Isolation of a bronchodilator molecule from Ficus trichopoda (Baker, 1883) (MORACEAE) leaves

The present work was undertaken to isolate a bronchodilator compound from Ficus trichopoda leaves used in traditional medicine to manage asthma symptoms in Madagascar. Isolated guinea pig trachea was used as experimental model. Bioassays fractioning followed by purification on preparative chromatography plate were used to isolate the active molecule. Bioassays reveal that the ethyl acetate fraction induced relaxation of histamine-contracted isolated trachea with an EC₅₀ of 61.31 µg/mL. Elution with dichloromethane-hexane-methanol (90:9:1 v/v/v) of this fraction gives 7 spots at Rf 0.24, 0.28, 0.37, 0.50, 0.61, 0.69, and 0.78. The molecule at Rf 0.50 relaxes isolated trachea precontracted with histamine (10 µM), with an EC₅₀ of 1.581 10-4 M. Chemical shifts (δH) and spin-spin coupling constants (JHH) data were compared with literature to identify the isolate as 2-(2,3-dihydroxyphenyl)- 4-ethylchroman-3,5,6-triol (MR23), belonging to the flavonoid family.

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Introduction

Asthma The genus Ficus is popular in the traditional medicine of many Asian and Middle Eastern countries. Triterpenes, flavonoids, polyphenols, alkaloids, sterols, coumarins, and other secondary metabolites present in the genus Ficus are responsible for its various pharmacological activities, like anti-diabetic, anti-microbial, antioxidant, anti-inflammatory and analgesic [1,2] . Compared to other species of the genus Ficus that have been the subject of broader reviews of their general pharmacological properties, studies on Ficus trichopoda remain limited. The few pharmacological studies in vitro and in vivo conducted on this species indicate that extracts from different parts of the plant, notably the leaves and bark, have shown notable efficacy against certain pathologies. As with other species of the same genus, F. trichopoda also possesses antioxidant, anti-inflammatory, and analgesic properties. Ajay A. M. et al. (2011) reported the anti-inflammatory and analgesic activities of the hydroalcoholic extract of F. trichopoda bark [3] . The bark extract also shows anti-bacterial activity [4] . Medicinal plants constitute valuable and distinctive sources of new therapeutic agents and are widely used in the treatment of various diseases. Many currently used bronchodilator drugs are of natural origin and mainly fall into three categories: anticholinergic agents such as tropane alkaloids and their semisynthetic derivatives, β₂-adrenergic receptor agonists, and xanthine derivatives, particularly theophylline [5,6] . In the search for plant-derived bronchodilator secondary metabolites from Malagasy flora, we have conducted an ethnobotanical survey in the High Plateau of Madagascar. This investigation revealed the traditional use of Ficus trichopoda leaves for the management of asthma symptoms. Furthermore, our previous studies demonstrated that the hydroalcoholic extract of F. trichopoda leaves exhibits a significant bronchodilatory effect against histamine-induced bronchoconstriction [7] . Therefore, the present study focuses on a bioactivity-guided phytochemical investigation aimed at the isolation and identification of bronchodilator secondary metabolites from the leaves of Ficus trichopoda.

Materials and Methods

Materials and Methods

Chemicals

All chemicals such as hexane, chloroform, ethanol, methanol and ethyl acetate (analytical grade) were purchased from LABOSI, 141 Rue de JAVEL, Paris 75. Deuterated methanol (MeOD), deuterated chloroform (CDCl3) and ingredients for making Kreb's Henseleit solution were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). Standard 5 mm, 7′′ NMR tubes (XR-55 series) were from Norell Inc. (Landisville, NJ, USA). Chromatographic preparative plates (Ref 4856-840) were purchased from Merck (39 Rte Industrielle de la Hardt, 67120 Molsheim, France).

Plant Materials

The leaves of Ficus trichopoda used in this work were collected from the high plateau region of Madagascar (Ambendrana in Amoron'i Mania Region and Sadabe in Analamanga Region). The authentication of the plant material was done by the taxonomist of the Botany Department of Botanical and Zoological Parc of Tsimbazaza (PBZT) Antananarivo, Madagascar. Sample preparation.

Animal Experimentations

Guinea pigs, of either sex, weighing between 250 and 300 g were used in this work. They were kept at the animal house of LPGPC, in standard conditions: 12/12 hours cycle of light and darkness and room temperature of 22 °C. Animals were supplied with tap water Ad libitum and commercial standard diet. Care and use of the animals were approved by the bioethics committee of the Sciences Faculty, University of Antananarivo, Antananarivo, Madagascar, registered as CE/Fac Sciences/04/2025.

Bioassays Fractioning

The plant material was dried under shade, at room temperature. The dried material was pulverized, and the powder (1180 g) was successively extracted by percolation in Soxhlet with hexane, dichloromethane, ethyl acetate and methanol till exhaustion. The different solvents were evaporated to dryness under reduced pressure using rotary vacuum evaporator. Each fraction obtained was then tested on isolated trachea precontracted with histamine to assess their bronchodilating activity [8] .

Isolation of a Bronchodilator Compound

The active compound was isolated from ethyl acetate fraction, using precoated preparative silica gel (GF254) glass base plates (Merck, 107747) of 1 mm thickness. Ethyl acetate fraction was deposited on preparative chromatographic plates and developed with dichloromethane-hexane-methanol (90:9:1 v/v/v) solvent system [9,10] . The plates were dried and separated compounds were detected under UV lamp (254 nm), retention factor (Rf) was calculated, and the compounds of each band were scraped off. The purity of the molecules was assured using 2-dimension-TLC on aluminium base Precoated Silica gel GF254 (Merck), developed with dichloromethane-methanol (92:8 and 98:2) (v/v) solvents. The different isolated molecules were tested on isolated trachea precontracted with histamine 10 µM to determine an active compound. The active compound dissolved in ethyl acetate was allowed to crystallize at room temperature and kept in hermetic container in dry environment.

Structural Determination

The structure of the isolated compound was elucidated using NMR spectral data (UV, 1D and 2D Nuclear Magnetic Resonance (NMR) using Bruker FT-NMR Avance-500 spectrometer (Ettlingen, Germany), and by mass spectroscopy using Q-TOF at the Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Ghana, Legon, Accra, Ghana. The experiment was conducted at a temperature of 300 K to ensure consistency and accuracy throughout the analysis. The 1H NMR spectrum was recorded at a frequency of 400 MHz, employing the "zg30" pulse sequence optimized for proton spectroscopy. Deuterated methanol (MeOD) from Sigma-Aldrich Inc. (St. Louis, MO, USA) and standard 5 mm, 7′′ NMR tube (XR-55 series) from Norell Inc. (Landisville, NJ, USA) were used. Measurement was taken on a 500 MHz Bruker spectrometer with AVANCE console and equipped with 5 mm TXI and TCI triple resonance inverse detection cryoprobes with z-axis pulse field gradient. The structure of the isolate was identified by concerted analysis of the 1H, 13C, HSQC, HMBC, NOESY, COSY NMR spectra and by comparison with data from the literature [11] .

Assessment of Broncho Dilating Activity of the Ethyl Acetate Fraction and the Isolate

Guinea pigs were sacrificed under thiopental sodium at the dose of 50 mg/kg, injected intramuscularly (Pentothal Sodium, Abbott, Turkey) and the tracheas were dissected out and immediately kept in Krebs-Henseleit solution, at room temperature (composition in mM: NaCl: 118, KCl: 4.7, MgSO4: 1.2, KH2PO4: 1.2, NaHCO3: 25, CaCl2: 2.5, and D-Glucose: 10.6) [12] . The tracheal tube was cut into small rings of 2-3 mm wide; each ring was opened by a longitudinal cut on the ventral side opposite to the smooth muscle layer to form tracheal strip with a central part of smooth muscle in between the cartilaginous portions on the edges. These tissues were mounted, under a tension of 1 g, in 5 mL tissue bath containing Kreb's solution, maintained at 37 °C and aerated with carbogen (a mixture of 95 % O2 and 5% CO2) prior to the experiments. Equilibration time of 1 hr elapsed before the addition of the used drugs. During this period, the organ bath was renewed every 15 min, and the tension was adjusted to 1 g. Histamine was injected in the bath to reach 10 µM to test the viability of the organ and to sensitize it. The preparation was rinsed and allowed to stabilize for 30 minutes, during which the bath was renewed twice. After this second period of stabilization, the organ was then contracted with histamine at 10 µM. After the sustained contraction was obtained, the relaxant effect of the ethyl acetate or isolate was assessed by cumulative addition till complete relaxation [13] . Isometric responses were recorded with software IOGA, developed by the Physics Department of the Sciences Faculty, University of Antananarivo, Antananarivo, Madagascar, to obtain concentration-dependent relaxation responses.

Structural Determination of the Bioactive Isolate

The chemical structure of the isolated compound was elucidated using spectroscopic methods including UV spectroscopy, 1D-and 2D-Nuclear Magnetic Resonance (NMR) spectroscopy (Bruker FT-NMR Avance-500 spectrometer, Ettlingen, Germany), and mass spectrometry (Q-TOF) at the Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Ghana, Legon, Accra, Ghana. All NMR experiments were conducted at a temperature of 300 K to ensure consistency and accuracy throughout the analysis. The 1 H NMR spectra were acquired at 500 MHz using the "zg30" pulse sequence optimized for proton spectroscopy. Measurements were performed using a 500 MHz Bruker spectrometer with AVANCE console equipped with 5 mm TXI and TCI triple resonance inverse detection cryoprobes with z-axis pulse field gradient. Spectral data processing was conducted using Bruker TopSpin software (version 3.6.3).

Data Analysis

The bronchodilation due to the isolate is expressed as a percentage relaxation in response to contraction induced by histamine. All results are expressed as Mean ± standard error mean (SEM) per experimental group, where 'n' represents the number of trachea strips used in the experiment. were then analysed by one way ANOVA (analysis of variance) followed by Student's 't' test to evaluate the significant differences between relaxation obtained with the different concentrations of the isolate. Differences were considered as significant when the 'p' value was less than 0.05.

Results and Discussion

In our search for bronchodilator natural compounds, we have carried out ethnobotanical surveys focused on plant used in the management of asthma symptoms. The ethanolic extract of F. trichopoda protects the animals against the dyspnoea induced by histamine pulverised in their cages, and relaxes isolated guinea pig tracheal muscles precontracted with histamine [9] . To identify a compound responsible for this bronchodilating activity, a bioassayguided fractionation followed by chromatographic purification on a preparative plate with silica gel was undertaken.

Bronchodilator Effect of the Ethyl Acetate Fraction

When injected cumulatively in the organ bath containing isolated guinea pig trachea precontracted with histamine at a concentration of 10 µM, the ethyl acetate fraction induced a concentration-dependent relaxation of the tissue. Complete (100%) relaxation was achieved at a concentration of 120 µg/mL. The EC₅₀ value, calculated by linear regression of the linear portion of the concentration-effect curve, is equal to 61.31 µg/mL (Figure 1). These results indicate that the ethyl acetate fraction contains bronchodilator compounds [14,15] . Multiple mechanistic pathways could be responsible for the bronchodilation induced by the isolate. Earlier studies conducted on natural products using guinea-pig trachea have reported the mechanisms involved in this bronchodilation such as anticholinergic [16,17] , Ca++ channel inhibition [18] phosphodiesterase inhibition [19,20] , potassium channel activation [21] , and/or leukotriene receptor blockade [22] . One of the limitations of the current finding is the preliminary screening of the isolate tested against histamine mediated tracheal constriction without exploring the detailed pharmacodynamics involved in the observed tracheal smooth muscle relaxation. In our future studies, we plan to study the detailed pharmacodynamics and draw more meaningful conclusions.

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Relaxation of isolated trachea induced by the ethyl acetate fraction injected, in a cumulative manner, in the bath containing the

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Conclusion

The current biologically guided chromatographic study enabled the identification of a bronchodilator compound from the ethyl acetate fraction of Ficus trichopoda leaves. Purification steps using different techniques enabled us to isolate a pure compound from a very complex fraction. Biological activity was very critical in tracking the active molecule belonging to the flavonoid family. This bronchodilator molecule from Ficus trichopoda leaves is reported here for the first time.

Acknowledgement

AcknowledgmentsThe authors acknowledge the laboratory of the Department of Pharmacognosy and Herbal Medicine, University of Ghana, Legon Accra, Ghana for enabling us to carry out the International Journal of Advanced Chemistry Research https://www.chemistryjournals.net structural determination of our sample, and the laboratory of pharmacology of the Pharmacology Department of Sciences Faculty of the University of Antananarivo, Madagascar for the pharmacological investigation, and all the staff of the two laboratories for their contribution.

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Relaxation of isolated trachea induced by the compound isolated from ethyl acetate fraction injected, in a cumulative manner, in the

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Structure of MR23 isolated from ethyl acetate fraction of Ficus trichopoda leaves (2-(2, 3-dihydroxyphenyl)-4-ethylchroman-3,5,6-

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