1. INTRODUCTION:

Lilium polyphyllum is a perennial, bulbous herb of Liliaceae and is found in North-west Himalaya in India to the westward of Afghanistan. The species of Liliaceae in Himalaya region exists in two extreme climatic conditions temperate and high altitude. The morphology of species in both habitats is more or less similar although they may be having variability at genetic level. Bulbs of the species have large number of medicinal properties like being used as refrigerant, galactagogue, expectorant, aphrodisiac, diuretic, antipyretic and tonic¹ also they can be used for culinary purposes².

There are nearly 100 species of the genus Lilium L. (Liliaceae) which are distributed throughout the cold and temperate regions of Northern Hemisphere³.

Most of the Lilium species mostly consist of fragrant, bulbous and perennial herbs, thus form an important group of flowering plant species in gardens.

The Lilium is used as an ornamental plant and this popularity is mainly due to the large size, showy flowers which bear strong fragrance⁴.

Inflammation is the response against noxious stimuli and conditions like infection and tissue injury^5,6. It has been extensively demonstrated that a strong interconnections occur between oxidative stress and the inflammatory response⁷. Endo-cellular alterations play a key role in the activation and/or dysfunction of immune cells. In line to these, the plant kingdom contains an immense number of secondary metabolites, in the form of phytochemicals, with significant redox-modulating properties which show effectiveness to modulate the inflammatory response⁸. First of all the ‘health regeneration’ by phytochemicals as free radical trappers and/or direct antioxidant effects on tissues⁹ such compounds are believed to intervene in cell functions by trapping and modifying the reactive species at the level of critical cell signaling pathways. Also, the interaction of these phytochemicals with the cellular molecules like enzymes, receptors, and transcription factors is a topic of emergence^10,11.

2. MATERIALS AND METHODS:

Plant materials:

All the chemicals used in this investigation were of analytical reagent (AR) grade and were purchased from Sigma Merck. De-ionized water was used in the whole study. All the glassware and equipment used for handling were stabilized properly prior to use.

The plant material was collected from Ladakh region of Jammu and Kashmir and was authenticated at FRI Dehradun. The collection process was preferably done in the dry condition. Plant was shade dried at room temperature and the plant samples were air dried and grounded into uniform powder with a grinder.

Extraction:

The extraction procedure was carried out first with DCM followed by ethanol and then with water based upon their polarity index. The extraction was done by Soxhlet extraction using thimble to get the pure form of extract. 75g of the plant material was used for extraction.

Anti-inflammatory Activity of Lilium polyphyllum plant extracts:

Inflammation is a complex physiological process mediated by a variety of signaling molecules produced by leukocytes, macrophages, and mast cells. Inflammation is a tissue response to injury characterized by increased blood flow to the tissue causing increased temperature, redness, swelling, and pain. Macrophages play an important role in inflammatory disease through the release of inflammatory mediators such as nitric oxide (NO), prostaglandin (PG) E2, and pro-inflammatory cytokines.

Preparation of Human Red Blood Cells (HRBC) Suspension:

Fresh whole human blood was collected and mixed with equal volume of sterilized Alsever solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid and 0.42 % sodium chloride in water). The blood was centrifuged at 3000rpm for 10 min and packed cells were washed three times with isosaline (0.85%, pH 7.2). The volume of the blood was measured and reconstituted as 10% v/v suspension with isosaline^12,13.

Heat Induced Hemolysis:

The principle involved here is stabilization of human red blood cell membrane by hypotonicity induced membrane lysis. The assay mixture contains 1ml phosphate buffer [pH 7.4, 0.15 M], 2ml hypo saline [0.36%], 0.5ml HRBC suspension [10% v/v] with various concentrations of plant extracts and standard drug Diclofenac sodium of various concentrations (50, 100, 200, 300, 400, 500μg) and control (distilled water instead of hyposaline to produce 100 % hemolysis) were incubated at 37⁰C for 30 min and centrifuged respectively. The hemoglobin content in the suspension was estimated using spectrophotometer at 560nm¹⁴.

The percentage of hemolysis of HRBC membrane can be calculated as follows:

% Hemolysis = (Absorbance of Test Sample / Absorbance of Control) x 100

The percentage of HRBC membrane stabilization can be calculated as follows:

% Protection = 100 – [(Absorbance by Test sample / Absorbance by Control) x 100]

3. OBSERVATIONS AND RESULTS:

The anti-inflammatory properties of various extracts of Lilium polyphyllum have been carried out, in which the lysis of cell was done by hypotonicity. The percentage hemolysis and correspondingly the percentage stabilization against the membrane hemolysis were carried out. The percentage stabilization was found to increase against the concentration increase.

The anti-inflammatory properties of extracts have been carried out and it was found that the reference compound (Diclofenac sodium) have been found to possess the highest % stabilization as shown in all tables and figures. Among the plant extracts it has been found that water extract of the plant possess the highest percentage stabilization followed by ethanol extract. The DCM extracts possess the least stabilization as compared to ethanol and water extracts as shown in the Tables (1, 2 and 3). The anti-inflammatory effect was also found concentration dependent, as with the increase in the concentration of the extracts the anti-inflammatory effects also increases as the stabilization potential increases.

Table-1: Report of anti-inflammatory effect of DCM extract of Lilium polyphyllum and Standard on HRBC membrane hemolysis and membrane stabilization

Conc. of Standard/Plant Extract (μg/ml)	% Hemolysis	% Stabilization	% Hemolysis of Diclofenac sodium	% Stabilization of Diclofenac sodium
0.5	43.16	56.84	46.37	53.63
1.0	39.11	60.89	32.69	67.31
1.5	35.21	64.79	23.43	76.57
2.0	30.55	69.45	9.53	90.47
2.5	28.15	71.85	5.12	94.88

Table-2: Report of anti-inflammatory effect of Ethanolic extract of Lilium polyphyllum and Standard on HRBC membrane hemolysis and membrane stabilization

Conc. of Standard/ Plant Extract (μg/ml)	% Hemolysis	% Stabilization	% Hemolysis of Diclofenac sodium	% Stabilization of Diclofenac sodium
0.5	40.39	59.61	46.37	53.63
1.0	37.19	62.81	32.69	67.31
1.5	33.31	66.69	19.43	80.57
2.0	28.18	71.82	9.53	90.47
2.5	23.27	76.73	5.12	94.88

Table-3: Report of anti-inflammatory effect of Water extract of leaf of Lilium polyphyllum and Standard on HRBC membrane hemolysis and membrane stabilization

Conc. of Standard/ Plant Extract (μg/ml)	% Hemolysis	% Stabilization	% Hemolysis of Diclofenac sodium	% Stabilization of Diclofenac sodium
0.5	35.14	64.86	46.37	53.63
1.0	27.11	72.89	32.69	67.31
1.5	20.32	79.68	19.43	80.57
2.0	14.92	85.08	9.53	90.47
2.5	11.19	88.81	5.12	94.88

The ethanol extract of the plant have been analysed by GC-MS analysis, C₁₃ and H¹NMR Spectroscopy. The various compounds have been identified as Methyl 2-furoate, 5-hydroxymethyl furfural, Methyl piperate, Piperine, 7, 10-Hexadecadienoic acid methyl ester, Palmitic acid, Methyl palmitate. The structure and the respective spectra of the identified compounds are shown below.

Compound Name: Methyl 2-furoate

Fig1: ¹H NMR of Methyl 2-furoate

Fig 2: ¹³C NMR of Methyl 2-furoate

Methyl 2-furoate: White crystals; ¹H NMR (CD₃OD, 400 MHz): δ_H 7.80 (1H, d, J= 1.2 Hz, H-5), 7.40 (1H, d, J= 3.6 Hz, H-3), 6.65 (1H, dd, J= 3.6 Hz and 1.6 Hz, H-4), 4.70 (3H, s, H-7); ¹³C NMR (CD₃OD, 100 MHz): δ_C 189.7 (C-6), 152.1 (C-2), 148.8 (C-5), 119.2 (C-3), 113.5 (C-4), 66.0 (C-6); GC-MS m/z 126.11 (cal. for C₆H₆O₃).

Compound Name: 5-hydroxymethyl furfural

Fig 3: ¹H NMR of 5-hydroxymethyl furfural

Fig 4: ¹³C NMR of 5-hydroxymethyl furfural

5-hydroxymethyl furfural: Yellowish oil; ¹H NMR (CDCl₃, 400 MHz): δ 9.51 (1H, s, H-7), 7.20 (1H, d, J= 3.6 Hz, H-3), 6.48 (1H, d, J= 3.6 Hz, H-4), 4.64 (2H, s, H-6); ¹³C NMR (CDCl₃, 100 MHz): δ 177.7 (C-7), 161.1 (C-5), 152.1 (C-2), 123.2 (C-3), 109.9 (C-4), 57.2 (C-6) HR-MS m/z 127.0392 [M+H]⁺ (cal. for C₆H₆O₃, m/z 126.0319).

Compound Name: Methyl piperate

Fig 5: ¹H NMR of Methyl piperate

Fig 6: ¹³C NMR of Methyl piperate

Methyl piperate: White powder; ¹H NMR (CDCl₃, 400 MHz): δ_H 7.42 (1H, dd, J=10.8, 15.2 Hz, =CH-, H-3), 7.00 (1H, d, J= 1.6 Hz, aromatic H, H-7), 6.92 (1H, dd, J= 1.6, 8.0 Hz, aromatic H, H-11), 6.80 (2H ), 6.70 (1H, dd, J=10.8, 15.2 Hz, =CH-, H-4), 5.99 (2H, s, -O-CH₂-O, H-12) 5.95 (1H, d, J= 15.2 Hz, =CH-, H-2), 3.77 (3H, s, OCH₃); ¹³C NMR (CDCl₃, 100 MHz): δ_C 167.5 (C-1), 148.6 (C-9), 148.3 (C-8), 144.9 (C-3), 140.2 (C-5), 130.6 (C-6), 124.5 (C-4), 122.9 (C-11), 120.0 (C-2), 108.5 (C-10), 105.9 (C-7), 101.4 (C-12), 51.4 (OCH₃); ESI-MS m/z 233.10 [M+H]⁺ (cal. for C₁₃H₁₂O₄,m/z 232.23).

Compound Name: Piperine

Fig 7: ¹H NMR of Piperine

Fig 8: ¹³C NMR of Piperine

Piperine: ¹H NMR (400 MHz, CDCl₃) δ: 1.60 (4 H, m, H-2′′, 4′′),1.67 (2 H, td, J = 7.5, 3.6 Hz, H-3′′), 3.59 (4 H, dt, J = 5.3 Hz, H-1′′, 5′′), 5.97 (2 H, s, -OCH₂O-), 6.43 (1 H, d, J = 14.6 Hz, H-2), 6.74 (1 H, m, H-4), 6.75 (1 H, m, H-5), 6.79 (1 H, d, J = 8.0 Hz, H-5′), 6.89 (1 H, dd, J = 1.7, 8.0 Hz, H-6′), 6.98 (1 H, d, J = 1.7 Hz, H-2′), 7.41 (1 H, ddd, J = 14.6, 7.7, 2.5 Hz, H-3); ¹³CNMR (100MHz, CDCl₃) δ: 24.5 (C-3′′), 25.5 (C-2′′), 26.5 (C-4′′), 43.1 (C-1′′), 46.8 (C-5′′), 101.1 (-OCH₂O-), 105.5 (C-2′), 108.3 (C-5′), 119.9 (C-2), 122.3 (C-6′), 125.2 (C-4), 130.8 (C-1′), 138.0 (C-5), 142.3 (C-3), 147.9 (C-4′), 148.0 (C-3′), 165.2 (C-1). ESI-MS m/z 286.20 [M+H]⁺ (cal. for C₁₇H₁₉NO₃,m/z 285.34).

Compound Name:

7, 10-Hexadecadienoic acid methyl ester

Fig 9: H NMR of 7, 10-Hexadecadienoic acid methyl ester

Fig 10: ¹³C NMR of 7, 10-Hexadecadienoic acid methyl ester

7,10-Hexadecadienoic acid methyl ester:¹H NMR (CDCl₃, 400 MHz) δ ppm: 5.33 (4H, m, H-8, H-9, H-11, H-12) 3.65 (3H, s, OCH₃) 2.30 (2H, t, J=7.6 Hz, H-2), 2.03 (2H, m), 1.60 (2H, m, H-3), 1.25 (18H, s), 0.88 (3H, t, J = 6.8 Hz, H-17); ¹³C NMR (CDCl₃, 100 MHz) δ ppm: 174.2 (C-2), 130.2 (C-8), 130.0 (C-9), 128.0 (C-11), 127.9 (C-12), 51.3 (OCH₃), 34.1 (C-3), 31.9 (C-4), 31.5 (C-16), 29.7-29.0 (C-5-7, C-13), 27.2 (C-10), 25.6 (C-14), 24.9 (C-15), 22.6 (C-16), 14.0 (C-17); ESI-MS m/z 265.20 [M-H]^-(cal. for C₁₇H₃₀O₂,m/z 266.42).

Compound Name: Palmitic acid

Fig 11: ¹H NMR of Palmitic acid

Fig 12: ¹³C NMR of Palmitic acid

Palmitic acid: ¹H NMR (CDCl₃, 400 MHz) δ: 2.34 (2H, t, J=8.0 Hz, CH₂), 1.63 (2H, t, J=8.0 Hz, CH₂), 1.26 (s, -(CH2)n-), 0.88 (3H, t, J= 8.0 Hz, CH₃). ¹³C NMR (CDCl₃, 100 MHz) δ: 14.0 (CH₃), 22.6 (CH₂), 24.7 (CH₂), 29.0 (CH₂), 29.2 (CH₂), 29.3 (CH₂), 29.4 (CH₂), 29.6 (CH₂), 29.64 (CH₂), 29.66 (CH₂), 29.68 (CH₂), 31.93 (CH₂), 34.07 (CH₂), 180.04 (C-1).

Compound Name: Methyl palmitate

Fig 13: ¹H NMR of Methyl palmitate

Fig 14: ¹³C NMR of Methyl palmitate

Methyl Palmitate: ¹H NMR (CDCl₃, 400 MHz) δ: 3.59 (3H, s, OCH₃), 2.23 (2H, t, J=8.0 Hz, CH₂), 1.55 (2H, t, J=8.0 Hz, CH₂), 1.19 (s, -(CH2)n-), 0.82 (3H, t, J= 8.0 Hz, CH₃). ¹³C NMR (CDCl₃, 100 MHz) δ: 14.01 (CH₃), 22.63 (CH₂), 24.93 (CH₂), 29.13 (CH₂), 29.22 (CH₂), 29.31 (CH₂), 29.41 (CH₂), 29.55 (CH₂), 29.66 (CH₂), 31.89 (CH₂), 34.07 (CH₂), 51.28 (OCH₃), 174.21 (C).

4. DISCUSSION:

An anti- inflammation refers to a reaction in which a particular substance in the form of either medicine or any other chemical or biochemical substance which may inhibit the process of inflammation or swelling. It had been found that nearly half of the analgesics are anti-inflammatory, which reduces pain by reducing inflammation of an organ or organism.

By taking into consideration the anti-inflammatory property, Lilium polyphyllum plant a well-known herb used as folk medicine in many parts of the world against various ailments and for the benefit of the human race is also used as a nontoxic herb in traditional medicine.

Various extracts of Lilium polyphyllum plant were analysed for their anti-inflammatory property and it had been found that the extracts possess a high level of effect against inflammation (Table 1,2,3). The medicinal properties associated with the plants are because of their inherent biochemical molecules, or secondary phytochemicals like Flavonoids, alkaloids etc. in which the most enhancing potential against the inflammation was noticed by heme-oxygenase. So the plants possessing heme oxygenase could be used as good anti-inflammatory medicines. Lilium polyphyllum plant extracts were analysed for the same property, in reference to standard Diclofenac sodium (anti-inflammatory drug). As first the cells were inflammated by hypotonicity induced membrane lysis, then stabilization against membrane induced hypotonicity was done and the percentage of stabilization was measured, and it had been that stabilization was found concentration dependent, and the stabilization percentage of crude drug Diclofenac sodium was found highest than the concerned plant extracts. Among the plant extracts the highest stabilization was found in case of water extract of (Tab 3) of plant, followed by ethanol extract of plant (Tab 2). Among the all plant extracts it has been found that the polar solvent extracts posses’ highest anti-inflammatory activity than the less polar solvent extracts. The results get agreed with the study, as most of the amino acids are anti-inflammatory in nature so are the polar plant extracts. These compounds have direct influence upon the inflammation and the plant extracts fulfill the job in highly influenced manner. As per the spectral studies various compounds have been identified in the ethanolic extract of the plant which include, Methyl 2-furoate, 5-hydroxymethyl furfural, Methyl piperate, Piperine, 7, 10-Hexadecadienoic acid methyl ester, Palmitic acid, Methyl palmitate mentioned above (Figures 1-14). It had been found that Palmitic acid acts a good anti-inflammatory agent as mentioned by [15], also the Palmitic acid [16], Methyl palmitate [17] are known medicines against inflammation as mentioned. So the anti-inflammatory property of the concerned plant could be because of the presence of these identified compounds in which Piperine, Palmitic acid Methyl palmitate etc are among few.

Thus Lilium polyphyllum plant extracts can be used as best anti-inflammatory medicines in addition to its already recognized properties.

5. CONCLUSION:

Any medicinal property which is associated with a particular plant is because of the presence of various biochemical species, similarly the plant Lilium polyphyllum does have anti-inflammatory property. As per the study in reference it could be concluded that the plant possess a good response against inflammation. The plant is enriched with many vital phytochemicals as identified by various spectral studies which can act as good anti-inflammatory medicines. So overall it can be concluded that nature had provided healing to every disease by means of other natural products.

6. REFERENCES:

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Received on 13.07.2020 Modified on 08.09.2020

Research J. Pharm. and Tech. 2021; 14(6):3195-3201.

DOI: 10.52711/0974-360X.2021.00557