Pharmacy and Biological Sciences 2016; 6(1): 77-86.
7. Xiao YJ, Chen YZ, Chen BH, Chen JH, Lin ZX and Fan YL: Study on cytotoxic activities on human leukemia cell line HL60 by flavonoids extracts of Scurrula parasitica from four different host trees. China Journal of Chinese Materials and Medicines 2008; 33(4): 427-32.
8. Soheil ZM, Muhamad NAK, Chim KC, Bey HG and Habsah AK. Phytochemistry and biology of Loranthus parasiticus Merr, a commonly used herbal medicine. The American J of Chinese Medicine 2014; 42(1): 23-35.
9. Mahajan NP, Joshi PP, Kondawar M, Senthil KKL and Vaidhyalingam V: Effect of methanol extract of Scurrula parasitica on blood sugar levels. Inventi Rapid: Ethnopharmacology 2013; 2: 1-3.
10. Laldingngheta J, Lalnundanga, Malsawmzuala and Lalhlenmawia H: Evaluation of the anti-diabetic activity of ethanol extract of leaves of Scurrula parasitica in streptozotocin-induced diabetic rats. Journal of Pharmacognosy and Phytochemistry 2019; 8(3): 2206-12.
11. Elizabeth M, Estabrook and John IY: Plant-plant communications: Rhizosphere Sharma H K, Chhangle L and Dolui A K. Traditional medicinal plants in Mizoram, India. Fitoterapia 2001; 72: 146-61.
12. Deliorman D and Ergun F: A new acyclic monoterpene glucoside from Viscum album Album. Fitoterapia 2001; 72(2): 101-05.
13. Paiva SR, Kaplan DE and Gottlieb OR: Academia Brasileira de Ciências 1995; 67: 461-66.
14. Stahl E: Thin Layer chromatography, Springer-Verlag, Berlin 1996; 494.
15. Skoog D, West D and Holler J: Fundamentals of Analytical Chemistry, 5^th Saunders College Publishing, New York 1988; 894.
16. Fried B and Sharma J: Thin layer chromatography technique and applications 3rdedition (Revised and expanded), Marcel Dekker, New York, USA 1994; 51.
17. Lala PK: Elements of Chromatography, Lina publishers, Kolkatta 1981; 25.
18. Edwards RL, Lyon T, Litwin SE, Rabovsky A, Symons JD and Jalili T: Quercetin Reduces Blood Pressure in Hypertensive Subjects. Journal of Nutrition 2007; 137: 2405-11.
19. Loke WM, Hodgson JM, Proudfoot JM, McKinley AJ, Puddey IB and Croft KD: Pure dietary flavonoids quercetin and (_)-epicatechin augment nitric oxide products and reduce endothelin-1 acutely in healthy men. American Journal of Clinical Nutrition 2008; 88: 1018-25.
20. Garcı´a-Lafuente A, Guillamo´n E, Villares A, Rostagno MA and Martı´nez JA: Flavonoids as anti-inflammatory agents: implications in cancer and cardiovascular disease. Journal of Inflammation Research 2009; 58: 537-52.
21. Egert S, Bosy-Westphal A, Seiberl J, Ku¨rbitz C, Settler U and Plachta-Danielzik S: Quercetin reduces systolic blood pressure and plasma oxidized low-density lipoprotein concentrations in overweight subjects with a high-cardiovascular disease risk phenotype: a double-blinded, placebo-controlled cross-over study. British Journal of Nutrition 2009; 102: 1065-74.
22. Pandey KB and Rizvi SI: Protection of protein carbonyl formation by quercetin in erythrocytes subjected to oxidative stress. Medical Chemistry Research 2010; 19: 186-92.
23. Joskova M, Franova S and Sadlonova V: Acute bronchodilator effect of quercetin in experimental allergic asthma. Bratislava Lekarske Listy 2011; 112: 9-12.
24. Jung CH, Lee JY, Cho CH and Kim CJ: Anti-asthmatic action of quercetin and rutin in conscious guinea-pigs challenged with aerosolized ovalbumin. Archives of Pharmacal Research 2007; 30: 1599-1607.
25. Moon H, Choi HH and Lee JY: Quercetin inhalation inhibits the asthmatic responses by exposure to aerosolized-ovalbumin in conscious guinea-pigs. Archives of Pharmacal Research 2008; 31: 771-78.
26. Kyle JA, Sharp L and Little J: Dietary flavonoid intake and colorectal cancer: a case-control study. British Journal of Nutrition 2010; 103: 429-36.
27. Han JJ, Hao J and Kim CH: Quercetin prevents cardiac hypertrophy induced by pressure overload in rats. Journal of Veterinary Medical Science 2009; 71: 737-43.
28. Perez-Vizcaino F, Duarte J and Jimenez R: Antihypertensive effects of the flavonoid quercetin. Pharmacological Reports 2009; 61: 67-75.
29. Geoghegan F, Wong RW and Rabie AB: Inhibitory effect of quercetin on periodontal pathogens in-vitro. Phytotherapy Research 2010; 24: 817-20.
30. Li M and Xu Z: Quercetin in a lotus leaves extract may be responsible for antibacterial activity. Archive of Pharmacal Research 2008; 31: 640-44.
31. Gonzalez O, Fontanes V and Raychaudhuri S: The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production. Hepatology 2009; 50: 1756-64.
32. Kaul TN, Middleton E Jr and Ogra PL: Antiviral effect of flavonoids on human viruses. Journal of Medical Virology 1985; 15: 71-79.
    

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    How to cite this article:

    Jerry L, Lalnundanga and Vabeiryureilai M: Phytochemical analysis and isolation of flavonoid compound from methanol extract of the leaves of Scurrula parasitica L. Int J Pharm Sci & Res 2021; 12(9): 4927-32. doi: 10.13040/IJPSR.0975-8232.12(9).4927-32.

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