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Journal of Stress Physiology & Biochemistry, Vol. 16, No. 4, 2020, pp. 107-112 ISSN 1997-0838 Original Text Copyright © 2020 by K. Shakila
ORIGINAL ARTICLE
Bioactive Flavonids of Spinifex littoreus (Burm. f.) Merr - Investigation By Elisa Method
K. Shakila
Arignar Anna Government Arts and Science College, Karaikal, Pondicherry, India.
*E-Mail: shakila.manickaraj@gmail. com
Received June 25, 2020
Spinifex littoreus Burm.f.Merr. Coastal shrubs used for the treatment of a wide variety of diseases such as galactogenic, blistering agent, antihelmenthic, antipyretic, antidiruetic, anti -haemorrhages, antileukaemias and anti-inflammatory. It is rich in flavonoids and known to possess various biological activities. Fresh leaves of Spinifex littoreus have been examined for their bioactive flavonoids. The flavonol Kaempferol, flavonol glycoside astragalin have been identified. The structure of isolated polyphenolic compounds were characterised with the help of physical methods like UV, 1H NMR, 13C NMR, Chemical reactions, Chromatographic techniques and hydrolytic studies The in-vitro studies showed that the isolated flavonol glycosides have been found to contain ample anti-oxidant property with the help of ELISA method.
Key words: Astragalin, Kaempferol, Poaceae family
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Spinifex littoreus Burm. f. Merr. (Syn.) Spinifex squarrosus L; Stipa spinifex L belongs to the family poaceae commonly called as Ravana - moustache. It is distributed throughout Fuijian, Guangdong, Guangri, Hainan - Taiwan, Cambodia, India, Indonesia, Malaysia, Myanmar, Philippines, Srilanka, Thailand and Vietnam (Wu & Raven 2006). It is used as galactogenic, blistering agent, antihelminthic, antipyretic, antidiruetic, anti - haemorrhages, antileukaemias and antiinflammatory (Muthukumaran et al., 2012). Leaves are found to contain analgesic activity (Yogamoorthi & Priya 2006) and antimicrobial activity (Thirunavukkarasu et al., 2010).
A biological system of antioxidant may preserve the cells from impairment caused by unstable molecules and the free radicals. Antioxidants terminate chain reactions (present in cell molecules) by removing free radical intermediates and inhibit other oxidation reactions being observed by them. They are believed to play a role in hindering the development of such chronic diseases as cancer, heart disease, stroke, arthritis and cataracts (Vinegar et al., 1969).
MATERIALS AND METHODS
Plant extraction and fractionation
Air dried leaves (800g) of S. littoreus collected from sea shore of chandrabady in Nagapattinam district were extracted with 85% EtOH (4x 500 ml) under reflux. The specimen for the S. littoreus is kept at Rapinat Herbarium and Centre for Molecular Systematics, St.Josheph's college (Campus), Tiruchirappalli- 620 002, the specimen number being SA 011.
The alc. extract was concentrated in vacuo and the aq. concentrate successively fractionated with benzene (3 x 250 ml), peroxide free Et2O (3x 250 ml) and EtOAc (4 x 250 ml).
The benzene and Et2O fractions did not yield any isolable material. Physical parameters
The EtOAc fraction was concentrated in vacuo and left in ice - chest for a day when a yellow solid was separated which was filtered and studied. When recrystallized from MeOH, it came out as long yellow
needles, m.p. 176 - 78° C. It was freely soluble in aq. NaOH, hot water, EtOH and EtOAc, but insoluble in Et2O, Me2CO and CHCL Chemical reactions
It gave a greenish - brown colour with alc. Fe3+, an intense yellow colour with NaOH, red colour with Mg -HCl and yellow precipitate with aq.lead acetate. It appeared dark purple under UV, which turned yellow on fuming with NH3. It answered Wilson's boric acid (Wilson, 1939), Gibb's, Shinoda (Shinoda, 1928) and Molisch's tests but did not respond to the Horhamer -Hansel test (Horhammer & Hansel, 1955). Spectral data
UV values of isolated compound had nm 264,
301sh, 350; +NaOMe 273, 324, 398; +AlCl3 with and without HCl 275, 304, 353, 397sh; +NaOAc 269, 305, 317sh, 353; +NaOAc/H3BO3 265, 301sh and 351. Chromatographic data
Sheets of 20x45cm Whatman no 1 were used. The papers were run in glass cylinders by the ascending technique, usually to a height of 22-30cm at 250C. The solvents used were H20, 5% HOAC, 15% aq. HOAC, 30% aq. HOAC, 60% aq. HOAC, n-BuOH:AOAC:H2O (4:1:5 upperphase), phenol saturated with water, BuOH:HOAC:H2O (3:1:1), Forestal (HOAC:Conc.Hcl: H2O 30:3:10), formic acid (EtOH:ACOOH:H2O 10:2:3). After through drying the chromatograms were dipped quickly into a saturated solution of ninhydrin. Rf values are varied greatly with the amount of flavonoid present and adsorption occurred at the origin of the chromatogram. It had Rf values as depicted in Table (I). The glycoside was identified as astragalin and that was confirmed by co- pc with an authentic sample of astragalin isolated from Cestrum noctunum (Barnabas, 1986).
Hydrolysis of the glycoside
The glycoside (0.05 g) dissolved in hot. aq. MeOH (2 ml, 50%) was hydrolysed with H2SO4 (5%) at 100° C for about 2h, and the hydrolytic products identified as described below.
Identification of aglycone (flavonol:Kaempferol)
The aglycone on recrystallisation from MeOH
afforded a yellow crystalline solid, m.p. 278 - 80 0C, which was identified as kaempferol by colour reactions, behaviour under UV. It was soluble in organic solvents but insoluble in water. It developed a reddish orange colour with Mg - HCl and yellow colour with NaOH. It appeared pale yellow under UV with or without NH3. It responded to Wilson's boric acid, Horhammer Hansel and Gibb's tests but did not answer the Molisch's test.
UV values, XMO" nm 253sh, 266, 294sh, 322sh, 367; +NaOMe 278, 316, 416(dec.); (+Alcy 260sh, 268, 303sh, 350, 424; (+AlCl3 - HCl) 256sh, 269, 303sh, 348, 424; +NaOAc, 274, 303, 387, and +(NaOAc +H3BO3) 267, 297sh, 320sh, 372. It has Rf values as depicted in Table (2). The structure has been ascertained by comparing the sample with an authentic sample isolated from Bauhunia acuminata (Barnabas & Nagarajan, 1979)
Identification of sugar
The aq. hydrolysate after the removal of the aglycone was neutralized with BaCO3 and filtered. The concentrated filtrate on PC gave Rf values corresponding to those of glucose. The identity of the sugar was confirmed by direct comparison with and authentic sample of glucose. DPPH Elisa method:
The antioxidant activity of the plant extract was estimated using a slight modification of the DPPH radical scavenging protocol given by Chang et al. (2001) DPPH free radical scavenging is one of the generally accepted mechanisms against lipid oxidation. The difference between DPPH free radical binding method and the other method is the short run time allowing rapid determination of the radical scavenging. The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating ability. The antioxidant activity of plant extracts were calculated according to the percentage inhibition in DPPH assay. It was optimized to be able to use micro titer plates, a multichannel pipet, and an ELISA reader, which makes it possible to analyze large numbers of samples in a run. With this method it was possible to determine the antiradical power of an antioxidant by measuring the decrease in the absorbance of DPPH at 517 nm. As the
result of the colour changing from purple to yellow the absorbance is decreased when the DPPH radical is scavenged by an antioxidant through donation of hydrogen to form a stable DPPH-H molecule (Matthäus, 2002). For a typical reaction, 2 ml of 100 цМ DPPH solution in ethanol was mixed with 2 ml of 100 |jg/ml of plant extract. The effective test concentrations of DPPH and the extract were therefore 50 цМ and 50 ng/ml, respectively. The reaction mixture was shaken vigorously and allowed to stand at room temperature and incubated in the dark for 15 min and thereafter the optical density was recorded at 517nm against the blank. For the control, 2 ml of DPPH solution in ethanol was mixed with 2 ml of ethanol, and the optical density of the solution was recorded after 15 min. The assay was carried out in triplicate. Then the absorbance was measured at 517 nm in an ELISA reader. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity (Oktay et al., 2003). The decrease in optical density of DPPH on the addition of test samples in relation to the control was used to calculate the antioxidant activity, as percentage inhibition (%IP) of DPPH radical:
(A control - A sample)
Radical scavenging (%) =...............................x 100
A control
(Galani et al., 2010)
The polyphenolic compounds and their percentage of inhibition of oxidation are listed in table 4.
RESULTS AND DISCUSSION
Characterisation of flavonoids from the active fraction of EtOAc fraction
The leaves of S. littoreus have been found to contain astragalin.
The band I absorption of the glycoside is at 350nm, which is an indicative of a flavonol skeleton. A comparison of band I absorption of the glycoside and the aglycone reveals that there may be 3 -glycosylation in the flavonol. A bathochromic shift of 48nm (band I) ascertained the presence of a free - OH at C- 4'. The AlCls spectra, with and without HCl showed four absorption peaks to reveal the presence of a 5 -OH group. They were absorption peaks by the bathochromic shift of 47nm on the addition of NaOAc. The H3BO3
spectrum is exactly same as that of MeOH indicating the absence of catechol type of substitution in B - ring.
In the 1H - NMR spectrum (400 MHz, DMSO - d6, TMS), the A- ring protons at C- 6 and C-8 appear separately as doublets at 56.21 ppm (J= 1.7Hz) and 56.21 ppm (J = 1.6 Hz) respectively. The 5 - OH proton resonates at 512.6ppm. In the B - ring, the protons at C-2', 6' and C-3' and 5' are due to the free rotation of phenyl ring appear as two pairs of ortho coupled doublets at 56.88ppm (J = 8.7Hz) and 58.03ppm (J =8.8 Hz). The H -1" signal of the glucose moiety appears at 55.46ppm (J = 7.2 Hz) found downfield from the bulk of the sugar protons. The remaining glycosyl protons appear in the range 53.1 to 53.7 ppm. The p - linkage of the glucose to 3 -OH is evident from the large coupling constant 57.2 Hz of H - 1".
Comparing 13C- NMR (100MHz, DMSO - d6, TMS) spectrum of the glycoside with that of the aglycone, the carbonyl carbon at C-4 of the glycoside appears at 1.59 ppm downfield than the aglycone. Due to glycosylation at C-3, the C-2 signal is shifted to 10.5 ppm downfield. The C-1" of the sugar appears at 5100.7 ppm and the rest of the sugar protons appear in the range 5 60.9 and 577.2 ppm.
A complete assignment of the 13C - NMR spectrum of the flavonol glycoside is available in Table (3). Based on these observations, the glycoside obtained from
EtOAc fraction of S.littoreus could be confirmed as astragalin( kaempferol - 3- O- glucoside). Investigation of antioxidant property
DPPH is one of the compounds that have a proton free radical with a characteristic absorption, which decreases significantly on exposure to proton radical scavengers (Yamaguchi et al., 1998). It is well accepted that the DPPH radical-scavenging by antioxidants is attributable to their hydrogen-donating ability (Chen & Ho, 1995; Wu et al., 2008). In this current study, the DPPH radical scavenging method was used to evaluate the antioxidant capacity of the yellow pigments isolated from the leaves, because the use of DPPH radical provides an easy, rapid and convenient method to evaluate the antioxidants and radical scavengers (Nickavar et al., 2007).
The antioxidant activities of the isolated polyphenolic compound was tested and assessed on the basis of radical scavenging effect of the stable DPPH free radical. The scavenging capacities of phenolic compound were compared with that of ascorbic acid 100 Mg/ml. These results imply that there are abundant antioxidative phytochemicals present in the plant species. The extract may exert its effect by reducing the oxidative damage due to free radicals. The EtOAc fraction exhibited the highest radical scavenging activity.
Table 1. Rf [x 100] values of the constituents of the leaves of S. littoreus (Whatman No : 1, Ascending, 30 ± 2° C)
Compound DEVELOPING SOLVENTS
a b c d e f g h
Glycoside from EtOAC fraction 13 40 42 68 77 70 71 53
Astragalin (authentic) 13 40 42 68 77 70 71 53
Table 2. Rf [x 100] values of the constituents of the leaves of S. littoreus (Whatman No : 1, Ascending, 30 ± 2° C)
Compound DEVELOPING SOLVENTS
a b c d e f g h i
Aglycone from Et2O fraction - - 5 15 49 93 67 62 87
Kaempferol (authentic) - - 5 15 48 93 67 62 87
Table 3. 13C NMR spectral data and their assignment for the glycoside from the flowers of S. littoreus
Compound C2 C3 C4 C5 C6 C7 C8 C9 C10 C'1 C'2 C'a C'4 C'5 C'6
Glycoside from EtOAc fraction (6 ppm) 156.75 133.06 177.41 161.62 98.71 164.14 93.60 156.35 104.10 121.04 130.70 115.06 159.81 115.06 130.70
Astragalin (from Literature) 156.75 133.06 177.41 161.62 98.71 164.14 93.60 156.35 104.10 121.04 130.70 115.06 159.81 115.06 130.70
Compound C''1 C''2 C''3 C''4 C''5 C''6
Glycoside from EtOAc fraction (5 ppm) 100.71 74.22 77.38 69.89 76.45 60.81
Astragalin (from Literature) 100.71 74.22 77.38 69.89 76.45 60.81
Table 4. Antioxidant activity of the isolated flavonoid glycosides
S.No Grouping Absorption Л at 517 nm % of inhibition of oxidation
1 Ascorbic acid 0.04 98%
2 CB 2 --
8 Astragalin 0.82 89%
CONCLUSION
Phytochemical studies of S. littoreus leaves led to the identification of bioactive flavonoids (Kaempferol, astragalin). So they have the valuable source of flavonoids with antioxidant activity.
ACKNOWLEDGMENT
I express my deep sense of gratitude to sophisticated instruments facility IISC, Banglore, for its help in recording the NMR spectra of some of the isolates.
My heartful thanks to the Ranipat Herbarium, St. Joseph College (Autonomous), Thiruchirapalli, in identifying the plant species of this examination.
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