INTRODUCTION:

Indigofera tinctoria belongs to the family of Fabaceae which is commonly known as Avuri (OR) Neeli in Tamil nadu. It has light green pinnate leaves and sheafs of pink or violet flowers. The plant is alegume, so it is rotated in to fields to improve the soil (1). The juice of Indigofera tinctoria leaves, flowers and indigo powder mixed with honey is used for enlargement of liver and spleen. Juice is also used in asthma, whooping cough, lung diseases and kidney disorders as in dropsy (2). Experimental evidence suggests that Indigofera tinctoria possesses anti-inflammatory, hepatoprotective, anti-epileptic, anti-cancer and neuroprotective properties (3) The methanol extract was concentrated in a rotary evaporator. The concentrated methanol extract was used for anti-inflammatory activity (4). The present work has carried out to compare the efficacy of phytochemically extracted the methanol Indigofera tinctoria fraction of and also to study its pharmacological effect has been anti-inflammatory activity by carrageenan model. Methanol fraction isorhamnetin 3-O-α-L-(6''-E-p-coumaroyl)-rhamnoside were identified (Fig. 1) using HPLC, UV, and NMR by comparison with the published data.

Isorhamnetin-3-O-α-L-(6¢¢-E-p-coumaroyl)-rhamnoside

Plant material:

Fresh flowers (2kg) of Indigofera tinctoria was collected during the August to December 2018 from the Cuddalore District in Tamilnadu, India. Dr. N. Ramakrishnan authenticated this species and the voucher specimen number is GUH7284, were deposited in Herbaium of the Botany Department, Government Arts College (Autonomous), Kumbakonam, Bharathidasan University, India.

Extraction and isolation Air dried flowers of Indigofera tinctoria (2.25kg) was extracted with 90% methanol (MeOH) in a Soxhlet apparatus. The hydro-alcoholic solution was concentrated under reduced pressure to dryness, and the residue was dissolved in hot water (1000mL) and kept in the cold overnight. After filtration, the clear solution was consecutively partitioned with petroleum ether, chloroform and ethyl acetate. Indigofera tinctoria flowers was reported on the identification isorhamnetin 3-O-α-L-(6''-E-p-coumaroyl)-rhamnoside in the methanolic crude extract was prepared by soaking a sample (50g) of powdered flowers material in 90% methanol (300mL) for 72 h. The extract was filtered using clean cloth and What man No. 1 filter paper. The filtrate was concentrated in vacuum at 30°C and stored in sterile sample containers at 4°C until further use. Double spot on TLC; Rf : 0.32,0.78

Spectroscopic methods:

Melting points were determined on a Fisher Scientific melting point apparatus and are uncorrected. The IR spectrum was measured on FT-IR spectrograph (Perkin Elmer Spectrophotometer, USA) with KBr tablets from 4000 to 400 cm^-1with resolution 2cm^-1. ¹H and NMR experiments have performed on a Bruker AMX 400 instrument (Bruker Company, Faelladen, Switzerland) standard pulse sequences running at 400 MHz for ¹H NMR and ¹³C NMR. Chemical shifts gave in δ (ppm) about TMS as internal standard material and the coupling constants (J) are in Hz. Column chromatography was performed on silica gel 60 as stationary phase (particle size 0.04 - 0.036mm, 230-400 mesh, ASTM E. Merck, Germany). The different solvent systems in volumetric ratios were employed (vol. ratios): CHCl₃/EtOAC/ MeOH (14:3:3). Flavonoids were visualized by UV light 363 nm, with NH3 vapors and by spraying with 1% AlCl₃ in MeOH. A sugar was detected by spraying with aniline phthalate solution in n-BuOH and heating at 105 ^oC. activated by heating at 110°C for one hour before to use. TLC was carried out on 0.25mm Brinkman percolated silica gel F254 plates (silica gel 60, 230-400 mesh, Merck, Germany). The different solvent systems were used for TLC analyses (Water: Chloroform: Methanol, 10:64:28v). spots were visualized by spraying with bromothymol blue in EtOH. A Shimadzu HPLC system (Columbia, MD), was used with UV detection at 280 - 350nm. A chromatographic system comprising a Spectra Physics P-200 series gradient pump (Fremont, CA, USA), a rheodyne injector fitted with a 20- FL loop, the C18 column (250× 4.6mm) (phenomenex, Torrance, CA, USA) was used.

Animals:

Male albino mice (30-40g) and male albino rats (100- 150g) of Wistar strain were procured from the animal house (5) 2014, Department of Zoology, Government Arts College (Autonomous), Bharathidasan University, Kumbakonam, Tamilnadu, India. Animals were fasted overnight and were divided into control, standard and different test groups each consisting of five groups (six animals each). They housed in cages and maintained under standard conditions at 26±2°C and relative humidity 44-56% and 10 h light and 14 h dark cycles each day for one week before and during the experiments. All animals were fed with the standard rodent pellet diet, and water adlibitum. Before starting the experiment on animals, the experimental protocol was subjected to the scrutiny of the Institutional Animal Ethics Committee (IAEC). (Approval No. BDU/IAEC/2011/31/29.03.2011).

Anti-inflammatory activity Carrageenan induced paw:

The anti-inflammatory activity of the test compounds were evaluated in Wistar rats employing the method. The different test concentration of isolated isorhamnetin 3-O-α-L-(6''-E-p-coumaroyl)-rhamnoside. Group I served as control, Group II standard diclofenac sodium 100 mg. Group III methanol extract 200 and 300 mg, Group IV petroleum ether extract 200 and 300 mg, Group V Chloroform extract 200 and 300 mg, methanolic extracts of Indigofera tinctoria were administrated to the animals in the test groups at the dose of 200 mg/kg by oral route(6). Animals in the standard group received Diclofenac sodium at dose of 100mg/kg, by oral route. Control group animals were received 1% DMSO at the dose of 10ml/kg body weight. The acute inflammation was induced by the sub-plantar administration of 0.1ml of 1% carrageenan in the right paw. Paw volume was measured by using digital plethysmometer (Ugo Basile-Italy) before administration of carrageenan and after 1, 2, and 3 hrs intervals (7). The efficacy of different drug was tested on its ability to inhibit paw edema as compared to control group.

Volume of edema = Final Paw Volume - Initial Paw Volume

The Percentage inhibition of paw edema was calculated by the formula as below.

% Inhibition of Paw edema = [(VC – VT) / VC] x 100

Where, VC = Paw edema of control group and VT = Paw edema of treated group

STATISTICAL ANALYSIS:

The experimental results were expressed as multiple comparisons of Mean ± SEM were carried out by one way analysis of variance (ANOVA) followed by Dunnet Multiple Comparisons Test and statistical significance was defined as P< 0.05

RESULTS AND DISCUSSION:

Chemical identification

Isorhamnetin-3-O-α-L-(6''-E-p-coumaroyl)-rhamnoside - Yellow amorphous powder; m.p. 200 - 202 ^oC;RT 26.3min;; IR νmax(KBr): 3272, 2912, 2840, 1692, 1641, 1622, 1517, 1237,1064 and 592 cm^-1 (8); ¹H NMR and ¹³C NMR (400 MHz,DMSO-d6, δ ppm, J, Hz) The UV spectrum of the flavones glycoside from the methanol fraction showed two absorption peaks at 260nm and 340nm. Chemical constituents- Spectral data of compounds. This compound also gave positive color reactions for a hydroxyl flavone with several reagents (9). 4'-trihydroxy-3'- methoxyflavonol. The ¹H NMR spectrum showed five aromatic protons signals at (δ 6.23, d, J = 2.1 Hz, H-6; δ 6.23, d, J = 2.1 Hz, H-8; δ 6.56, d, J = 2.1 Hz, H-2'; δ 7.80, d, J = 2.1 Hz, H-5'; δ 6.90, dd, J = 8.5 are typical of an AX system in B ring and their corresponding carbon signals appear at δ 98.5, 93.2, 114.6, 115.4, 121.7, respectively. Also methoxy protons signal was present at δ 3.80 ppm (3H, s) which showed with the carbon resonance at δ 145.1 (C-3'). These data clearly confirmed the characteristic pattern of isorhamnetin as aglycone (13). In addition, an anomeric α- rhamnose proton was recognized in this spectrum as a doublet at δ 5.48 ppm with respect to the question of α-or β-linkage of the sugar moieties, it has been found the coupling constant J = 1.61 Hz. corresponded to the anomeric proton of α-linked rhamnose (14). The methyl protons of the sugar rhamnose appear at δ 1.15 ppm which is therefore assigned to a 6-deoxy sugar (rhamnose) and rest of the sugar protons appear in the range δ 3.48 (10).

The ¹H-NMR spectrum of 4 also showed signals ascribed to sugar moieties and a p-coumaroyl residue (Table 1). The arrangements of the sugar units were assigned after hydrolysis of 4 compared to those of reliable sugar samples. The lower field shifts of H₂-6''' (δ 7.36, J = 8.2 Hz) of one glycosyl unit suggested the substitution site of the pcoumaroyl unit. Also the signals at 6.36 and 7.61 ppm (both d, JAB = 15.8 Hz) assigned to Trans olefinic protons suggested the presence of p-coumaric acid as the acyl moiety. In the ¹³C NMR spectrum, the signal at 167.6 ppm (s, C=O) supported this proposal. The ¹H NMR is suitable method to distinguish between both Z, E- isomerism of cinnamic acid. The E-cinnamoyl residue is detectable by a pair of doublets with shift values of 6.36 ppm for C₈-H and 7.61 ppm for C₇-H, the corresponding large coupling constant is about 16 Hz, which agrees well with findings of this study (11) The ¹³C NMR spectrum contained 31 carbon signals, 15 of them has assigned to the flavonol aglycone and one was methoxy carbon signal verified the isorhamnetin, and remaining 15 signals has attributed to sugar rhamnose with addition of p-coumaroyl unit. The sugar moiety has proved to be acylated at C- to achieve 3 of the aglycone as deduced from the anomeric proton at δH 5.48 and δC 131.1 ppm, which were in close agreement compound 4 as isorhamnetin-3-O-α-L-(6''-E-p-coumaroyl)- rhamnoside.

Table 1. ¹H and ¹³C NMR data of 3 and 4 (DMSO-d₆, 400 MHz)

Position	δ_C in ppm	δ_H [J (Hz)] in ppm
2	157.6
3	134.2.
4	177.5
5	160.8
6	98.5	6.29 (d, J=2.1 Hz)
7	164.4
8	93.2	6.56 (d, J=2.1 Hz)
9	156.7
10	103.8
1′	121.9
2′	114.6	7.80 (d, J=2.1 Hz)
3′	145.1
4′	148.4
5′	115.4	6.90 (d, J=8.5 Hz)
6′	121.7	7.62 (dd, J=2.1, 8.5 Hz)
1¢¢	100.0	5.48 (brd, J=1.58 Hz)
2¢¢	70.2	3.48 (dd, J=3.3, 1.58 Hz)
3¢¢	70.7	3.56 (dd, J=9.5, 3.3 Hz)
4¢¢	72.3	3.37 (d, J=9.5 Hz)
5¢¢	68.8	3.48 (dq, J=9.5, 6.2 Hz)
6¢¢	17.9	1.15 (s)
3-OH		9.38 (s)
5-OH		12.56 (s)
7-OH		10.78 (s)
4¢-OH		9.72 (s)
3¢-OMe	55.4	3.80 (s)
1′′′	126.9
2′′′	131.1	7.38 (d, J=8.2 Hz)
3′′′	116.7	6.80 (d, J=8.7 Hz)
4′′′	161.3
5′′′	116.7	6.80 (d, J=8.7 Hz)
6′′′	131.1	7.36 (d, J=8.2 Hz)
7′′′	146.9	7.61 (d, J=15.8 Hz)
8′′′	114.3	6.36 (d, J=15.8 Hz)
9′′′	167.6

Anti-inflammatory activity:

Inflammation is a response of living tissue to injuries that involve activation of various enzymes,
mediator release, cell migration, tissue breakdown and repair (12). Carrageen an-induced hind paw edema is a suitable experimental animal model of acute inflammation (13). Carrageenan induced paw edema takes place in three phases, in the first phase (1 h after carrageenan induce) involves the release of serotonin and histamine from mast cells, in a second phase (2 h) was provided by kinins and the third phase (3 h) was mediated by prostaglandins, the cyclooxygenase and lipoxygenase products (14) .As shown in the results (Table 2), restraint of paw edema (after 3 h) for test compound (200mg) with 2.82±0.28mL paw volume, respectively. It shows the methonlic extracts compounds had a significant anti-inflammatory effect, and the results were compared with standard diclofenac sodium 100 mg/kg and showed the paw volume reduction of 2.62±0.13 without statistical significance (p > 0.05) between methonlic extracts compounds and diclofenac sodium. Results were also reported in % restraint of edema (protection against inflammation) after three-hour treatment in comparison with the control group (Table 2). It showed a maximum percentage reduction (58.23%) in paw edema at 3 hours. methonlic extracts compounds at the dose of 200mg/kg body weight showed the percentage of inhibition of paw edema at 3 h 49.45%, respectively. All the test and standard groups have reduced the thickness of edema of the hind paw in different percentages compared to the control group .The maximum inhibition (3.11±0.24; p < 0.001) elicited by the chloroform extract was recorded at 3 hours. In the carrageenan isorhamnetin 3-O-α-L- (6''-E-p-coumaroyl)-rhamnoside showed significant inhibitory effect on the oedema formation. This effect started from the first hour and was maintained in all the inflammatory phases, suggesting that the main mechanism of action of the tested compound may involve prostaglandin biosynthesis pathway and may influence other mediators of inflammation.

Table 2. Anti - inflammatory activity of isorhamnetin 3-O-α-L- (6''-E-p-coumaroyl)-rhamnoside From Indigofera tinctoria

Groups

Initial paw volume

Paw volume at different time interval (in ml )

Control (1% DMSO)

2.28 ± 0.19

2.61 ± 0.04

2.90 ± 0.14

2.81 ±

0.15

Diclofenac Sodium

(100 mg/kg)

2.26±

0.24

2.52 ± 0.07

2.70 ± 0.11

2.62 ±

0.13

Methanol extract

(200 mg/kg)

(300 mg/kg)

2.27±

0.18

2.49±

0.32

2.92±

0.24

2.82±

0.28

2.29±

0.15

2.40 ± 0.12

2.59 ± 0.07

2.72 ±

0.8

Petroleum ether extract

(200 mg/kg)

(300 mg/kg)

2.25±

0.27

2.87±

0.26

3.08±

0.13

2.99±

0.11

2.26±

0.14

2.72 ± 0.02

2.98 ± 0.14

2.91 ±

0.12

Chloroform extract

(200 mg/kg)

(300 mg/kg)

2.27±

0.19

2.86±

0.28

3.31±

0.30

3.11±

0.24

2.28±

0.19

2.73 ± 0.03

3.13 ± 0.23

2.97±

0.05

All values are expressed in Mean ± SEM

Values are expressed in Mean ± Standard Deviation (n=6)

One-way ANOVA (Dunnetts method) Means for groups in homogeneous subsets are displayed. Subset for alpha = 0.05 level.

Phytochemical Screening:

The phytochemical screening of plant for the presence of glycosides, flavonoids, phenols, resin and tannins. This analysis revealed that the flower contained higher value of fat, protein, fiber and minerals (15).

Phytochemical Test	PE	CE	EE	ME	WE
Carbohydrates/glycosides	(-)	(-)	(-)	(+)	(+)
Alkaloid	(-)	(-)	(-)	(-)	(-)
Flavonoids	(-)	(-)	(-)	(+)	(+)
Saponins	(-)	(-)	(-)	(-)	(+)
Tannins	(-)	(-)	(-)	(+)	(-)
Phenolics compound	(-)	(-)	(-)	(+)	(+)
Protein and amino acid	(-)	(-)	(-)	(+)	(+)

PE= Pt. ether Extract; CE=Chloroform Extract ;

EE= Ethyl acetate Extract; ME= Methanolic Extract;

WE= Water Extract

CONCLUSIONS:

The present investigation, we confirm isorhamnetin 3-O-α-L- (6''-E-p-coumaroyl)-rhamnoside isolated from methanol crude extract of Indigofera tinctoria was better anti-inflammatory

Group:

Bioactive substances from this plant were employed to develop drugs for treating.

Inflammation:

This effect may be due to its flavonoids, quercetin, isorhamnetin and their

Glycosides composition.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Amrithpal Singh. Medicinal Plants of the World, Special indin edition, Oxford and IBH” Publishing Co.Pvt Ltd, , New delhi. 2006:168.

2. Nadkarni K M, Indian meteria medica, 3rd revised enlarged edition, Ramdas Bhatkat for Popular Prakashan Pvt. Ltd Mumbai. 2002; 680-681.

3. Asuntha G, Prasannaraju Y, Prasad KVSRG. Effect of ethanol extract of Indigofera tinctoria Linn. (Fabaceae) on lithium/pilocarpine-induced status epilepticus and oxidative stress in Wistar rats.Trop J Pharm Res, 2010; 9: 149–156.

4. Tyagi PK, Rai VK, Pahria AK, Kumar SS, Singh Y, Sharma M, Goval M. Preliminary phytochemical screening and evaluation of antiinflammatory activity of ethanolic extract of leaves of Indigofera tinctoria Linn. J Curr Pharm Res. 2010; 3: 47–50.

5. Sivagnanam Ilayaraja, Kalaivanan Prabakaran, Rajamanickam Manivannan Evaluation of Anti- Bacterial, Analgesic and Anti-Inflammatory activities of Oncocalyxone A isolated from Prenanthes sarmentosus Journal of Applied Pharmaceutical Science. 2014; 4 (10): 88-91.

6. Mossa, J.S., Rafatullah, S., Galal, A.M., Al-Yahya, M.A. Pharmacological Studies of Rhusretinoeehoea. International J Pharmacognosy. 1995; 33:242-246.

7. Vinegar R, Schriber W, Hugo R. Biphasic development of carrageenan edema in rats. Journal of Pharmacology and Experimental Therapeutics. 1969; 166: 96-103.

8. Harborne. JB(1988). The Flavonoids, Advances in Research
Sciences. 980. New York: Chapman and Hall.

9. Harborne JB, Mabry TJ, Mabry H (1975) The Flavonoids. New
York: Academic Press.

10. Markham K.R.; Geiger, H. ¹H nuclear magnetic resonance spectroscopy of flavonoids and their glycosides in hexadeutero dimethyl sulfoxide: Advances in Research since 1986, ed; J.B. Harborne. Chapman and Hall; London, 1994, pp 441-497.

11. R. Manivannan and R. Shopna Isolation of Quercetin and Isorhamnetin Derivatives and Evaluation of Anti-microbial and Anti-inflammatory Activities of Persicaria glabra Natural Product Sciences. 2015, 21(3) : 170-175 .

12. Mohini A. Phanse, Manohar J. Patil, Konde Abbulu, Pravin D. Chaudhari and Bhoomi In-vivo and in-vitro screening of medicinal plants for their anti-inflammatory activity: an overview Patel Journal of Applied Pharmaceutical Science. 2012; (07): 19-33.

13. Turner, R.A. Analgesic. In: Turner, R.A., Ed., Screening Methods in Pharmacology, Academic Press, London, 100 (1965)

14. Mahesh S, Patil MB and Kumar R: Evaluation of anti-inflammatory activity of ethanolic extract of Borassus flabellifer L. Male flowers in experimental animals, Journal of Medicinal Plants Research. 2009; 3(2): 49-54.

15. Subhash Chandra, Sarla Saklani, Abhay P. Mishra, Ganesh Rana. Nutriional antinutrional profile and phytochemical screening of flowers of Indigofera tinctoria from garhwal himalya. International Journal of Herbal Medicine. 2014; 1 (5): 23-27.

Received on 19.08.2019 Modified on 24.10.2019

Research J. Pharm. and Tech 2020; 13(5): 2187-2191.

DOI: 10.5958/0974-360X.2020.00393.5