Chemical and Biological Profiling of Three Ferulic Acids Alkyl Esters Isolated from Jatropha pandurifolia (Family: Euphorbiaceae) Stem Bark.

The study's objectives include phytochemical profiling and biological (antioxidant, thrombolytic and cytotoxic) analysis of pure chemicals from Jatropha pandurifolia stem bark ethyl acetate extract. Five different compounds including octacosanyl cis ferulate (1), hexacosyl (E)-ferulate (2) triacontyl ferulate (3), ß-sitosterol (4) and stigmasterol (5) are elucidated. Their structures determine through 1HNMR analysis and comparison to published data, while three ferulic acid alkyl esters (1-3) were isolated for the first time from J. pandurifolia. Compounds 1, 2, and 3 all have significant thrombolytic potential with respective values of 68.92% ±1.17 (**P<0.01), 66.56% ±2.35 (**P<0.01) and 70.81%±0.98 (**P<0.01) with comparison to standard streptokinase (73.6%±0.76). When compared to BHT (6.82± 0.99 µg/ml) the IC50 (DPPH assay) values were 16.26±1.07 (**P<0.01), 14.12±1.23 (**P<0.01), and 13.16±1.70 µg/ml (**P<0.01). Comparing the three compounds to the reference vincristine sulphate (LC50: 0.52±0.18 µg/ml), of compound 1 (1.56±0.35 µg/ml) (**P<0.01), compound 2 (1.3±0.78 µg/ml) (**P<0.01) and compound 3 (1.29±0.33 µg/ml) (**P<0.01). The results can therefore be interpreted as a concept of isolated molecules having potential for application in additional pharmaceutical research.

The class of organic molecules known as phytochemicals, which includes tannins, sugars, triterpenoids, alkaloids, flavonoids, steroids, and others, is rife in medicinal plants (plant chemicals). These phytochemicals are fantastic for offering health advantages and acting as a protection, avoiding a number of diseases 1 .
Excessive generation of reactive oxygen species (ROS) leads to oxidative stress 2 that in turn trigger several diseases such in inflammation, hypertension, atherosclerosis, AIDS, DM and cancer etc. Antioxidative enzymes such as peroxidase, superoxide dismutase (SOD) is concealed by the over production of free radicals that consequences cellular mutation and damage 3 . The brine shrimp bioassay is linked to cytotoxic act in living tissue and represents pesticidal activity 4, 5 . The mechanism of a thrombolytic drug is to liquefy blood clot (thrombus) in acutely occluded coronary arteries, pulmonary embolism, atrial fibrillation, thrombosis and prosthetic heart valves 6 that ensures sufficient blood supply to myocardium and to develop prospects with blood loss reduction. Market available thrombolytic agents like urokinase, alteplase, streptokinase, anistreplase, tissue-type plasminogen activator (tPA) offer some restrictions like side effects with associated bleeding history 7, 8 . Due to the presence of phytochemical elements, medicinal plants are essential in the prevention of various diseases, and their use and promotion fit into all current prevention efforts. Jatropha (Family: Euphorbiaceae, a castor family) having 175 species of succulent, caudiciform, herbaceous perennials and woody plants including Jatropha pandurifolia ( Jatropha integerrima) that is a tiny, evergreen tree or shrub with lustrous leaves with star-shaped red, pink, or vermilion blooms and also known as peregrine or spicy jatropha 9 . In Asia, Latin America, and Africa, several plant parts including the leaves, stem, bark, roots, and oil, have long been used for a variety of ailments as styptic agent, emetic agent, antidote 10,11 , purgative 12 , in the treatment of warts, toothaches, ringworm, gastric emptying, gum bleeding, rheumatic pain and skin diseases 13,14 . Numerous phytochemicals holding potential medicinal norms were reported from this plant including alkaloid (jatrophine, jatropham and curcin) 15 , diterpene (jatrophone, jatrophatrione, jatropholone A-B) 16,17 , sitosterol, glycoside, lignin, tannin, saponin 18 , fatty acids (palmitic, oleic, and linoleic acids), carotenoid, flavonoid etc 19, 20 . This plant has been linked to antiinflammatory, antituberculosis, antiplasmodial, cytotoxicity 21,22 , anticancer 23,24 , antimicrobial 25-27 , antifungal antioxidant potentials 28 according to previous studies. Following several chromatographic methods structure elucidations was carried out by HNMR spectroscopy. The chemical investigation revealed very first isolation of three ferulic acid alkyl esters from this species. ²-sitosterol and stigmasterol were also isolated concurrently. In the current study the obtained pure three ferulic acid alkyl esters were subjected to antioxidant, thrombolytic and cytotoxic investigation.

overall experimental measures
Whilst performing VLC (vacuum liquid chromatography) fine VLC-grade silica like Kiesel gel 60H was used in the column. With the aid of dichloromethane, ethyl acetate, and methanol, the elution was carried out while gradually increasing polarity. An appropriate solvent system was used to elute TLC plates that are pre coated with Silica gel 60, F254. The TLC plates were sprayed with vanillin-H 2 SO 4 , heated for 5-10 minutes at 110°C, and UV light at short wavelength (254) and long wavelength (366) nm were used to visualize the spots. NMR spectra were transcribed in CDCl 3 on a 400 MHz Ultra shield NMR Spectrophotometer.
In August 2019, J. pandurifolia stem barks were collected from premises of University of Dhaka, Bangladesh. The plant was identified in National Herbarium, Dhaka and an identification no. was preserved (DACB Accession No. 65545). The air dried and powdered stem-barks of J. pandurifolia (3.0 kg) in ethyl acetate over the period of 15 days and filtered through a cotton pad then by Whatman filter paper. The extract was then evaporated using a Buchii rotary evaporator to be concentrated under reduced pressure.

extraction and isolation
An aliquot part of the crude ethyl acetate extract (40g) was partitioned by VLC technique in increasing order of polarity to provoke adsorption and seperation like a normal-phase column chromatography. Used solvents were petroleum ether, ethyl acetate and ethanol in sequence. VLC fractions were numbered from 1 to 41. White crystals were appeared in fraction VLC 10, 13 and 25. The crystals were washed with n-hexane, collected separately and were purified by PTLC (preparative TLC) with a convenient solvent system (1% ethyl acetate in toluene). By recrystallization technique JP-10 (Compound 1, colorless crystals, 5 3.93 (singlet, 3H) and 5.85 (singlet with broad signal) for OCH3 and OH-7 groups.
Structure elucidation of C-4 and C-5 were also performed by 1 HNMR analysis and compared spectral data were compared to the reference data.

Protocol for biological investigation
Isolated pure compounds were taken for in vitro investigations according to established methods.

thrombolytic activity
The percentage of human blood clot lysis was estimated 29 in comparison to Streptokinase (100¼l equivalent to 30,000 I.U) as positive control and distilled water (100 µl) as negative control. The calculation as follows: % of thrombolytic activity = (wt. of clot after treatment / wt. of clot before treatment) × 100 antioxidant activity (dPPh free radical scavenging assay) According to Brand-Williams et al. 30 DPPH assay was carried out in presence of different concentration by serial dilution technique. Butylated hydroxyl toluene (BHT) and methanolic DPPH solution were used as positive control and negative control accordingly. IC 50 values were calculated from the regression equation by plotting concentrations vs percentage of scavenging the free radicals. I % = 1-{A1 / A0} × 100 A0: absorbance of the control and A1: absorbance of test samples/standard. The experiment was repeated in triplicate at each concentration.

Brine shrimp lethality bioassay
The cytotoxic test was anticipated by brine shrimp lethality bioassay 31 of pure compounds at different concentrations. The percentage of mortality was calculated and plotted against LogC Then lethal concentrations (LC 50 ) were calculated to measure cytotoxic activity. Saline water and vincristine sulphate (VS) were employed as negative and positive controls, respectively. % mortality = (no. of dead nauplii/initial total no. of live nauplii) × 100 statistical analysis The results of every experiment were shown as mean SEM. Using SPSS 20 software, one-way analysis of variance (ANOVA) was used to compare the study groups to one another. A p value of 0.05 or less was considered statistically significant.

structure elucidation
Modern chromatographic purification and separation of the ethyl acetate-stem bark extract of J. pandurifolia provided a total of three compounds (1-3), the structures were elucidated by 1H NMR analysis.

Compound 01 (JP-10)
Colorless crystal. Black and bluish spots were observed under the both 254 nm and 356 nm UV light accordingly on TLC and deep purple spot with vanillin-H 2 SO 4 and heating for 1 min at 110 0 C. Three aromatic protons were seen in the 1H  6.79 (J = 12 Hz) respectively are close to the carboxylic group. A triplet was appeared at ´ 4.11 representing a methylene group. The spectrum also revealed two multiplets at ´1.27 (50H) and 1.64 (-CH 2 group) and a triplet at ´ 0.88 for a -CH 3 group. A broad singlet (´3.93) and doublet (´5.83) indicate methoxy and hydroxyl groups consecutively on the phenyl ring. Compared NMR data with those of compound-1 was determined to be octacosanyl cis ferulate 32 .  Three aromatic protons were seen in the 1H NMR spectrum at ´ 7.03 s, 6.91 d (J = 8 Hz) and 7.07 dd (J = 8.1 Hz) assignable to H-5, H-8 and H-9 of phenyl ring respectively. Protons at position 2 and 3 having doublets at 6.29 (J = 16 Hz) and 7.61 (J = 16 Hz) respectively are close to the carboxylic group. A triplet was appeared at ´ 4.18 representing a methylene group. The spectrum also revealed two multiplets at ´1.28 (46H) and 1.64 (-CH2 group) and a triplet at ´ 0.88 for a-CH3 group. A singlet (´3.92) and broad singlet (´ 5.93) indicate methoxy and hydroxyl groups consecutively on the phenyl ring. Compared NMR data with those of compound-2 was determined to be hexacosyl (E)-ferulate 33 .

Compound 03 (JP-25)
Colourless crystal. Black and bluish spots were observed under the both 254 nm and 356 nm UV light accordingly on TLC and deep purple spot with vanillin-H 2 SO 4 and heating for 1 min at 110°C. The 1H NMR spectrum displayed three aromatic protons at ´ 7.03 s, 6.91 d (J = 8 Hz) and 7.07 dd (J = 8.1 Hz) assignable to H-5, H-8 and H-9 of phenyl ring respectively. Protons at position 2 and 3 having doublets at 6.29 (J = 16 Hz) and 7.61 (J = 16 Hz) respectively are close to the carboxylic group. A triplet was appeared at ´ 4.18 representing a methylene group. The spectrum also revealed two multiplets at ´1.28 (46H) and 1.68 (-CH2 group) and a triplet at ´ 0.88 for a-CH3 group. A singlet (´ 3.93) and broad singlet (´ 5.85) indicate methoxy and hydroxyl groups consecutively on the phenyl ring. Compared NMR data with those of compound-3 was determined to be triacontyl ferulate 34 . Values are expressed as mean ± SD (n = 3); Significance level among different groups at P d"0.05 (*P<0.05; **P<0.01, ***P<0.001); Test groups were compared to standard group; EASBJP represents ethyl acetate stem bark of J. pandurifolia

Biological investigations of J. pandurifolia
The isolated pure crystals confirmed various degrees of secondary metabolites when subjected to thrombolytic, antioxidant and brine shrimp cytotoxic activities.

thrombolytic activity
Tissue plasminogen activator (t-PA), streptokinase, alteplase, reteplase, tenecteplase and urokinase etc. are commonly prescribed thrombolytic medication for the treatment of various CVD, myocardial infarction, cerebral thrombosis etc. Several secondary metabolites such as alkaloids, tannins, flavonoids, saponins exert thrombolytic properties as a natural supply 35 .

antioxidant activity
Other than superoxide dismutase, catalase enzymes or glutathione transferase diverse phytochemicals like polyphenolic compounds such as, flavonoids, phenolics, tocopherols, tannins etc. are to blame for inhibiting oxidation that may cause cell damage in living organism 36   the isolated compounds that could be an innate reserve of polyphenolic compounds. Turkmen et al. (2006) hypothesized that solvent polarity may be a factor in the effectiveness of various extracts at scavenging free radicals 37 .

Brine shrimp lethality bioassay
Brine shrimp lethality bioassay is a quick and dynamic technique to compute the cytotoxicity of any compounds that might be noxious to health 38. The pure compounds showed significant brine Earlier research has reported the cytotoxic and antimicrobial activity from the n-hexane stem bark extract of this plant 22 44 . Latex and leaf of J. curcas 45 and J. gossypiifolia 46 were turned up as blood thinner with probable estimation in clotting time reduction which could be correlated to thrombolytic property of the current plant. Biological investigations of the pure compounds from this plant strongly prove the abundance of ethno pharmacological activity. Further investigations of the isolated compounds are required specially, biological and molecular docking studies for unveiling probable activities.

ConClusion
The biological investigation found in this research justifies the pharmacological activity of this plant for curing miscellaneous malady. Different chain length of the ester increases the antioxidant potential of ferulic acids. Mild to strong thrombolytic, antioxidant and cytotoxic activities were unveiled by the pure compounds of ethyl acetate fraction that would be remarkable investigation of unrevealed ethno-pharmacological potency. The present study suggests exploring extensive research on this plant to discover many potent bioactive molecules.

aCknowledgeMent
The authors are grateful to the University of Dhaka, Bangladesh for funding the research project under Centennial Research Grant (CRG) 2021-22 and also thankful to Khozirah Shaari, University Putra Malaysia for her support in taking NMR spectra of the isolated compounds. Also expressing gratitude to the Faculty of Pharmacy, University of Dhaka, Bangladesh for providing all the research amenities to bring out this work successfully.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.

Funding source
The research project was funded by