Phytochemical analysis of the ethyl acetate extract of the stem bark of Sesbania grandiflora (L.) led to the isolation of seven compounds including, a tetracyclic diterpene, kaurenoic acid (1). Other isolated compounds include two pentacyclic triterpenes β-amyrin (2) and lupeol (3), two steroidal ketones, stigmata-4,22-dien-3-one (4) and stigmast-4-en-3-one (5) stigmasterol (6) and a fatty acid, linoleic acid (7). Structures of these compounds were characterized by extensive NMR analysis and by comparing their spectral data with the published values. Among these compounds 1, 2, 3, 4 and 5 are isolated from S. grandiflora for the first time. Chemotaxonomic significance of the isolated compounds is also described herein. A comparative biological study for antioxidant, antimicrobial, thrombolytic and cytotoxic activities of the leaf and bark extracts of the plant was also conducted. Crude methanol extracts of both leaf and bark demonstrated strong antibacterial activity against Bacillus megaterium and Aspergillus niger as compared to the standards kanamycin and ketoconazole. The dichloromethane and ethyl acetate extract of both the bark and leaf showed very mild to moderate activities against the five microorganisms studied. The petroleum ether extract of the leaf showed no activity against most of the microorganisms studied. In the cytotoxic activity assay using brine shrimp and tamoxifen as a standard, the methanol extracts of the bark indicated the highest lethality as compared to the leaf extracts. Among all the extractives the ethyl acetate extracts of leaf showed moderate antioxidant activity using DPPH free radical scavenging method with butylated hydroxyl anisole and ascorbic acid as standards whereas different bark extracts showed very mild activities. The ethyl acetate and methanol extracts of the leaf exhibited significant thrombolytic activity as compared to the standard Streptokinase.

Keywords: Sesbania grandiflora, kaurenoic acid, β-amyrin, stigmata-4,22-dien-3-one, stigmast-4-en-3-one, antioxidant, cytotoxic, thrombolytic

Sesbania grandiflora (L.) Pers. commonly known as vegetable hummingbird, agati or hummingbird tree¹ is a small tree belonging to the family Leguminoseae. This plant is native to tropical Asia and is widely available in Malaysia, Indonesia, Philippines, and India.² It has widely been used in the herbal system of medicine to treat various diseases such as for the remedy of a large number of diseases like smallpox, inflammation, dysentery, rheumatism, leprosy, fever, gout, stomatitis and headache.^3,4 The flowers and leaf of S. grandiflora were described to contain anxiolytic,⁵ anticancer⁶ and antioxidant⁷ activities. The bark extract demonstrated potent acute inflammation as well as adjuvant-induced arthritis inhibitory activity in rats.⁸ Qualitative analysis of the plant is carried out using standard chemical methods. The results revealed the presence of alkaloids, carbohydrates, phenolic compounds, tannin, flavonoids and saponins in plant extracts.⁹ Although S. grandiflora was previously studied extensively for its phytopharmacological potential, no comparative biological studies have been performed before among different extracts of the leaf and stem bark of this plant. We also report herein the isolation of seven compounds including a diterpene, two triterpenes, three steroids and, a fatty acid. The compounds are kaurenoic acid (1), β-amyrin (2) lupeol (3), stigmata-4,22-dien-3-one (4) stigmast-4-en-3-one (5) stigmasterol (6) and linoleic acid (7) respectively among which compounds 1, 2, 3, 4 and 5 are being reported for the first time from Sesbania grandiflora. Previous studies claimed that kaurenoic acid (1) showed significant hemolytic activities,¹⁰ whereas compounds lupeol (3)¹¹ and stigmasterol (6)¹² showed potential cytotoxic activities on cancer cell lines.

Plant materials

The plant samples were collected from Gazipur district, Dhaka, Bangladesh in May 2017. A twig containing a flower, a fruit and, a dried pod was sent to Bangladesh National Herbarium (BNH) for identification (Accession number: DACB-47383).

Chemicals and solvents

2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich Co., USA. Silica gel and silica gel F₂₅₄ plates were purchased from Macherey-Nagel, Germany. Nutrient agar media, standard disc of kanamycin and ketoconazole were purchased from Hi media, India. All the chemicals and solvents used were of analytical grades.

General experimental procedures

Buchi rotary evaporator was used to concentrate the soluble extracts. The Vacuum liquid chromatography (VLC) column was dry-packed with Kiselgel 60H under suction. Sephadex LH-20 was used to perform gel permeation chromatography. All the solvents were distilled before use. Analytical and preparative thin-layer chromatography (TLC and PTLC) were performed both on aluminum sheets and glass plates coated with silica gel (Kiselgel 60 F₂₅₄). The TLC spots were confirmed under UV light at 254 nm and 366 nm and also by spraying with vanillin/H2SO4 reagent followed by heating. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker Avance 400 MHz Ultrashield NMR Spectrophotometer equipped with broadband and selective inverse probes. Chemical shifts are presented in δ (ppm) using Tetramethylsilane (TMS) as internal standard and coupling constants (J) are expressed in hertz.

Extraction for isolation

The air-dried and coarsely powdered stem barks of S. grandiflora (1.8 kg) were soaked in ethyl acetate (5 L) for 15 days at room temperature with occasional shaking and then double filtered using clean cotton plug. Each of the filtered extracts was concentrated with a rotary evaporator at 45^oC to yield 55 g of crude extract. An aliquot of the crude extract was fractionated by vacuum liquid chromatography (VLC) using silica gel 60H. The solvent system was used in the following order: petroleum ether > petroleum ether-dichloromethane > dichloromethane-ethyl acetate > ethyl acetate > ethyl acetate-methanol, and methanol in increasing order of polarity. The VLC fractionation yielded 45 fractions.

Following TLC screening, VLC fraction No. 13 and 14 (eluted with 90% dichloromethane in petroleum ether) were subjected to gel permeation chromatography over lipophilic Sephadex (LH-20). Compound 1 (8.0 mg) and 7 (11.0 mg), were obtained from the Sephadex column fraction eluted with 70% petroleum ether in chloroform and were purified by pTLC. R_f values were found 0.33 and 0.36 respectively (in 0.5% ethyl acetate in toluene).

VLC fraction No. 16, 17 and 18 (eluted with 100% dichloromethane) were subjected to gel permeation chromatography over lipophilic Sephadex (LH-20).

Compound 2, 3, 4 and 5 were obtained by mixing of similar Sephadex column fractions eluted with 100% chloroform and then purified by pTLC. Compound 2 and 3 (10.0 mg; Rf value: 0.51) and compound 4 and 5 were obtained as solid mixture (15.0 mg; Rf value: 0.49). The R_f values of these mixtures were 0.51 and 0.49 respectively (in 10% ethyl acetate in toluene). Compound 6 (5.5 mg) was obtained as off-white crystals, from the Sephadex column fraction eluted with 90% petroleum ether in chloroform and was purified by pTLC. The R_f value was 0.27 (in 10% ethyl acetate in toluene).

Spectroscopic data

Kaurenoic acid (1).¹³ Off white amorphous powder, ¹H NMR (400 MHz, CDCl₃): δ 0.84 (1H, m, H-1), δ 1.90 (1H, m, H-1), δ 1.47 (1H, m, H-2), δ 1.92 (1H, m, H-2), δ 1.04 (1H, m, H-3), δ 2.19 (1H, m, H-3), δ 1.09 (1H, m, H-5), δ 1.87 (2H, m, H-6), δ 1.48 (1H, m, H-7), δ 1.55 (1H, m, H-7) δ 1.09 (1H, m, H-9), δ 1.62 (1H, m, H-11), δ 1.64 (1H, m, H-11), δ 1.50 (1H, m, H-12), δ 1.63 (1H, m, H-12), δ 2.66 (1H, m, H-13), δ 1.18 (1H, m, H-14), δ 2.01 (1H, m, H-14), δ 2.08 (2H, m, H-15), δ 4.73 (1H, s, H-17), δ 4.79 (1H, s, H-17), δ 1.23 (3H, s, H-18), δ 0.94 (3H, s, H-18).

¹³C NMR (100 MHz, CDCl₃): δ 40.7 (C-1), 19.1 (C-2), 37.8 (C-3), 43.7 (C-4), 57.1 (C-5), 21.8 (C-6), 41.3 (C-7), 44.2 (C-8), 55.1 (C-9), 39.7 (C-10), 18.4 (C-11), 33.1 (C-12), 43.9 (C-13), 39.7 (C-14), 49.0 (C-15), 155.9 (C-16), 103.0 (C-17), 29.0 (C-18), 183.2 (C-19), 15.6 (C-20).

β-amyrin (2).¹⁴ Off-white crystal; ¹H NMR (400 MHz, CDCl₃): δ 3.14 (1H, m, H-3), 5.11 (1H, t, J = 3.6 Hz, H-12), 0.78 (3H, s, H-23), 0.95 (3H, s, H-24), 0.82 (3H, s, H-25), 0.92 (3H, s, H-26), 1.12 (3H, s, H-27), 0.98 (3H, s, H-28), 0.86 (3H, s, H-29), 0.86 (3H, s, H-30).

Lupeol (3).¹⁵ Off white crystal; ¹H NMR (400 MHz, CDCl₃): δ 3.14 (1H, m, H-3), 0.95 (3H, s, H-23), 0.75 (3H, s, H-24), 0.82 (3H, s, H-25), 1.01 (3H, s, H-26), 0.93 (3H, s, H-27), 0.77 (3H, s, H-28), 4.62 (br. s), 4.50 (br. s, H-29), 1.67 (3H, s, H-30).

Stigmata-4, 22-dien-3-one (4).¹⁶ White crystal; ¹H NMR (400 MHz, CDCl₃): δ 5.71 (1H, s, H-4), 0.71 (3H, s, H-18), 1.17 (3H, s, H-19), 1.00 (3H, m, H-21), 5.11 (1H, m, H-22), 5.03 (1H, m, H-23), 0.79 (3H, m, H-26), 0.81 (3H, m, H-27), 0.83 (3H, m, H-29).

Stigmast-4-en-3-one (5).¹⁶ White crystal; ¹H NMR (400 MHz, CDCl₃): δ 5.71 (1H, s, H-4), 0.71 (3H, s, H-18), 1.17 (3H, s, H-19), 0.90 (3H, d, J=6.6, H-21), 0.79 (3H, m, H-26), 0.81 (3H, m, H-27), 0.83 (3H, m, H-29).

Stigmasterol (6).¹⁷ Off-white crystal; ¹H NMR (400 MHz, CDCl₃): δ 3.50 (1H, m, H-3), 5.30 (1H, d, J = 5.4, H-6), 4.97 (1H, dd, J = 15.2, 8.5, H-22), 5.12 (1H, dd, J = 15.2, 8.5, H-23), 1.01 (3H, s, Me-18), 0.70 (3H, s, Me-19), 1.02 (3H, d , J = 7.4, Me-21), 0.80 (d, J = 6.9, Me-26), 0.84 (d, J = 6.3, Me-27), 0.81 (t , J = 7.5, Me-29).

Linoleic acid (7).¹⁸ Straw colored oily liquid; ¹H NMR (400 MHz, CDCl₃): δ 2.37 (2H, t, J=7.5 Hz, H-2), 1.66 (2H, m, H-3), 1.27-1.33 (14H, m, H-4,5,6,7,15,16,17), 2.06 (4H, m, H-8,14), 5.35 (4H, m, H-9, 10, 12, 13), 2.79 (2H, m, H-11), 0.91 (3H, m, H-18).

Biological investigation

The leaf and bark samples of S. grandiflora were air-dried for several days and ground into powder separately. About 400 g of the powders from both leaf and bark parts were soaked separately in 1.2 L of methanol at room temperature for 7 days with occasional stirring and shaking. The resultant mixtures were filtered through cotton plug and concentrated with a rotary evaporator at 45°C temperature to yield the crude methanol extracts of S. grandiflora (MEF-L: 14.62 g and MEF-B: 13.24 g ). Finally, a portion of the concentrated methanol extracts (10 g from each plant part) were fractionated by the modified Kupchan partitioning Method¹⁹ into petroleum ether (PEF-L and PEF-B), dichloromethane (DCMF-L and DCMF-B) and ethyl acetate (EAF-L and EAF-B) fractions. Subsequent evaporation of solvents yielded 2.76 g (PEF-L), 2.85 g (DCMF-L) and 2.94 g (EAF-L) extracts from leaf part and 2.26 g (PEF-B), 2.41 g (DCMF-B) and 2.57 g (EAF-L) extracts from bark part, respectively. All the dried extracts were kept in the tight containers in the refrigerator for further studies.

Antioxidant activity

The DPPH scavenging method²⁰ was used to evaluate the antioxidant potential of the leaf and bark extracts of S. grandiflora. The calculated amount of extracts (about 1.6 mg each) were dissolved in methanol to get different concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125 and 1.562µg/mL) in test tubes by serial dilution technique. In each test tube, 2.0mL of the sample solution was mixed with 2.0 mL of a DPPH (1, 1-diphenyl-2-picrylhydrazyl) in methanol solution (20µg/mL) to obtain the above-mentioned concentrations. Each sample was kept in dark chamber at room temperature for about 30 minutes. The absorbance of each sample was measured against methanol as negative control by UV spectrophotometer. Butylated Hydroxy Anisole (BHA) and ascorbic acid were used as reference solutions as well as positive control. The degree of decolorization of DPPH from purple to yellow indicated the scavenging activity of the extract. The percentage of radical scavenging activity was calculated by the following equation:

Radical scavenging (%) = [(A₀-A₁/A₀)×100].

Where,

A₀ = Absorbance of the control solution (DPPH solution without sample) and

A₁ = Absorbance of the sample extracts (DPPH solution with sample).

The extract concentration that produces 50% inhibition (IC₅₀) was calculated from the plot of percentage inhibition against extract concentration. A lower IC₅₀ value corresponds to a higher antioxidant activity of the extract.

Antimicrobial activity

In vitro antimicrobial activity of the crude methanol extracts and other fractions were assessed against two Gram-positive: Staphylococcus aureus (ATCC 25923) and Bacillus megaterium (ATCC 28318) as well as two Gram-negative bacteria; Pseudomonus aeruginosa (ATCC 27833) and Escherichia coli (ATCC 28739) and one fungal strain Aspergillus niger. The antimicrobial activity was tested by the standard disc diffusion method.²¹ The samples were dissolved separately in a specific volume of dichloromethane or methanol depending on their solubility. Kanamycin (30μg/disc) and ketoconazole (30μg/disc) were used as standards for antibacterial and antifungal screening respectively.

Cytotoxic activity

In brine shrimp lethality bioassay,²² adequate amount of fractions of test samples were dissolved in DMSO to obtain solutions of varying concentrations (400, 200, 100, 50, 25, 12.50, 6.25, 3.125, 1.563, 0.781µg/mL) by serial dilution technique in premarked glass test tubes (containing 5 mL of seawater with 10 matured shrimps in each test tube). DMSO was used as a solvent and negative control while tamoxifen served as the standard and positive control. The mortality of brine shrimp was observed after 24 hours of treatment for each of the concentrations. An approximate linear correlation was observed by plotting logarithm of concentration versus percentage of mortality in triplicate test samples.

Thrombolytic activity

For this test about 5 mL of blood was drawn from healthy human volunteers (n=5) (aged 25-40 years) who did not have a history of oral contraceptive or anticoagulant therapy. 500µL blood was transferred to each of the previously weighed and marked Eppendorf tubes (n = 8) and allowed them to form clots. 5 mL of 0.9% sodium chloride (NaCl) was added to the commercially available lyophilized S-Kinase™ (Streptokinase) vial of 15, 00,000 I.U. and mixed properly. This solution was used as a stock from which 100µL was used for in vitro thrombolytic assay.²³ Streptokinase (SK) solution was used as the standard and positive control. As a negative control, 100µL of distilled water was added to the control tube. All tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, the released fluids were discarded from each tube and the tubes were weighed again to observe the difference in weight after clot disruption.

The differences in weights taken before and after clot lysis were expressed as the percentage of clot lysis as shown below:

% of clot lysis = (weight of released clot /clot weight) × 100.

Statistical analysis

All statistical analysis was done using Microsoft Excel. Statistical values were considered significant when P<0.05. Antioxidant, antimicrobial, cytotoxic and thrombolytic activity was conducted in triplicate and expressed as mean ± standard deviation.

Structure elucidation

Compound 1 was obtained as an off-white amorphous powder. The ¹H NMR spectrum (Table 1) displayed the characteristic exomethylene group at δ 4.73 s and 4.79 s and two methyl groups at δ 1.23 and 0.94. The ¹³C NMR spectrum exhibited 20 carbons including a carbonyl carbon at δ 183.2. In the HMBC experiment (Table 1) the methyl at δ 1.23 (Me-18) and the proton δ 1.05 (H-5) showed ³J correlation to the carbonyl carbon, thus placing the carboxyl group at C-19. The structure of the compound was established as Kaurenoic acid by analysis of NMR data including HSQC and HMBC correlations and was further confirmed by comparison of the ¹D NMR data published in the literature. Kaurenoic acid, a diterpene, not reported from this plant before or even from the genus Sesbania (Figure 1).

Figure 1 Compounds (1-7) isolated from S. grandiflora.

Biological activity

Antioxidant activity

Different organic fractions of S. grandiflora were subjected to evaluation of antioxidant activity. The IC₅₀ value of standard BHA and ascorbic acid were 9.26±0.10μg/mL and 8.58±0.13µg/mL, respectively. Among all the extracts, ethyl acetate and methanol fractions showed significant inhibitory activity with IC₅₀ values of 52.52±0.11 and 73.37±0.09µg/mL, respectively in leaf and 71.29±0.16 and 89.20±0.18µg/mL, respectively in bark part. The results are summarized in Table 2.

Antimicrobial activity

Antimicrobial activity of different fractions of S. grandiflora was tested against two gram-positive and two gram-negative bacterial species. During the evaluation, ethyl acetate and methanol fractions showed significant activity against B. megaterium with ZOI of EAF-L: 18 and MEF-L: 21 in leaf extracts and ZOI of EAF-B: 12 and MEF-B: 20 in bark extracts. The ethyl acetate and methanol fractions also showed activity against the fungal species A. niger with ZOI of EAF-L: 12 and MEF-L: 19 in leaf extracts and ZOI of EAF-B: 11 and MEF-B: 17 in bark extracts. The other solvent fractions showed poor to moderate inhibitory activity against all the bacterial and fungal strains. In this test, Kanamycin was used as antibacterial standard and Ketoconazole was used as antifungal standard. The results are summarized in Table 3.

Cytotoxic activity

The median lethal concentration (LC₅₀) of the test samples after 8 hours were obtained by a plot of percentage of the shrimps killed against the logarithm of the sample concentration (toxicant concentration). Ethyl acetate and methanol fractions showed very potent cytotoxic activity in both leaf (LC₅₀ values were 0.6799±0.10 and 0.6564±0.04µg/mL, respectively) and bark extracts (LC₅₀ values were 0.6509±0.03 and 0.6874±0.10µg/mL, respectively), in comparison to the standard (LC₅₀ value 0.3019±0.05µg/mL). The results are shown in Table 4.

Thrombolytic activity

The leaf and bark extracts of S. grandiflora showed significant thrombolytic activity. Among all the fractions, ethyl acetate and methanol soluble fraction seemed to possess the highest clot lysis activity in leaf extract (51.89±1.81% and 49.13±1.29%, respectively) and bark extract (47.23±1.16% and 45.42±1.38%, respectively) compared to the standard Streptokinase (79.68±1.43%). Distilled water showed a negligible lysis of clot (3.69±1.21%). The results are shown in Table 5.

Chemotaxonomic Significance

Compounds 1 and 2 are the first isolation from the Sesbania genus, and the first report of compound 3 in this species. Compounds 1 and 2 were previously isolated from the genus Copaifera langsdorffii²⁴ and Caesalpinia bonduc²⁵ within the same family. Compound 3 was isolated from another Sesbania species Sesbania aegyptiaca.²⁶ This might be considered as further chemical markers to distinguish S. grandiflora from other Sesbania species. Compounds 4 and 5 are reported for the first time in Leguminoseae family, previously isolated from several plant families i.e., Asteraceae,²⁷ Compositae,²⁸ Simarubaceae^29,30 and Scrophulariaceae.³¹ Therefore, steroids 4 and 5 may be an important addition of the Leguminoseae family as chemotaxonomic markers. Compounds 6 and 7 were isolated from the same species earlier.^32,33

Among the isolated compounds, kaurenoic acid (1), β-amyrin (2) lupeol (3), stigmata-4,22-dien-3-one (4) stigmast-4-en-3-one (5) and stigmasterol (6) are being reported for the first time from Sesbania grandiflora. From the biological investigation among various fractions of leaf and bark of S. grandiflora (L.), it is evident that ethyl acetate and methanol extracts have significant antioxidant, antibacterial, cytotoxic and thrombolytic activities. It is strongly believed that the detailed information as reported in this study might be used as additional data for future extensive research, development, and utilization of S. grandiflora in various diseases. Further work is to be carried out to explore the other chemical constituents and biological activity of pure compounds. 

The authors are thankful to BCSIR, Dhaka Lab and King’s College, London for performing the NMR spectroscopy of the isolated compounds in order to elucidate the structures.

Authors declare that there is no conflict of interest.

1. "Sesbania grandiflora". Natural Resources Conservation Service; PLANTS Database. USDA; 2015.
2. Anantaworasakul P, Hamamoto H, Sekimizu K, et al. In vitro antibacterial activity and in vivo therapeutic effect of Sesbania grandiflora in bacterial infected silkworms. Pharmaceutical Biology. 2017;55(1):1256‒1262.
3. Das J, Das MP, Velusamy P. Sesbania grandiflora leaf extract mediated green synthesis of antibacterial silver nanoparticles against selected human pathogens. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2013;104:265‒270.
4. Joshi SG. Leguminoseae: text book of medicinal plants. New Delhi: Oxford and IBH Publishing Co. Pvt Ltd; 2000. 130 p.
5. Kasture VS, Deshmukh VK, Chopde CT. Anxiolytic and anticonvulsive activity of Sesbania grandiflora leaf in experimental animals. Phytotherapy Research. 2002;16(5):455‒460.
6. Sreelatha S, Padma PR, Umasankari E. Evaluation of anticancer activity of ethanol extract of Sesbania grandiflora (Agati Sesban) against Ehrlich ascites carcinoma in Swiss albino mice. Journal of Ethnopharmacology.2011;134(3):984‒987.
7. Gowri SS, Vasantha K. Free radical scavenging and antioxidant activity of leaf from Agathi (Sesbania grandiflora) (L.) Pers. American-Eurasian Journal of Scientific Research. 2010;5(2):114‒119.
8. Patil RB, Nanjwade BK, Manvi FV. Effect of Sesbania grandiflora and Sesbania sesban bark on carrageenan-induced acute inflammation and adjuvant-induced arthritis in rats. Pharma Science Monitor. 2010;1(1):75‒89.
9. Ghani A. Medicinal plants of Bangladesh: Chemical constituents and uses. 2nd ed. The Asiatic Society of Bangladesh, Dhaka. 2003;17:63‒438.
10. De S Vargas F, DO de Almeida P, Aranha E, et al. Biological Activities and Cytotoxicity of Diterpenes from Copaifera spp. Oleoresins. Molecules. 2015;20(4):6194‒6210. 
11. Moriarty DM, Huang J, Yancey CA, et al. Lupeol is the Cytotoxic Principle in the Leaf Extract of Dendropanaxcf. querceti. Planta Medica. 1998;64(04):370‒372. 
12. Nasrin M, Afroz F, Sharmin S, et al. Cytotoxic, Antimicrobial and Antioxidant Properties of Commelina diffusa Burm. F. Pharmacology & Pharmacy. 2019;10(2):82‒93.
13. Henriete SV, Jacqueline AT, Alaíde BO, et al. Novel Derivatives of Kaurenoic Acid: Preparation and Evaluation of their Trypanocidal Activity. Journal of the Brazilian Chemical Society. 2002;13(2):151‒157.
14. Mostafa AA, Al-Askar AA, Almaary KS, et al. Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases. Saudi Journal of Biological Sciences. 2018;25(2):361‒366.
15. Silva ATM, Magalhães CG, Duarte LP, et al. Lupeol and its esters: NMR, powder XRD data and in vitro evaluation of cancer cell growth. Brazilian Journal of Pharmaceutical Sciences. 2017;53(3):1‒10.
16. Jibril S, Sirat HM, Zakari A, et al. Isolation of Chemical Constituents from n-Hexane Leaf Extract of Cassia singueana del. (Fabaceae). ChemSearch Journal. 2019;10(1):20‒24.
17. Pateh UU, Haruna AK, Garba M, et al. Isolation of stigmasterol, β-sitosterol and 2-hydroxyhexadecanoic acid methyl ester from the rhizomes of Stylochiton lancifoliusPyer and Kotchy (Araceae). Nigerian Journal of Pharmaceutical Research. 2009;8(1):19‒25.
18. Alexandri E, Ahmed R, Siddiqui H, et al. High Resolution NMR Spectroscopy as a Structural and Analytical Tool for Unsaturated Lipids in Solution. Molecules. 2017;22(10):1663.
19. Kupchan SM, Tsou G, Sigel CW. Datiscacin, a novel cytotoxic cucurbitacin 20-acetate from Datisca glomerata. Journal of Organic Chemistry. 1973;38(7):1420‒1421.
20. Schwarz K, Bertelsen G, Nissen LR, et al. Investigation of plant extracts for the protection of processed foods against lipid oxidation: Comparison of antioxidant assays based on radical scavenging, lipid oxidation and analysis of the principal antioxidant compounds. European Food Research and Technology. 2001;212(3):319‒328.
21. Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology. 1999;86(6):985‒990.
22. Meyer BN, Ferrigni NR, Putnam JE, et al. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica. 1982;45(5):31‒34.
23. Prasad S, Kashyap RS, Deopujari JY, et al. Effect of Fagonia arabica (Dhamasa) on in vitro thrombolysis. BMC Complementary and Alternative Medicine. 2007;7(1):36.
24. Fabiano de SV, Patricia DO de A, Elenn Suzany PA, et al. Biological activities and cytotoxicity of diterpenes from Copaifera spp. oleoresins. Molecules. 2015;20(4):6194‒6210.
25. Zhaohua W, Yongyi W, Jian H, et al. A new cassane diterpene from Caesalpinia bonduc. Asian Journal of Traditional Medicines. 2007;2(4):135‒139.
26. Saxena VK, Mishra LN, et al. Analysis of the oil from the seeds of Sesbania aegyptiaca Poir. Asian Journal of Chemistry. 2003;15(3-4):1811‒1813.
27. Achari B, Esahak A, Dastidar, et al. Brine shrimp: a convenient general bioassay for active plant constituents. Journal of the Indian Chemical Society. 1974;51(3):419‒422.
28. Antonio GG, Jesus GD, Dominguez B, et al. Triterpenes and steroids from Eupatorium adenoforum Spreng. Revista Latinoamericana de Quimica. 1987;18(1):51‒52.
29. Mandal S, Das PC, Joshi PC, et al. Steroidal constituents of Ailanthus excelsa Roxb. Journal of the Indian Chemical Society. 1999;76(10):509‒510.
30. Malek, Nurestri BHA, David ID. The steroidal components of Eurycoma apiculatta. Sains Malaysiana. 1982;11(2):87‒103.
31. Halina RB, Bozena KO, Eliza LZ. Chemical and biological investigation of lipophilic fraction of Linaria vulgaris Mill. Bulletin of the Polish Academy of Sciences: Biological Sciences. 1996;43(3-4):179‒184.
32. Shareef H, Rizwani,HG, Zia-ul-Haq M, et al. Tocopherol and phytosterol profile of Sesbania grandiflora (Linn.) seed oil. Journal of Medicinal Plants Research. 2012;6(18):3478‒3481.
33. Tiwari RD, Garg SP. Examination of the component fatty acids of the oil from the seeds of Sesbania grandiflora. Journal Oil Technologists Association of India. 1960;16:35‒37.