Isolation and Characterization of Flavonoids of the Ethanolic extracts of stems of Mimosa hamata (Willd.) by Chromatographic techniques
Khan N.I.1*, Hatapakki B.C.2, Tamboli A.M.1
1Department of Pharmacology, Sahyadri College of Pharmacy, Methwade, Maharashtra, India – 413307.
2Department of Pharmacognosy, P.S.P.S.'s Indira Institute of Pharmacy,
Sadavali (Devrukh), Maharashtra, India – 415804.
*Corresponding Author E-mail: naziya.aara@gmail.com
ABSTRACT:
Since there are many bioactive compounds present in plant material containing various multi-component mixtures, their separation and determination is important to identify the active phytoconstituents responsible for pharmacological activity. Practically most of them have to be separated by column chromatographic techniques. The present study deals with the identification and characterization of bioactive principles from the stems of Mimosa hamata. The isolated fractions from the ethanolic extracts of stems of Mimosa hamata was carried out by column chromatography. For separation of a bioactive compound, the solvent system tried for column chromatography was Chloroform: Methanol in various ratios like 90:10, 80:20, 70:30, and 60:40 amongst these we could separate the first fraction at 80:20 and second fraction at 70:30. Two flavonoids compounds were isolated from the ethanolic extracts of stems of the medicinal plant Mimosa hamata (Willd). Based on chemical and spectral analyses their structures were elucidated as Quercetin and Cirsimaritin. From the above study it reveals the presence of flavonoids in the ethanolic extracts of stems of Mimosa hamata (Willd.) was isolated using column chromatography further subjected to characterization of isolated compounds including UV, IR, NMR and Mass spectroscopic study for elucidating the structure of the two separated compounds. The interpreted data concluded that, both isolated compounds are flavonoids i.e. Cirsimaritin and Quercetin. The pharmacological effect of Mimosa hamata stems may be due to the presence of its phytoconstituents flavonoids.
KEYWORDS: Mimosa hamata, Quercetin, Cirsimaritin, Chloroform, Ethanolic, Flavonoids, Methanol.
INTRODUCTION:
In ancient Indian literature, it is mentioned that every plant on this earth is useful for human beings, animals and other plants.1 Many of the modern drug mainly based on synthetic chemical compounds, however have been found to have harmful side effects on the human system. This has triggered off extensive research and development in the field of herbal medicine. In fact, there is a growing demand for herbal medicines in most of the developed and developing countries of the world today.2 Mimosa hamata (Willd.) (Fig.1) (Family Mimosaceae) (Genus Mimosa) commonly known as Alai is a flowering shrub of pea family and is native to arid regions of Indian subcontinent.
Genus Mimosa (family: Mimosaceae) has about 400 species which are mainly shrubs and small trees in tropics. About 8 species are found in India, with medicinal importance3 while some are of ornamental use. Mimosa pudica the curious plant in the genus is a creeping form. Because of the way it folds its leaves when touched, it is known as touch-me-not plant. Mimosa hamata also folds its leaves when touched4. Mimosa hamata is a much straggling shrub occurring in tropics and widely distributed in India and Pakistan5. The plant is used for urinary complaints and as a tonic against general weakness. A paste of leaves is applied over glandular swellings and is used in dressing for sinus, sores and piles6. Its roots possess contraceptive efficacy while seeds are used as blood purifier7. Various bioefficacies viz., antifungal activity of deprotenized leaf extract8,9. Antibacterial activity of alcoholic extract of aerial parts, antiviral activity of methanolic extract of roots3 and Antioxidant activity10 have been reported. The major phytoconstituents present in Mimosa hamata (Willd.) include 4-ethylgallic acid from fresh flowers, triterpene saponin B (3-O-Larabinosyl-D-glucosyl morolic acid), mimonoside A, B, C and saponin A (3-O-D-glucosyl-L-rhamnosyl morolic acid) from the roots, ethylgallate and gallic acid from leaves.11
The plant Mimosa hamata (Willd.) belonging to the family Mimosaceae is being selected for phytochemical investigations to pin point the pharmacological activity. The said plant has been reported to possess antibacterial, antiviral and antioxidant properties.8,9,3,10.
MATERIAL AND METHODS:
Plant material:
For the present study stems of M. hamata was collected from Methwade, Tal. Sangola, Dist. Solapur (Maharashtra) and plant was authenticated at Botanical Survey of India, Pune. The plant material was dried out at room temperature for about two weak. The dried plant samples were powdered by grinder and sieved to give particle size 40- 100mm.
Extraction:
Preparation of Alcoholic extract:
The collected stems of Mimosa hamata (Willd.) were shade dried, reduced to a coarse powder in a mechanical grinder to obtain of desired particle size (40# sieve). About 200gms of powdered material was subjected to extraction with 90% of alcohol in a Soxhlet extractor at a temperature of 60 – 700C, concentrated on a rotary flash evaporator at 500C (Superfit, India), and finally to dry powder. Some part of the total extract was reserved for phytochemical investigation and rest of the extract was used for biological activity.
Preliminary phytochemical analysis:
The ethanolic extracts of stems of Mimosa hamata (Willd.) was then subjected to preliminary phytochemical analysis to assess the presence of various phytoconstituents, it revealed the presence of flavonoids, carbohydrate, glycosides, and tannins. Preliminary Thin layer chromatography studies also confirmed the presence of phytoconstituents12.
ISOLATION:
Fractionates of the plant extract were obtained by column chromatography on the basis of TLC.13-16
Column chromatography:
Column preparation:
The column was mounted on the top and the base on a circular support with two finger clips. The glass wool was soaked (~ 0.1g) in a small amount of chloroform and pushed to the base of the column using a large glass stirrer. The column tube was firmly locked and closed, followed by the addition of sufficient chloroform to the column reaching a height of about 15cm. sufficient sand was added to the column which reached a height of 1cm above the glass wool. The column was gently tapped on the sides with the stirring rod and ensured that the sand stabilized in the same way. A sufficient quantity of silica was gently added to the column and a height of about 10 cm was obtained. Again, 1cm of sand was added to the top of the column in the same way. The column was filled with sufficient chloroform at the height of the last addition of sand. The clamp knob was opened to drain excess chloroform into the crystallization plate while waiting for the ether level to be just above the top layer of sand. The ethanolic extracts of stems of Mimosa hamata (Willd.) was subjected to column chromatography using different solvent systems. The column was first eluted with 100% chloroform. The polarity of solvent was gradually increased with methanol. The collected fractions were dried. The dried fractions were kept in container with suitable label and kept for further use. A total of 4 fractions were collected from the column chromatography.
Preparation of the Chromatographic Fractions:
Based on the solvent used for elution in the Column chromatography, and based on the colour of the elutes, the eluted fractions were used for further analysis.
Qualitative Test of Fractions:
The qualitative tests were performed for all the four fractions isolated from the ethanolic extracts of stems of Mimosa hamata (Willd.) Perform the qualitative test of four fractions amongst the four fractions, fourth fraction shows positive test for flavonoids. The fourth fraction was further analysis and those fractions that were not rich in flavonoids could be excluded from the succeeding steps of the present research, because this research would be directed towards the isolation and identification of flavonoids in particular.
30 ml of the fractions which were rich in flavonoids i.e fourth fraction this fraction was chromatographed over silica gel column (100 – 200 mesh, 100grams). The fraction was packed on a silica gel column (Merck, India) and eluted with Chloroform: Methanol in the ratio of 90:10, 80:20, 70:30 and 60:40. Based on TLC profile, the elutes were pooled into different fractions. The yield of the fractions is as follows:
Sub-fraction-1 (270mg, 80: 20),
Sub-fraction-2 (310mg, 70: 30).
To perform the test of above two fractions isolated from the ethanolic extract of stems of Mimosa hamata for the presence of flavonoids. The tests for above two fractions revealed the presence of flavonoids.
Fig. 1 Isolated fractions
Characterization:
The UV spectrum 200-800nm region was obtained to determine the λ max. This was carried out to determine the absorbance region which could provide some information about the chromophore and nature of the extract.17
TLC characterization of ethanolic extract of stems of Mimosa hamata: The principle of partioning is either partition or adsorption. The constituent which is having more attraction for mobile phase moves with it, while the constituent which is having more attraction for stationary phase gets adsorbed on it. This technique various compounds appear as a band on the TLC plate, having dissimilar RF values. The ethanolic extract of stems of Mimosa hamata was exposed to thin layer chromatographic studies for the parting and identification of their components.18
Gas Chromatography- Mass Spectroscopy (GC-MS): The obtained fraction was diluted in 2ml of ethanol and poured into the GC vial and inserted into the GC-MS docks.19
IR Samples were prepared in KBr disks using a hydrostatic press at 6-8 tons pressure.20
1H spectra is recorded for isolated fraction in CDCl3 in order to establish the protonation site.21
Characterization of Flavonoids:
Compound-I:
Cirsimaritin:
4′,5-Dihydroxy-6,7-dimethoxyflavone, Skrofulein, Cirsimaritin.
The ethanolic extract of stems of Mimosa hamata (Willd). (Fabaceae) afforded 4′,5-Dihydroxy-6,7-dimethoxyflavone, Skrofulein, Cirsimaritin, M.F: C17H14O6, RF:0.92 (Chloroform: Methanol = 8:2)
The elemental analyses of the compound are consistent with its molecular formula, C17H14O6which is in excellent agreement with the appearance of its molecular ion peak at m/z 315 in the mass spectrum. The compound bears one carbonyl group two methyl group and two hydroxyl groups.
UV spectrum:
Extensive literature survey relating to UV-VIS absorption behavior of 4′,5-Dihydroxy-6,7-dimethoxyflavone compounds, the absorption maxima at λmax255 and 430 nm indicated the presence of a 4′,5-Dihydroxy-6,7-dimethoxyflavone nucleus within the molecule.
1H NMR Spectrum of Compound I:
The 1H NMR Spectrum (Fig. 2) of the compound was studied in CDCl3 with Bruker 400 MHz instrument using TMS as internal standard.
Fig.2 1H NMR Spectrum of isolated Compound I
Hydroxyl groups are unlikely in the aromatic ring were observed in the 1H-NMR spectrum for the protons attached to each –OH at δ-0.88 and also no splitting was observed for the two protons attached to this aromatic ring; besides, the whole aromatic region would observe at δ-7.27 and δ-8.11 show two singlet also 1H-NMR spectrum displayed signals for two methoxy proton at δ-1.21 and δ-1.26 This clearly indicates the substitution pattern of rings A and B in the structure of compound 1.
As expected, the 13C-NMR spectrum (CDCl3, 100 MHz; 1H-decoupled; (Fig.3) of the 4′,5-Dihydroxy-6,7-dimethoxyflavone derivative recorded signals for carbons at 129 and 133 ppm for aromatic carbon atom, 165ppm for carbonyl carbon atom and 62 ppm for Methoxy carbon atom in which their nature get identified. These 1H- and 13C- NMR spectral data for obtained from the high-resolution NMR spectrophotometer are fully in agreement with those reported elsewhere.
GC-MS Spectrum of Compound-I
Fig. 4GC-MS Spectrum of Compound I
A typical GC/MS chromatogram is presented in Figure 3. The molecular ion [M]+ m/z 315 for all of the derivatives is a prominent peak in the mass spectrum. Generation of the [M-19] fragment (loss of water via a-cleavage) and the [M-16] fragment (subsequent loss of OH after rearrangement). Then [M-39], is also a fragmentation pattern by Loss of (CH2)2. Derivatives possessing loss of group on the benzene ring, such as hydroxyl group produce the [M-32] fragment. Also go on to increase radiation the molecular ion peak get converted in to fragment Ion like m/z 156, 127, 113, 9985, 71 and finally last fragment ion observe at 57.
Quercetin:
2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one
The ethanol extract of stems of Mimosa hamata (Willd). (Fabaceae) afforded 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one, M.F: C15H10O7, M.P: 316 °C, RF:0.82 (Chloroform: Methanol = 8:2)
Molecular Formula and Functional Groups
The elemental analyses of the compound are consistent with its molecular formula, C15H10O7 with the appearance of its molecular ion peak at m/z 303 in the mass spectrum. The compound bears one carbonyl group and four hydroxyl groups.
UV Spectroscopy was helpful in the structural identification of unidentified compounds. It relates to an assessment of the absorption spectrum or reflectivity spectroscopy in fragment of the visible ultraviolet and adjuvant region. In this region of the electromagnetic spectrum, atoms and molecules undergo electronic transition. The isolated compound of UV spectra shows peak at 369 nm.
The IR Spectra of Quercetin observe near about 3600/cm-1is because of Phenolic Hydroxyl group in molecule. The intense absorption peak at 1666/cm-1 for carbonyl group. The peak observed at 1476/cm-1is due to the aromatic ring and 2816/cm-1for C-H Group.
Fig. 5H1 NMR Spectrum of isolated Compound II
In H1 NMR Spectrum of Compound II NMR Peak and Probable assignments observe in above H1 NMR Spectrum 7.5ppm peak for aromatic proton at downfield, 12.16ppm (OH) Hydroxyl group proton is of shown highly de-shielding effect, 7ppm, 6.1ppm, 6.3ppm all are shown hydroxyl proton. These peaks matched exactly with standard Quercetin as given in the literature. Thus, it can be confirmed that the isolated compound is Quercetin.22
C13 NMR Spectrum of Compound II
In C13 NMR Spectra in Fig.No:6 probable peak at C4 Carbon atom shown at 178.65 ppm, C7 Carbon atom at 165.73ppm, C5 Carbon atom at 163.03ppm, C3 and C5 Carbon atom at 145.97 ppm, C2 Carbon atom at 148.03 ppm, C3 Carbon atom at 135.63 ppm, C1 Carbon atom at 123.49 ppm, C2 and C6 Carbon atom at 105.50 ppm, C10 Carbon atom at 103.23 ppm, C6 Carbon atom at 98.23 ppm.
GC-MS Spectrum of Compound II
Fig. 7GC-MS Spectrum of isolated Compound II
The GC-MS Shown Mass spectra at 303 indicate for Quercetin compound.
GC-MS analysis of the Mimosa hamata (Willd.) resulted in identification of various phytochemicals having medicinal and pharmaceutical importance.23In GC-MS when compound in vapor form injected molecule get produce molecular ion by ejecting one proton from outer most orbit observe at m/e 303 and this molecular ion get fragmented in to fragment ion like m/e 257, m/e 229, m/e247, m/e201, m/e153, m/e 95.
RESULTS AND DISCUSSION:
In the present study, column chromatography and TLC eluted four different fractions further we performed the qualitative phytochemical test of these four fractions, it revealed the presence of flavonoids in the fourth fraction of Mimosa hamata (Willd.). From the fourth fraction two flavonoids were isolated further subjected to characterization of isolated compounds with UV, IR, NMR, and Mass spectroscopic study for elucidating the structure of the two separated compounds. The interpreted data and Chemical test concluded that, both isolated compounds were flavonoids i.e. Cirsimaritin and Quercetin. The pharmacological effect of Mimosa hamata stems may be due to the presence of its phytoconstituents i. e. flavonoids. In the field of medicine, flavonoids are part of every medicinal scientist’s resources and play an important role in treating various diseases. They are also a vital part of the successful régimes that have led to major therapeutic triumphs in chemotherapy. In pharmaceutical sciences, they serve as raw materials in the formulation of new and effective drugs.
CONCLUSION:
With reference to the above study the the stems of Mimosa hamata (Willd.) contains a higher percentage yield of Flavonoids as it exceeds the lowest yield of any medicinally useful Flavonoid ever produced on a commercial basis. Two Flavonoids were isolated from the stems of Mimosa hamata (Willd.) using Chloroform: Methanol as solvent system. The crude extract and flavonoids-rich fraction may be used as a source of alternative medicine. This study would serve as a guide for the science teachers and researcher in their research on the use of indigenous materials in their community. Likewise, this study would help the students in working on their scientific activities, thereby preparing and assisting them to participate in school, regional, national and even international science fairs. Moreover, the research procedures employed in this study would give an avenue to facilitate an appreciation and application of the topic in science.
ACKNOWLEDGEMENTS:
I would like to thank Dr. D. M. Ingawale, Dr. B.C Hatapakki, for providing the necessary facilities during my entire work. I would also like to thank my colleagues Dr. M. S. Patil, A. M. Tamboli for their continuous support and encouragement.
CONFLICT OF INTEREST:
Nil.
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Received on 20.06.2020 Modified on 04.09.2020
Accepted on 09.10.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(9):4602-4608.
DOI: 10.52711/0974-360X.2021.00800