LCMS/MS analysis and evaluation of anti-inflammatory and antioxidant activities of the polyphenol fraction of Litsea quinqueflora (Dennst.) Suresh

The main aim of the work was to scientifically prove the anti-inflammatory property of the polyphenol-rich fraction of hydro-alcoholic leaf extract of Litsea quinqueflora (Dennst.) Suresh by protein denaturation and free radical scavenging activity. The polyphenol-rich fraction of hydroalcoholic leaf extract was obtained via acid-alkali hydrolysis, followed by fractionation with chloroform and ethyl acetate. HPTLC profiling of the finally obtained ethyl acetate fraction and consequent derivatisation with aluminium chloride revealed the presence of flavonoids in a more purified form. LCMS/MS analysis tentatively identified the presence of bioactive polyphenolic compounds such as gallocatechin, sinapic acid, pinocembrin, paeonol and umbelliferone in the separated fraction. The polyphenol-rich fraction of hydro-alcoholic extract of leaves showed anti-denaturing activity in heatinduced bovine serum albumin denaturation with an IC50 value of 23.59 μg/ml and was statistically significant at 0.1% level. The antioxidant property of the polyphenol-rich fraction determined by its free radical scavenging ability against DPPH and ABTS showed IC50 values 122.98 and 135.44 μg/ml respectively and was also statistically significant at 0.1% level. Hence, the traditional use of Litsea quinqueflora as an anti-inflammatory agent can be attributed to the presence of polyphenols.


Introduction
Plant-derived medicines attracted the attention of researchers due to the presence of different phytochemicals having a multitude of health benefits to humans (1). Among these phytochemicals, polyphenols are of much importance owing to their specific biological properties. They are the organic foods produced by plants as secondary metabolites. Polyphenols are involved in the physiological and other diverse functions such as lignification, growth, predator resistance etc. (2). These inevitable plant products play an important role in human health as they regulate metabolism, weight, cell proliferation and chronic diseases (3). Biological properties such as anti-inflammatory, immunomodulatory, antioxidant, cardioprotective and anticancer activities can be attributed to polyphenols (4). The diverse structure of phenolic compounds influences the anti-inflammatory activity of a drug. Even though a variety of nonsteroidal anti-inflammatory drugs (NSAIDs) are available, natural phenolic compounds are also equally good and can inhibit the pro-inflammatory mediators such as interleukins, cyclooxygenase, lipoxygenase, nuclear factors etc. Traditional healers could identify the efficacy of plant-derived compounds, and they used them widely even before the advent of allopathy drugs (5).
The genus Litsea belongs to the family Lauraceae, which possess various pharmacological properties and have been used in traditional medicines to treat influenza, stomachaches, inflammatory diseases, bruises, insect bites etc. (6). Phytochemical analysis of different Litsea species identified flavonoids and terpenoids as their major constituents (7). Flavonoids obtained from different Litsea species such as Litsea cubeba Pers., Litsea glutinosa (Loureiro) Robinson and Litsea coreana H. Lev. act as anti-inflammatory and antioxidant compounds and inhibited different inflammatory pathways (8). Flavonoids like pinocembrin chalcone and kaempferol 3,4`-di-O-Lrhamnopyranoside were isolated from the leaves of Litsea fruticosa (Hemsl.) Gamble through column chromatography (9). Leaves of Litsea quinqueflora (Dennst.) Suresh was selected as the test material in this study as its leaf paste has been frequently used by the local healers of Kerala as an anti-inflammatory drug. The anti-inflammatory property of crude methanolic extract of L. quinqueflora leaves was reported using the HRBC membrane stabilisation method under induced hypotonic conditions and preliminary screening revealed the presence of different biologically active phytochemicals (10). Different leaf extracts of L. quinqueflora possessed flavonoids and phenols and exhibited antioxidant properties also (11). All these studies used crude extracts of leaves, but a hydro-alcoholic solvent system was reported suitable for the extraction of polyphenol compounds (12). Hence, the present investigation was aimed to isolate, characterise and evaluate the anti-inflammatory and antioxidant properties of polyphenolic fraction (PLE) of the hydro-alcoholic leaf extract of L. quinqueflora. A more purified fraction of the hydroethanolic leaf extract was obtained via acid-alkali hydrolysis and fractionation using chloroform and ethyl acetate. This purified extract was then subjected to HPTLC followed by LCMS/MS analysis to get a clear picture of its phytochemical background.

Collection of plant and processing
Leaves of Litsea quinqueflora (Dennst.) Suresh was obtained from the Kurianad area of Kottayam district, Kerala, India. The flowering and fruiting twig of the plant is presented in Fig 1. The plant specimen was identified and authenticated by Dr Sujanapal. P, Kerala Forest Research Institute (KFRI), Kerala, India. The voucher specimen was deposited in the National Herbarium of KFRI (Accession No. 13057). Leaves from female plants were separated from twigs and washed thoroughly in water, and kept for shade drying. It was then powdered and used for further studies.

Isolation of polyphenols
Extraction of polyphenol was done by the acid-alkali hydrolysis method (13), followed by fractionation with chloroform and ethyl acetate (14,15). Powdered plant material (10 gm) was added to 100 ml 80 % ethanol followed by agitation at 200 rpm for 5 hrs at room temperature in a shaking incubator (211DS, Labnet, NJ, USA). The filtrate thus obtained was concentrated at 40 ºC in a rotary evaporator (Hei-VAP Core, Heidolph, Germany). The pH changes were made by adding NaOH and HCl. The chloroform layer was separated and discarded. Ethyl acetate fraction of the acidified extract was collected and concentrated at 110 rpm. The concentrated fraction was blended at 120 rpm for 5 hrs with 50 ml NaOH. This alkaline fraction was acidified with HCl, and the separated ethyl acetate layer was concentrated to dryness (13).

HPTLC fingerprint profiling
High-Performance Thin Layer Chromatography (HPTLC) was performed with 5 ×10 cm aluminium plates pre-coated with silica gel F254 (Merck, Germany). The polyphenol sample thus isolated was dissolved in methanol, and 2 µl was spotted to the pre-activated plate using automatic TLC applicator Linomat-V with continuous N2 flow (CAMAG, Switzerland). The plate was placed in the twin glass chamber for development in the mobile phase consisting of toluene, ethyl acetate and methanol in the ratio 7:3:1. Visualisation of the oven-dried plate was done in CAMAG TLC visualiser under UV 254 nm, 366 nm and visible light. HPTLC profiles were obtained after derivatisation using AlCl3 (1 % ethanolic solution of aluminium chloride) (16,17). Densitometric scanning of these plates was carried out in CAMAG TLC scanner III using a deuterium lamp with Camag WIN CATS software.

Anti-inflammatory assay: Inhibition of protein denaturation
Inhibition of protein denaturation was done as per the method of Mizushima and Kobayashi (19). The reaction mixture (pH 6.3) contained 0.45 ml of 1 % aqueous solution of bovine serum albumin (BSA) with 0.05 ml of leaf extracts (62.5, 125, 250, 500 µg/ml). The reaction mixture was incubated at 37 °C for 20 min, and later the temperature was raised to 57 °C for 3 min. It was then allowed to cool at room temperature. Then added 2.5 ml of phosphatebuffered saline (PBS) of pH 6.3 and measured the optical density (OD) at 660 nm using UV-Vis spectrometer (Shimadzu -UV 1800) with PBS as blank. Diclofenac sodium was used as the standard anti-inflammatory drug. Test control was mixed with distilled water instead of extract, and in product control, distilled water was added instead of BSA.

DPPH radical scavenging assay
Evaluation of radical scavenging activity of PLE against DPPH was done with 0.1 mmol/L of DPPH based on the standard method (21). The reaction was done with 1 ml DPPH, and 0.5 ml of PLE of various concentrations (12.5, 25, 50 and 100 μg/ml) and test control was taken without sample. The reaction solution was finally adjusted to 3 ml by adding ethanol and incubated in the dark for 20 min. Ascorbic acid was used as the standard drug to compare with the test, and absorbance was measured at 517 nm.

Statistical analysis
The anti-inflammatory and antioxidant assays were performed in triplicates, and results were expressed in mean with standard deviation. IC50 values were calculated using Microsoft Excel 2013. The inhibitory percentages obtained with each concentration of each assay were statistically analysed through oneway analysis of variance (ANOVA) followed by post hoc Tukey test using IBM SPSS statistics 25 (23, 24).

Results and Discussion
The plant phenols exist mainly in conjugated forms, either as esters or as glycosides (25). Phenolic compounds, especially flavonoids, are mainly extracted through alcoholic solvents, and further pH changes of the extracts lead to the frequent hydrolysis of flavonoids and increased the separation of the maximum amount of phenolic constituents (25). Acid and alkaline hydrolysis of the extract leads to the separation or glycosylation of phenolic glycosides and resulted in the formation of aglycones. The easy absorption and enhanced solubility of phenolic aglycones than bound forms were evaluated and proved in earlier studies in Ginkgo biloba L. flavonoids in rat plasma (26). Methods of extraction of polyphenols included isolation of aglycones from different polyphenol compounds such as isoflavones, flavanones, anthocyanins etc. (27). Different hydrolytic methods like heating enabled to break down glycosides and separated aglycones from soybeans (28) and grapefruit (29).

Isolation of polyphenols and HPTLC fingerprint profiling
The hydroethanolic filtrate of L. quinqueflora leaves after acid-alkali hydrolysis and fractionation with chloroform and ethyl acetate yielded 0.04% polyphenol fraction of extract from leaf powder. The maximum separation of polyphenols was obtained with the mobile phase toluene, ethyl acetate and methanol in the ratio 7:3:1. The Rf values ranged from 0.01-0.92. The bands prominent at 254 nm were not so prominent at 366 nm. But no bands were obtained under visible light. There were four main peaks obtained under 366 nm. Densitometry estimation revealed that Rf value 0.09 was predominant with 42 % area followed by Rf values 0.17 and 0.33. HPTLC profiling with densitometric estimation and derivatisation is shown in Fig. 2 and peak values in Table 1 Table 2 and the spectrum of each compound is displayed in Fig. 3.
The LCMS analysis revealed the presence of different polyphenolic compounds, which were considered natural anti-inflammatory agents.     (54). Umbelliferone was isolated from the rhizome of Potentilla evestita L. and evaluated its anti-inflammatory and antinociceptive properties in animals (55). Hence, the tentatively identified polyphenolic compounds in the present study are efficient biomolecules with antioxidant, antiinflammatory, anti-apoptotic, antinociceptive, antimicrobial and antianxiety activities and can be considered the contributing factor for the traditional use of L. quinqueflora leaves.

Anti-inflammatory assay: Inhibition of protein denaturation
Polyphenol portion of leaf extract showed inhibition against protein denaturation, a well-documented process during inflammation. The isolated phenolic portion (PLE) showed an increase in inhibitory percentage with an increase in concentration and was statistically significant at 0.1% level (p<0.001) ( Table 3). It inhibited protein denaturation with an IC50 value of 23.59 µg/ml, almost near the standard drug diclofenac.
Denaturation of proteins can be induced due to different stimuli such as heat, acid, alkali, detergents, alcohol etc. (56). In the present investigation, heat was used as a stimulus. Denatured protein can act as an antigen, affect our system's immunity, and lead to chronic inflammatory conditions like rheumatoid arthritis (57) and amyloidosis (58). Recent studies in ethanolic fraction of fruits of Spondias mangiferra Willd. and aqueous extract of bark of Ficus benghalensis L. reported protein denaturation under in vitro conditions (59,60). Denaturation of proteins is as a serious cause of rheumatoid arthritis and other inflammatory conditions and their activity against protein denaturation considered them as anti-arthritic drugs. Hence, a drug with antidenaturant activity can be considered an antiinflammatory agent. Many plant extracts exhibit antidenaturant activity, where polyphenols, especially flavonoids, are mainly considered as antiinflammatory agents (61). Anti-inflammatory study of a polyphenol-rich extract of Petroselinum crispum (Mill.) Fuss, Apium graveolens L. and Coriandrum sativum L. showed 50 % inhibition of protein denaturation at 118, 227 and 247 µg/ml concentrations, respectively (62). Comparing those IC50 values with PLE depicted the better efficacy of polyphenolic fraction of leaf extract of L. quinqueflora as an anti-inflammatory agent. The inhibitory potential of PLE in protein denaturation assay was noticeably higher than that of sequentially isolated leaf extracts (63) and silver nanoparticles capped with methanolic extract (64) of L. quinqueflora. So, polyphenols can be considered as more potent phytoconstituent of L. quinqueflora. Phenolic compounds are demonstrated as thermal stability enhancers of proteins. Protein-phenol interaction enabled proteins to withstand heatinduced denaturation for a certain time (65). This corroborated the anti-denaturation activity of PLE, especially the polyphenol compounds present in PLE and thus the anti-inflammatory activity.

Antioxidant properties
Polyphenol portion of PLE exhibited effective radical scavenging activity against free radicals. The IC50 value in the DPPH assay was 122.98 µg/ml, and the ABTS assay was 135.44 µg/ml. The gradient of radical scavenging activity is proportional to the increase in concentration. The inhibitory percentages obtained with different concentrations of DPPH (Fig. 4) and ABTS (Fig. 5) were statistically significant at 0.1% level (p<0.001) (represented with asterisks).
The plant extracts with polyphenol contents exhibit good radical scavenging activities. The polyphenolic extracts of leaves of Ichnocarpus frutescens (L.) R.Br. inhibited free radicals at IC50 value of 163.38 µg/ml (66), and polyphenol-rich extract of fruits of Mallotus philippensis (Lam.) Müll.Arg. showed effective scavenging activity against ABTS and DPPH radicals (67). The radical scavenging activity of PLE was found to be higher than these earlier reports on other plants. Bark and leaf extracts of Litsea glutinosa (Lour.) Robinson, Litsea monopetala (Roxb.) Persoon, L. assamica Hook. f. and L. laeta (Nees) Hook. f. reported the antioxidant activity, especially the scavenging activity against DPPH free radical (68). Based on IC50 value, the antioxidant activity of the polyphenol fraction of L. quinqueflora was higher than that of these four species. When compared with the free radical scavenging activities of root extract of Litsea martabanica (Kurz) Hook.f. and stem bark extracts of Litsea petiolata Hook.f. (69,70), PLE exhibited less inhibitory activity against free radicals. Polyphenol compounds isolated from leaves of L. quinqueflora can be considered as a potential natural antioxidant source. The hydroxyl groups present in the phenolic constituents of plant extract assumed to be the active centre behind the radical scavenging activity.

Conclusion
Hydro-alcoholic extract of L. quinqueflora (Dennst.) Suresh leaves yielded polyphenols in the form of aglycones. They exhibited higher anti-inflammatory and antioxidant activities than that of crude extracts, as evidenced by previous publications. The compounds present in the polyphenol fraction were tentatively identified as gallocatechin, sinapic acid, pinocembrin, paeonol and umbelliferone and are known for their anti-inflammatory property.
Polyphenol extract of L. quinqueflora exhibited antiinflammatory and antioxidant properties in protein denaturation and free radical scavenging assays, respectively and the results were statistically significant. This provides strong support for its dosedependent inhibitory activity. Hence, the present investigation can be assumed as preliminary scientific evidence for the traditional use of Litsea quinqueflora leaves as an anti-inflammatory drug.