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Spectroscopic Analysis of Flavonoid Quercetin from Methanol Extracts of Emblica Officinalis Fruits
Aditi Gupta
Department of Chemistry, Govt. Degree College, Kathua, J&k, (India)
ABSTRACT
A brown coloured flavonoid has been isolated from the column chromatography of methanol extracts of Emblica officinalis seeds. The structure of this brown powder was fully elucidated with the help of detailed spectroscopic study and chemical analysis like UV, IR, 1H-NMR, 13 C-NMR and MS. Quercetin is used for the treatment of allergies, cancer, cardiovascular diseases, inflammations and also act as antihistaminic compound.
Quercetin, a flavonol, is one of the most potent antioxidants among polyphenols.
Keywords:
Emblica officinalis, Methanol extracts, Brown powder, Flavonoid, Quercetin
I. INTRODUCTION
Emblica officinalis Gaertn. (Phyllanthus emblica Linn.), also known as amla, has been used in Ayurveda, the ancient Indian system of medicine. According to the main classic texts on Ayurveda, Charak Samhita and Sushruta Samhita, amla is regarded as the “best among rejuvenative herbs”, and the “best among the sour fruits”
[1]. It belongs to family euphorbiaceae and is also known as Indian gooseberry [2]. The fruit also forms an important constituent of many Ayurvedic preparations such as chyvanprash and triphala and is regarded as “one of the best rejuvenating” herbs [3]. It is the richest source of antioxidants like vitamin C, emblicanin A and B, punigluconin, pedunculagin, catalase, gluthathione peroxidase, tannin, trigalloyl, polyphenols, flavonoids, ellagic acid, phyllembic acid, gallic acid and tannic acids[4-6].
II.EXPERIMENTAL
2.1 General
The melting point was determined on Lab fit melting point apparatus. A UV spectrum in ethanol was obtained on SHIMADZU UV-1800 UV spectrophotometer. An IR spectrum was recorded on SHIMADZU FTIR-8400S (Fourier Transforms infrared spectrophotometer). 1H-NMR (400MHz) and 13C-NMR were recorded in MeOD on Bruker, Avance 400 MHz NMR spectrometer. Chemical shifts are given as δ with TMS as internal standard. A HR-mass spectrum was recorded on Agilent, 6540, Q-TOF (HR-MS) mass spectrometer.2.2 Plant material
Fruits of Emblica officinalis were purchased from a specific seed shop of Jammu’s district and classified systematically by Dr. Gurdev Singh of the botany department at Lovely Professional University.2.3 Extraction and isolation
The dried and crushed fruits (one kg) of Emblica officinalis were soaked in methanol for 120 hours. The crude extract of methanol was subjected to column chromatography and 5:3 Pet.ether: DCM fraction after keeping for around 2 months results into shiny, brown powder. The brown powder thus separated and recrystallised with ethanol thrice for the identification of secondary metabolite.
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III. RESULTS AND DISCUSSION
The shiny brown powder obtained from methanol extracts of Emblica officinalis fruits was found to be a flavonoid on performing Shinoda test and Zinc Hydrochloride reduction test [7]. The flavonoid framework was also supported by UV and IR spectroscopy. The melting point was found out to be 315⁰C, which is very close to that of quercetin [8]. TLC of the powder which were recrystallised using ethanol showed Rf value equals to 0.99 that is similar to Rf value of quercetin observed in literature was 1.02 [9] thus powder obtained may be of quercetin.
3.1
The UV spectral Analysis
UV Spectra peak observed at 368 nm is very close to 370 nm corresponds to flavonoid as shown in Fig. 1.Fig. 1. UV spectrum of Brown powder from Emblica officinalis fruits
3.2 IR Spectral Analysis
IR spectral peaks shows a band at 3564 cm-1 which is due to free O-H, at 3082 cm-1 showed the presence of intra molecular hydroxyl groups, 1618 cm-1 is due to carbonyl group (C=O), 1510, 1550, 1610 cm-1 showed the presence of C-C stretching of aromatic ring and 1111 cm-1 due to ether group as shown in Fig. 2.Fig 2. IR Spectra of brown powder
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3.3 1H-NMR Spectral Analysis
The 1H-NMR (DMSO) shows values at δ 6.69(1H, d, J=1.7Hz), at δ 6.71(1H, d, J=1.7Hz) are due to meta-coupled protons of A-ring (H-6 and H-8) of a flavonoid nucleus.Signals at δ = 6.92 d, 7.4 and δ = 7.29 dd are assigned to H-5’, H-2’ and H-6’ of the ring. Aromatic protons show various peaks in the region of 6 – 9.
1 2
3 4
5 6
7 8
9
10 ppm
1.21772.08122.50912.54722.74392.75862.87352.95862.98003.09893.15233.19143.40613.41403.43043.44813.46493.51603.59993.64513.65713.74033.80293.84113.95104.03014.04674.09824.13054.15944.19724.26604.27524.29774.31584.37404.40246.69116.71336.92536.93696.99797.02127.10197.29997.4608
8.16538.31108.3335 0.411.433.480.511.261.000.462.100.33
Current Data Parameters NAME Apr08-2013 EXPNO 390 PROCNO 1 F2 - Acquisition Parameters Date_ 20130408 Time 23.43 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT DMSO NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 362 DW 41.600 usec DE 6.00 usec TE 296.4 K D1 1.00000000 sec TD0 1
======== CHANNEL f1 ========
NUC1 1H P1 10.90 usec PL1 -3.00 dB SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300000 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 EO (1)
BRUKER
AVANCE II 400 NMR Spectrometer SAIF
Panjab University Chandigarh
Fig. 3 1H-NMR Spectra
3.4 13C-NMR Spectral Analysis is shown in Fig. 4.
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
38.8739.0839.2939.5039.7039.9140.12
78.4378.7679.09
107.39108.69110.22112.28
120.41
136.25137.80139.74145.22148.06
159.11
Current Data Parameters NAME Apr22-2013-Administrator EXPNO 591 PROCNO 1 F2 - Acquisition Parameters Date_ 20130423 Time 6.11 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zgpg30 TD 65536 SOLVENT DMSO NS 1024 DS 4 SWH 29761.904 Hz FIDRES 0.454131 Hz AQ 1.1010548 sec RG 1030 DW 16.800 usec DE 6.00 usec TE 292.8 K D1 2.00000000 sec d11 0.03000000 sec DELTA 1.89999998 sec TD0 1
======== CHANNEL f1 ========
NUC1 13C P1 9.60 usec PL1 -2.00 dB SFO1 100.6228298 MHz
======== CHANNEL f2 ========
CPDPRG2 waltz16 NUC2 1H PCPD2 80.00 usec PL2 -3.00 dB PL12 14.31 dB PL13 18.00 dB SFO2 400.1316005 MHz F2 - Processing parameters SI 32768 SF 100.6128193 MHz WDW EM SSB 0 LB 1.00 Hz GB 0 PC 1.40
EO BRUKER
AVANCE II 400 NMR Spectrometer SAIF
Panjab University Chandigarh
Fig. 4 13 C-NMR Spectrum
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Justification of various peaks in 13 C-NMR Spectrum
Position
13C NMR
2 147.8
3 136.8
4 176.9
5 159.11
6 98.8
7 167.0
8 94.0
1’ 120.41
2’ 112.28
3’ 145.22
4’ 148.7
5’ 116.2
6’ 120.6
3.5 Mass Spectral Analysis :
Mass Spectrum [10] of brown powder is shown belowIV. CONCLUSIONS
On the basis of above spectral analysis the brown powder may be classified as flavonol Quercetin as shown in Fig. 5.
Serial No. Peaks Justification of Peaks
1 301 [M-H]
-2 302 [M+H]
+3 303 [M+2H]
+4 317 [M+OH+H]
+5 300 [M-2H]
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C6 C5 C8
C4
C3 C2 O1
OH HO
O
OH C1'
c2' c3' C4' c5'
c6'
OH
OH
Fig. 5 Structure of Quercetin
V. ACKNOWLEDGEMENTS
The author is thankful to LPU and Dr. Gurdev Singh for identification.
REFERENCES
[1] D. Arora, R. Shri, S. Sharma and A Suttee, Phytochemical and Microscopical investigations on Emblica officinalis Gaertn, International Journal of Pharmacognosy and Phytochemical Research, 4(4), 2012, 1-4.
[2] K.H Khan, Roles of Emblica officinalis in Medicine - A Review, Botany Research International, 2(04), 2009 218-228.
[3] S.K Soni, N Bansal and R Soni, Standardization of conditions for fermentation and maturation of wine from Amla (Emblica officinalis Gaertn.), Natural Product Radiance, 8(4), 2009, 436-444.
[4] A Kumar, Protective effect of amla on oxidative stress and toxicity in rats challenged dimethal hydrazine, Nutritional Research, 24, 2004, 313 – 319.
[5] S Kumar et al., Free and bound phenolic antioxidants in amla (Emblica officinalis) and turmeric (Curcuma longa), Journal of Food Composition and Analysis, 19, 2006, 446 – 452.
[6] E Manju, A Thangavel and V Leela, Effect of dietary supplementation of amla and grape seed powders on antioxidant status in the seminal plasma of Broiler breeder cocks, Tamilnadu J. Veterinary & Animal Sciences, 7(5), 2011, 229-233.
[7] S De, Y.N Dey, A.K Ghosh, Phytochemical investigation and chromatographic evaluation of the different extracts of tuber of Amorphaphallus paeoniifolius (araceae), International Journal on Pharmaceutical and Biomedical Research, 1(5), 2010, 150-157.
[8] C. H Ice, S.H Wender, Quercetin and its Glycosides in Leaves of Vaccinium myrtillus, JACS, 75(1), 1953, 50-52.
[9] C. I Sajeeth, P.K Manna, R. Manavalan, C.I Jolly, Quantitative estimation of Gallic Acid, Rutin and Quercetin in certain herbal plants by hptlc method, Der Chemica Sinica, 1(2),2010, 80-85.
[10] Z Guvenalp, R.L.O Demireze, Flavonol Glycosides from Asperula arvensis L., Turk J Chem, 29, 2005, 163-169.
