Identification of bioactive compounds by GCMS, quantification of total alkaloid, flavonoid, phenols and evaluation of anti-inflammatory activity in extracts of Firmiana colorata

Introduction

Herbal plants are used as sources of medicine by mankind since time immemorial. Around 80% of the world's population is reportedly dependent on traditional medicine today [1] . It has been hypothesized that the pharmacological properties of plants are due to the variety of phytochemicals present in them [2] . Phytochemicals are an overwhelming variety of organic compounds that vary remarkably in their qualitative and quantitative distribution along different parts of the plant, species and geographical area. They form an integrated part of the defence system of plants and therefore, most of these constituents are potent bioactive compounds [3] . Pharmaceutical industries are using bioactive compounds for the synthesis of useful drugs and as potent agents in the treatment of many diseases. Hence studies on pharmacological activities of these bioactive compounds have received wide attention in the scientific community. World Health Organisation has also emphasized the need to ensure the quality of medicinal plant products using modern controlled techniques and suitable standards [4] .

Consequently, the identification and quantification of bioactive constituents in plants is the need of the day. Furthermore, methods used in the identification of bioactive compounds should be simple, reliable and repeatable. The screening of medicinal plants by spectrophotometric and chromatographic techniques has proven extremely useful in providing basic information on the phytochemical makeup of the plant. And, Gas Chromatography-Mass Spectrometry (GC-MS) has become a firmly established, compatible, reliable and commonly used key technique for phytochemical profiling of plant species [5] . Gas chromatography-mass spectrometry (GC-MS) is one of the fastest and most accurate techniques to detect various compounds including alcohols, long-chain hydrocarbons, esters, amino acids alkaloids, nitro compounds, organic acids and steroids. The best advantage of the technique is that it requires small volumes of plant extracts and identifies compounds in a single step using a mass detector and GC-MS libraries [6] . The most important bioactive compounds of plants identified by GCMS, analysed and intensively studied are alkaloids, flavonoids, tannins, glycosides and phenolic compounds. These bioactive compounds possess numerous health effects such as analgesic, antiseptic, antimicrobial, antioxidant, antithrombotic, vasodilatory activities and many more. Studies have also inferred the beneficial facet of plant-based drugs as good sources of anti-inflammatory agents [7] . Plant-based drugs are preferred owing to the fact that nonsteroidal anti-inflammatory drugs (NSAID) used to treat and manage inflammation in contemporary medicine have not been therapeutically successful in all conditions. Moreover, adverse side effects associated with NSAIDs can lead to haemorrhage and gastric ulcers [8] . Therefore, screening indigenous plants for developing anti-inflammatory drugs is gaining momentum. Plant extracts used to reduce inflammation are characterized by either an overproduction of inflammatory mediators such as Reactive Oxygen Species (ROS), nitric oxide, interleukin-6 and proinflammatory cytokines or downregulation of proinflammatory cytokines, cyclooxygenases, reduced translocation of NF-kB to the nucleus and suppression of the nuclear factor-kappa B (NF-kB) signaling pathway. Plant extracts have also exhibited anti-inflammatory effects by interfering with lipopolysaccharide binding to Toll-like receptor 4 (TLR4), signaling and activating the antioxidative nuclear factor erythroid 2-related factor 2 (Nrt2) [9] . Oils have also been commonly used in traditional medicine to treat and manage inflammation and pain. They are used generally for topical application (often combined with massages). Essential oils are composed of low molecular weight volatile compounds which are mainly responsible for their distinct aroma and therapeutic activities. Essential oils can be extracted through hydro distillation from herbs, shrubs and leaves, flowers, fruits, roots, seeds and bark of trees [10] . Leaves of a deciduous tree, Firmiana colorata, were used in the current study to extract oil and identify the bioactive compounds present in it by GCMS. Firmiana colorata belongs to the Malvaceae family and genus Firmiana. Most of the Firmiana species are grown for their attractively shaped flowers [11] . Furthermore, Firmiana also finds a place in traditional medicine and several species of Firmiana were observed to possess antimicrobial, antiinflammatory, anticancer, neuroprotective and hepatoprotective properties. One of its species is Firmiana colorata, used in traditional medicine to treat digestive ailments, jaundice, general weakness, fever and intestinal dysfunction. Firmiana colorata is known to possess antimicrobial properties against several microorganisms. (12) Despite the ethnobotanical importance of F. colorata, adequate studies on its anti-inflammatory properties, qualitative and quantitative accounts of phytochemicals are not available. The present study aimed to analyse leaf, leaf oil, bark and flower extracts of F. colorata through gas chromatography-mass spectrometry (GC-MS) to elucidate their chemical composition and to determine their potential applications. An effort is also being made to investigate the In vitro anti-inflammatory activities in ethyl acetate and ethanol extracts of leaves, bark and flowers of F. colorata. The present study can provide substantial information about the bioactive compounds of F. colorata that may further be utilized in the pharmacological industries.

Materials and Methods Preparation of Plant extracts

Firmiana colorata leaves, bark and flowers were collected from the Indian Institute of Science, Bengaluru, India (geographical coordinates: 13° 1' 1" North, 77° 34' 1" East) and authenticated from Central Ayurveda Research Institute, Ministry of Ayush, Bengaluru, India. The voucher specimen number provided was SMPU/CARI/BNG/202021/1052. Collected samples were washed under running tap water and shade-dried for seven days at room temperature and ground into a coarse powder. Firmiana colorata leaf, bark and flower crude extracts (hereafter referred to as LE, BE, FE prefixed with EA-ethyl acetate or Eth-ethanol) were obtained by hot percolation extraction method using a Soxhlet apparatus. Sample powder weighed about 150 grams was packed in a Soxhlet apparatus and extracted with ethyl acetate as the first solvent followed by ethanol. A rotary evaporator was used to concentrate the filtrate under vacuum. A total of 6 extracts viz., EA-LE, EA-BE, EA-FE, Eth-LE, Eth-BE and Eth-FE were obtained using Soxhlet apparatus. All six extracts were used for the determination of TAC, TPC, TFC, AAPH-induced anti-haemolytic assay, lactate dehydrogenase assay and protein denaturation assay.

Determination of total phenolic content (TPC)

Folin-Ciocalteu (FC) method by spectrophotometry was employed to determine the total phenolic content in ethyl acetate and ethanol LE, BE and FE (1mg/ml) of F. colorata. l ml of each extract was taken in respectively labelled tubes followed by addition of 2ml of reaction mixture. FC reagent and 35% Na 2 CO 3 in a ratio of 1:1 was combined to make the reaction mixture. All tubes were incubated for 45 minutes at room temperature. The optical density of the test was measured against a reaction mixture blank at 760 nm spectrophotometrically. TPC in each extract was determined using a calibration curve of standard gallic acid solution generated. The results are provided as micrograms of gallic acid equivalent per milligram of extract [13] .

Determination of total flavonoid content (TFC)

The total flavonoid content in ethyl acetate and ethanol LE, BE and FE of F. colorata was determined by the aluminium chloride method by spectrophotometry. 1ml of LE, BE and FE (1mg/1 ml) were taken in three different tubes. 0.3 mL of 5% NaNO 2 was added to all the tubes, and incubated for 5 min at 25 °C followed by the addition of 10% methanolic solution of aluminium trichloride and 2 ml of 1N NaOH. After mixing thoroughly, the mixture was incubated for 15 minutes at room temperature. The absorbance of the test against the methanol blank was measured at 510 nm using a spectrophotometer. A calibration curve for standard rutin solution was generated and TFC was calculated in each extract. Results are represented as micrograms of rutin equivalent per milligram of extract [14] .

Determination of Total Alkaloid content (TAC)

Bromocresol green (BCG) method by spectrophotometry was used to determine the total alkaloid content in ethyl acetate and ethanol LE, BE and FE of F. colorata. 1ml of each extract (1 mg/ml) was taken in respective tubes followed by addition the of 5 ml BCG solution and 5 ml phosphate buffer (pH 4.7). The mixture was transferred to a separating funnel and thoroughly agitated. The resultant complex was extracted using chloroform and the optical density of the complex was measured at 470 nm. A calibration curve for standard caffeic acid generated was used to determine TAC in each extract and results are provided as micrograms of caffeic acid equivalent per milligram of extract [15] .

Extraction of essential oil from leaves of Firmiana colorata

Leaves of Firmiana colorata were washed, dried and ground to coarse powder. 200 grams of leaf powder was subjected to hydro distillation for 6 hours using a Clevenger apparatus with a graduated column to enable the measurement of oil yield directly. Anhydrous sodium sulphate was added to remove moisture content in it. The yield was calculated in % volume/gram of leaf powder. The oil samples were stored in air-tight containers and refrigerated at < 4 0 C until GC-MS analysis [16] .

Identification of volatile compounds by GC-MS Analysis

GC-MS analysis was performed to determine the bioactive compounds in leaf, leaf oil, bark and flower extracts of Firmiana colorata. The GC-MS system was equipped with a Perkin Elmer Elite-5MS column (30 m length × 0.25 mm internal diameter × 1 micron in thickness). The carrier gas helium was maintained at a flow rate of 2ml/min and the split ratio was 10:1. The initial temperature of the GC oven was operated at 80 0 C with 2-minute initial hold followed by a ramp:1 increase of 10 0 C/min up to 150 0 C with a hold time of 1.00 minute and ramp:2 increase of 15 0 C/min up to a final temperature of 250 0 C with a total hold time of 25.00 minutes for leaf oil, LE and BE. Whereas the total hold time for FE was 45 minutes. The samples were ionized with 70 eV GCMS radiation. The input temperature was 250 °C and the source temperature was 230 °C. The mass of the compounds was scanned from 29 to 800 amu. Identification of volatiles was performed by comparing their mass spectra with those of NIST/EPA/NIH Mass Spectral Library 2014 coupled with the GC-MS system.

Determination of Anti-inflammatory activity Preparation of Erythrocyte Suspension

Whole blood samples were collected from physiologically normal adult human subjects who had not taken NSAIDs for at least 7 days prior to the blood withdrawal. This was the prime criterion for the selection of human subjects. The blood samples were centrifuged at 3000 rpm for 10 minutes in heparinized centrifuge tubes to remove the plasma and separate packed red cells. It was washed thrice with normal saline. Blood volume was measured and reconstituted with 10 mM phosphate buffer solution (pH 7.4) to obtain 40% RBC suspension. (17) This was used as an erythrocyte stock suspension. 10% erythrocyte suspension was prepared from stock and further used for AAPH-induced anti-haemolytic assay and Lactate dehydrogenase assay.

a. Human red blood cell (HRBC) membrane stabilization assay

Haemolysis was induced in 10% erythrocyte suspension by adding an equal volume of 0.2 M AAPH [2,2ʹ-Azobis (2amidinopropane) dihydrochloride]. Different concentrations (0-100 µg/mL) of EA and Eth LE, BE and FE of F. colorata were immediately added to the reaction mixture taken in respective labelled tubes. Erythrocyte suspension served as a positive control, reaction mixture served as a negative control and 0.2% Triton (in PBS) with 100% haemolysis was taken as reference control. All tubes were incubated at 37 °C for 2 hours. 8 volumes of PBS were added to all tubes post-incubation for dilution and centrifuged at 2000 rpm for 10 min. The supernatant was collected and the degree of haemolysis was assessed by measuring the optical density at 540 nm. The percentage of haemolysis or membrane stabilization was calculated, and results were expressed as IC 50 value in comparison with L-ascorbic acid as standard [18] .

b. Lactate dehydrogenase (LDH) assay

The amount of LDH released by the damaged erythrocytes was calculated using the method described by Lenfant (2000) with slight modifications. 500 µl of 10% erythrocytes were incubated for 2 hours with an equal volume of 100 mM AAPH solution that contained EA and Eth -LE, BE and FE of F. colorata respectively. Whilst tubes without extracts served as a negative control and 40 µg/ml of L-Ascorbic acid served as a positive control. 200 µl of the reaction mixtures were diluted in 400 µl of PBS following the incubation period and centrifuged at 3000 rpm for 5 minutes. The amount of LDH released from 200 µl of supernatant was measured using an LDH estimation kit (Cayman Chemical -601170, India) with the results represented as percent LDH released [19] .

c. Protein denaturation assay

Effect of EA and Eth -LE, BE and FE of F. colorata extracts on inhibition of protein denaturation was determined as per the procedure described by Naz et al., with some modifications. Different concentrations of each of the extracts (0-800 µg/ml) were taken in separate tubes, labelled promptly and mixed well with 1% BSA (bovine serum albumin). The pH of the reaction mixture was adjusted using a small amount of 1N HCl. The reaction mixture was incubated for 10 minutes at 37 0 C and later placed in a water bath maintained at 70 0 C for 5 minutes. The tubes were cooled at room temperature and turbidity was measured spectrophotometrically at 660 nm. (20) Diclofenac sodium was used as a reference drug. The assay was performed in triplicate. The percentage of inhibition of protein was determined with respect to control using the following formula.

Statistical Analysis

TAC, TPC and TFC spectrophotometric determinations were replicated thrice and quantitative results are reported as mean±standard deviation. HRBC membrane stabilization, Lactate dehydrogenase, Protein denaturation assays were also done in triplicates and results are reported as mean±standard deviation. Microsoft Excel 2019 was used for all statistical and graphical evaluations of results.

Results and Discussion

Several biological activities have been reported on genus Firmiana, viz; antidiabetic, antimicrobial, antioxidant, anticancer, anti-inflammatory and others. (21,22,23) On the other hand, a review of current literature has traced less information dealing with the biological activities of the selected plant species Firmiana colorata. Limited data available on Firmiana colorata has revealed that the seed oil of Firmiana colorata contains Cyclopropenoid fatty acids including sterculic, malvalic, myristic, palmitic, stearic, oleic and linoleic acids. (24) Two new coumarin derivatives isolated from Firmiana colorata namely 4-(3,5dihydroxyphenyl)-3,5,7-trihydroxy-2H-chromen-2-one and 4-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-(((2R,3S,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy)-2H-chromen-2-one with significant immunomodulatory and anticancer activities have been reported [25] . The data reported by Soumita Bhattacharjee et al. [26] and Alia Yassin Ragheb Abd Al-Rahman [27] on a few bioactive compounds present leaf extract of F. colorata may differ from the current study data. In view of the fact that biosynthesis and composition of bioactive compounds varies across different species and even within the same species due to geographical distribution of the plant, environmental conditions, abiotic constraints such as water, stress level of pollution, local climatic and seasonal factors [28] . The present study appears to be the first ever to identify the bioactive compounds present in the leaf, leaf oil, bark and flower extracts of F. colorata through GCMS. Evaluation of anti-inflammatory activity possessed by LE, BE and FE involving HRBC membrane stabilization assay, LDH assay and Protein denaturation assay is also reported for first time. The results of the study have been tabulated. The concentration of TAC, TPC and TFC present in ethyl acetate and ethanol LE, BE and FE of F. colorata is given in Table 1 and presented in figures 1-3 which show that phenolic and flavonoid content was more in FE than BE and LE. Also, alkaloid content was higher in BE when compared to LE and FE. A similar study reports that leaf extract of F. colorata has high phenolic and flavonoid content [29] . While limited data is available on the alkaloid, flavonoid and phenolic content of F. colorata, there is sufficient data available for other species of Firmiana and their medicinal potency has been substantiated. In view of the same, alkaloids, flavonoids and phenolic content of F. colorata will presumably contribute to its anti-inflammatory activity. The results of GCMS analysis of leaf oil, LE, BE and FE of F. colorata are given in Tables 2-5. 49 compounds were identified via GCMS. Retention time (RT), peak area (PA), molecular formula, molecular weight, hit spectra and known bioactivities of each compound have been tabulated and chromatograms incorporated as figures 4-7. Details of 11 compounds present in LE of F. colorata are provided in Table 2. LE contains pharmacologically important bioactive compounds which include Neophytadiene, Stearic acid, 3, 7, 11, 15-tetramethyl-2hexadecen-1-ol, Palmitic acid, ethyl ester, Eicosanoic acid, ethyl ester and n-Hexadecanoic acid. Table 3 provides the details of 23 compounds identified in leaf oil of F. colorata. The molecular weight of the compounds ranged from 214 to 604 g/mol. 2-Piperidinone, N-[4-bromo-n-butyl],

Tetradecane,2,6,10-trimethyl, Nonadecane, Phytol and 2-Hexyl-1-octanol are a few of them. Presence of such compounds presumably contributed to the anti-inflammatory effects exhibited by leaf oil. Tetracontane, 3,5,24-trimethyl, Carbonic acid, eicosyl vinyl ester, Heptadecane,2,6-dimetyl, 1-Decanol, 2-octyl-, Heptadecane,2,6-dimetyl, Silane, trichlorodocosyl-have also been identified but bioactivity of these compounds has not been reported in the literature. There is scope for further research on these compounds to develop new drugs. 8 compounds viz., Pterine-6-carboxylic acid, Spiro [Androst-5-Ene-17,1'-Cyclobutan]-2'-One, 3-Hydroxy-, (3.β,17β)-, 9-Octadecenoic acid(Z)-, phenyl methyl ester, Panaxjapyne A, Nonadecane, β Carotene, 1-Iodo-2methylundecane were present in the bark extract of F. colorata (table 4). Majority of the compounds identified in BE have been reported to possess medicinal properties. FE showed the presence of Ribitol,1,3:2,4-di-Obenzylidene-, Santochlor, Undecane, Ribitol, 1,3:2,4-di-Obenzylidene-, Palmitic acid, methyl ester, 10-Octadecenoic acid, methyl ester and Lauric acid, methyl ester as mentioned in table 5. Most important among them is Palmitic acid, methyl ester which is known to inhibit neuroinflammation [30] . Among the 49 identified compounds, Neophytadiene, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, Stearic acid, Eicosanoic acid, ethyl ester, 2-Piperidinone, N-[4-bromo-nbutyl]-, Tetradecane,2,6,10-trimethyl, Spiro[Androst-5-Ene-17,1'-Cyclobutan]-2'-One, 3-hydroxy-, (3.β,17β)-, 9-Octadecenoic acid(Z)-,phenyl methyl ester, Phytol and Ribitol, 1,3:2,4-di-O-benzylidene-are known to possess potent anti-inflammatory activity (tables 2-5). These Phytochemicals may exhibit different mechanisms of antiinflammatory effects. They may act on the three main inflammatory signaling pathways viz., NF-κB, STATs and MAPKs which in turn play crucial roles in inducing, promoting and regulating inflammatory responses in the body [31] . Additionally, RBC membrane is similar to the lysosomal membrane, inhibition of RBC haemolysis may provide insights into the inflammatory process. Stabilization of these cell membranes may retard the lysis and subsequent release of the cytoplasmic contents which minimizes the tissue damage and hence the inflammatory response [32] . Therefore, substances that contribute significant protection to cell membranes against injurious substances are important in the event of inhibiting inflammation. Further, it is possible that plant extracts may affect the ratio of surface area/volume of the cells by expansion of membrane or the shrinkage of cells and interaction with membrane proteins [33] . Therapeutic strategies targeting these inflammatory signaling pathways may be used to treat inflammatory diseases and various phytochemicals modulating different inflammatory cascades can be exploited for adjunctive therapy. The bioactivity of several identified compounds has not been reported in the literature. It is presumed that these compounds have medicinal potency on the grounds of the antimicrobial, anti-haemolytic, anti-inflammatory and antioxidant properties exhibited by Firmiana colorata. Hence, isolation and in-depth study of these compounds is necessary to identify their biological activities to enable the discovery of a new class of anti-inflammatory agents and other novel drugs. The results of HRBC membrane stabilization, LDH and Protein denaturation assays are given in Tables 6 and7 and figures are also provided (figures 8.8 -8.9). The antihaemolytic activity was found to be high and the release of LDH was least in ethyl acetate and ethanol flower extracts. LE showed the least anti-haemolytic activity and ethyl acetate bark extract had moderate anti-inflammatory activity. Inhibition of protein denaturation was also exhibited more by flower extract (figure 8.10). Overall, antiinflammatory activity was more in flower extracts of F. colorata and it may be attributed to Ribitol,1,3:2,4-di-Obenzylidene-. It could also be due to Cyclopentane tridecanoic acid, methyl ester, which lacks data on its bioactivity. Results of the present study clearly demonstrate that extracts of Firmiana colorata have potent anti-inflammatory activities owing to the considerable quality and quantity of phytochemicals present in them. Also, GC-MS analysis is the stepping stone toward understanding the nature of bioactive compounds present in F. colorata. Further detailed studies on these compounds may throw light on the mechanism of actions at the molecular level and substantiate the results of the bioactivity profile of F. colorata. Anti-inflammatory, antithermogenic (40) 11 18.77 10.7 n-Hexadecenoic acid C16H32O2 256 saturated fatty acid Antioxidant hypocholesterolemic nematicide, antiandrogenic, hemolytic inhibitor, pesticide, lubricant, 5-Alpha reductase inhibitor, antipsychotic. (41) Anti-HIV Antioxidant, antibacterial, antifungal, antimalarial (53) 8 20.90 9.2 1-Iodo-2-methylundecane C12H25I 296 Iodoalkane Antimicrobial, enhance reproductive activities (54) Antibacterial, antifungal, antioxidant, decrease blood cholesterol [57]

Conclusion

Firmiana colorata may be recommended as a plant of phytopharmaceutical importance as it contains several and a variety of bioactive compounds. The results of the current study can accelerate the development of a new class of plant-based anti-inflammatory agents to treat several inflammatory diseases.

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