Abstract
Background
The Aim of the present research article is to proposing a conservative approach for the Cassia fistula by using of small branches instead of stem bark because of plant has many important chemical constituents which show different medicinal activity so consumption of plant is high. We studied here Comparative preliminary phytochemical screening test of the ethanol extract and aqueous extract of the stem bark and small branches of Cassia fistula obtained by cold maceration process. Physicochemical analysis of Cassia fistula was done to ascertain the quality of the raw material used in the study. Successive soxhlet extraction method used for the successive extraction of stem bark and small branches with different solvents for comparative chemical profile study by HPLC, LCMS, and GCMS. Molecular Docking Interaction of Abundant Medicinal Phytochemicals in the Liquid Chromatography−Mass Spectrometry (LC−MS) Analysis Data of C. fistula with the L. donovani Drug Target Proteins and Pancreatic lipase colipase target protein.
Result
The pH of the small branches was found slightly higher as compared to stem bark and the percentage of other parameters like total ash content, acid insoluble ash, loss on drying at 105 °C, water soluble extractive and alcohol soluble extractive values were found fewer in the small branches as compare to stem bark of the plant. It was observed that the number of peaks in stem bark and small branches of the plant sample were almost similar and the retention time of each peak in stem bark was coincide with the retention of small branches of the sample. Therefore, similarity was observed in stem bark and small branches of the Cassia fistula plant in HPLC, LC-MS and GC-MS. The results obtained from HPLC analysis shows that stem bark contains 0.0084% and small branches having 0.0257% of rhein in Cassia fistula. Compounds 3, 9 and 12 are present in Stem bark as well as small branches of C. fistula and Compounds 22, 32 and 37 are present in small branches only. All the compounds have very good binding energy (Kcal/mol) with the respective target proteins.
Conclusion
The small branches have more active chemical constituents than stem bark against particular target proteins.
Keywords: Golden shower, Ayurvedic Pharmacopoeia of India, API, Docking, Target protein, Rhein, HPLC, Medicinal plants
Golden shower; Ayurvedic Pharmacopoeia of India; API; Docking; Target protein; Rhein; HPLC; Medicinal plants.
1. Introduction
Cassia fistula Linn. commonly known as the Golden Shower belongs to the family Fabaceae. It is a deciduous tree with greenish grey bark, compound leaves, leaf lets are each 5–12 cm long pairs. A semi-wild tree known for its beautiful bunches of yellow flowers and also used in traditional medicine for several indications. A fruit is cylindrical pod and seeds many in black, sweet pulp separated by transverse partitions. The long pods which are green, when unripe, turn black on ripening after flowers shed [1]. Pulp is dark brown in Colour, sticky, sweet and mucilaginous, odour characteristic, and somewhat disagreeable [2, 3]. Drug occurs in flat or curved thick pieces; outer surface smooth to rough with warty patches; greenish grey to red; inner surface rough, reddish with parallel striations; fracture, laminate; odour, sweet and characteristic; taste, astringent [4]. It is a medium size tree which is native of tropical Asia. It is widely cultivated in South Africa, East Africa, Brazil, India, West Indies, China, Mexico, etc. All parts (see stem bark and small branches of plants in Figure 1) of the plant have medicinal properties so it is a very valuable medicinal plant which is utilized in the traditional system of medicine.
Figure 1.
Raw Plant material of Cassia fistula Linn.
1.1. Taxonomic classification
Kingdom - Plantae
Subkingdom – Tracheobinota
Super Division - Spermatophyta
Division - Mangoliophyta
Class – Magnoliopsida
Sub Class - Rosidae
Order - Fabales
Family - Fabacae
Genus - Cassia
Species – fistula
1.2. Chemical constituents
Cassia fistula was reported to have important classes of phyto constituents like Anthraquinone glycosides, cardiac glycosides, phenolic compounds, carbohydrate, protein, fats, alkaloids, tannins, saponins, steroids, ter-penoids and phloba-tannins, linoleic acid, oleic acid, stearic acid, rhein glycosides fistulic acids, sennosides A, B, anthraquinones, flavanoid-3-olderivatives, ceryl alcohol, kaempferol, bianthraquinone glycosides, fistulin, essential oils, volatile components, phytol (16.1%), 2- hexadecanone (12%), crystals and 4-hydroxy benzoic acids hydrate etc. [5, 6, 7, 8].
Lupeol, β-sitosterol, hexacosanol, 5,7,3,4′-tetrahydroxy-6, 8-dimethoxyflavone-3-O-α-arabinopyranoside,5,7,4′-trihydroxy-6,8,3′-trimethoxyflavone-3-O-α-L-rhamnosyl (1→2) -O-β-D- glucopyranoside and 1,8-dihydroxy-3, 7-dimethoxyxanthone-4-O- α-L-rhamnosyl (1→2)-O-β-D-glucopyranoside are present in the stem bark of the plant (see chemical structure in Figure 2).
Figure 2.
A) Chemical Structure of chemical constituents present in both stem bark and small branches of Cassia fistula. 1. Betaine, 2. Nicotinic acid, 3. Butein, 4. 4-Methoxycinnamic acid, 5. 4-Hydroxycoumarin, 6. Caffeic acid, 7. (E)-parinaric acid, 8. Oleanolic acid, 9. Lup-20(29)-en-28-al, 3beta-hydroxy, 10. (22E)-Stigmasta-5,22-dien3-ol, 11. Erucamide, 12. Betulin. B) Chemical Structure of chemical constituents present only in stem bark of Cassia fistula. 13. Abietic acid, 14. Nervonic acid, 15. Epicatechin, 16. Aloin A, 17. Quercetin, 18. Luteolin, 19. Rhamnetin, 20. (-)-Epigallocatechin. C) Chemical Structure of chemical constituents present only in small branches of Cassia fistula 21. Apigenin, 22. Kaempferol, 23. 4-Piperidone, 24. Vanillin, 25. Quinine, 26. b-Asarone, 27. (E)-Ferulic acid, 28. 3-Hydroxypyridine, 29. 6-Gingerol, 30. 10-Gingerol, 31. 8-Hydroxyquinoline, 32. (±)-Naringenin, 33. 7-Ethoxycoumarin, 34. (+/-)-Methoprene, 35. (E)-4-Methoxycinnamic acid, 36. Asiatic acid, 37. Lupa-12,20(29)-dien-3-one, 38. Aloe-emodin, 39. (+)-ar-Turmerone, 40. Adipic acid.
A major anthraquinone derivative called rhein and 1,8-dihydroxy-3-anthraquinone found in fruit pulp of the plant. Fistucacidin, an optically inactive leucoanthocyanidin which is a phenolic compound from the heart wood of the plant. A bianthoquinone glycoside, fistulin together with kaempferol and rhein found in the flowers of the plant. A major carbohydrate called galactomannan consisting of 8 different type of sugar moieties are reported from the seeds of the plant. The anthraquinones like Rhein, Chrysophanol and Physcion, 9-(-)-epiafzelechin, 3-O-B-D-Glucopyranoside, 7 bioflavonoids and two triflavonoids together with (-) – epiafzelechin, (-)-epicatechin and procyanidin B-2 from the leaves of the plant. 3B-hydroxy-17-norpimar- 8(9)-en-15-one, 3-formyl-1-hydroxy-8- methoxy anthraquinone and fistulic acid from pods of the plant. The young and old leaves of the plant contain highest amount of phenolic, flavonoid and proanthocyanidin contents. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is a lipophilic anthraquinone extensively found in medicinal plants (see chemical structure in Figure 2). It is a major bioactive compound reported in Cassia fistula for many therapeutic activities [4, 9, 10, 11, 12, 13, 14].
The aim of the present study was to recommend the suitable substituent for those drugs which are uses in huge amount. Stem bark or root of plant like Aragvadha (Cassia fistula) stem bark is mentioned as ingredient in some ayurvedic formulations. It is difficult to get huge amounts of roots from the big trees without uprooting. Removal of the stem bark from the trunk of the tree makes the plant weak and susceptible to damage by insects and natural elements. The usage of roots and barks of the trunk is therefore forbidden with an aim to conserve and protect the medicinal plants from extinction and make them available for future generation.
2. Material and methods
Cassia fistula Linn. stem bark and small branches were procured from Regional Ayurveda Research Institute, Central Council for Research in Ayurvedic Sciences, Ministry of AYUSH, Government of India, Gwalior Road, Jhansi, Uttar Pradesh.
2.1. Molecular docking studies
AutoDock Vina was used for the virtual screening of the phytochemical compounds and target proteins of L. donovani [15] and pancreatic lipase target protein. The target protein was changed into a macromolecule, which converted the atomic coordinates into pdbqt format. The grid box was selected around the crystal structure while other parameters were left as default for molecular docking by AutoDock Vina [16]. The binding affinity was used to analyze the results of molecular docking, and then all possible docked conformations were generated for different constituents. The detailed interactions, including their types such as hydrogen bonding, van der Waals, alkyl, pi-alkyl, and halogen interactions, between different constituents and the target proteins were analyzed by BIOVIA Discovery Studio [17]. The most favorable binding poses of the compounds were analyzed by choosing the lowest free energy of binding (ΔG).
2.2. Receptor and ligand preparation
The crystal structure of Nucleoside hydrolase, Sterol 24-c-methyl transferase and Pancreatic lipase were downloaded from PDB (ID: 5TSQ, 5WP4 and 1LPB) [18, 19]. The proteins were finally prepared by Discovery Studio keeping all of the parameters at default. The X, Y and Z coordinates in the 5TSQ proteins were 10.14 A°, 31.63 A° and 18.52 A° and radius was 5.0 A°. The X, Y and Z coordinates in the 5WP4 proteins were 10.23 A°, -1.58 A° and 33.49 A° and radius was 8.89 A°. The X, Y and Z coordinates in the 1LPB proteins were 9.82 A°, 23.49 A° and 50.87 A° and radius was 10 A°. The critical residues of the binding pockets were identified from the native catalytic pockets of the available crystal structure of proteins and Discovery Studio. The 3D structure of the constituents of the Stem bark and Small branches extract of C. fistula was retrieved from the PubChem database in SDF format [20]. Aloin A, Erucamide, and (22E)-Stigmasta-5,22-dien3-ol were sketched by Chem draw 16.0. The atomic coordinates of all of the ligands were changed to pdbqt setup using Open Babel GUI, an open-source chemical toolbox for the interconversion of chemical structures. MMFF94 was used for energy minimization.
2.3. Preliminary phytochemical analysis
Preliminary phytochemical screening results showed the presence or absence of certain phytochemicals in the Cassia fistula sample. 4g of the sample was taken in a glass stoppered 250 ml flask. 100 ml of absolute ethanol was added. The flasks were shaken occasionally for 6 hours and allowed to stand for 18 hours. The extract was filtered and evaporated to dryness. The same procedure was followed for aqueous extraction. The extracts were collected, dried, weighed and stored separately for preliminary phytochemicals screening. The tests performed using alcoholic extract and aqueous extract to different types of qualitative test for the identification of phytoconstituents present in the stem bark and small branches of Cassia fistula [21, 22, 23].
2.4. Physicochemical parameters
Physicochemical analysis was done to ascertain the quality of the raw material used in the study. Various type of physicochemical parameters performed like loss on drying, total ash content, acid insoluble ash, water extractive value, alcohol soluble extractive value and pH (10% w/v aqueous solution) [24, 25].
2.5. HPLC, LCMS and GCMS chemical profile
The chromatographic profiling of Cassia fistula was performed using three different techniques (HPLC, LC-MS & GC-MS) for the comparison between stem bark and small branches of the plant. The dried powdered stem bark and small branches of Cassia fistula were successively extracted with 200 ml of each solvent in the increasing order of polarity i.e. hexane, chloroform, ethyl acetate and ethanol by using soxhlet apparatus for 24 hrs. The extracts were evaporated to dryness under reduced pressure. The same procedure was followed for total ethanol extraction. The obtained extracts were collected, dried, weighed and stored separately for further studies. The different extracts of the Cassia fistula stem bark and small branches were weighed and dissolved in appropriate solvents and filtered through 0.22 μ membrane filters and used for HPLC profiling and compared under the same chromatographic conditions like Column type: ZORBAX Eclipse XBD- C18 (4.6 mm × 150 mm), 5μm particle size, Mobile Phase: Acetonitrile: Water (67:33), Acetonitrile: Phosphate Buffer (75:25), Acetonitrile: Phosphate Buffer (60:40), Acetonitrile: Phosphate Buffer (50:50), Acetonitrile: Water (60:40) used in the analysis of hexane, chloroform, ethyl acetate, ethanol, total ethanol extract, VWD Detector @ 254nm used in the detection of hexane, chloroform, ethyl acetate extract peak, DAD Detector @ 254nm used in the detection of ethanol extract and total ethanol extract peak, Flow Rate: 0.5 mL/min in the hexane and chloroform extract analysis and 1.0 mL/min in the ethyl acetate, ethanol and total ethanol extract analysis, Injection Volume: 10 μl in each analysis. For the LCMS analysis the test solution was prepared by dissolving 10 mg of each ethanol extracts of Cassia fistula stem bark and small branches in 2 ml methanol. It was filtered and sent for LC-MS analysis. For the GCMS analysis the volatile content of Cassia fistula stem bark and small branches were dissolved in 2 ml chloroform. It was filtered and sent for GC-MS analysis.
3. Result and discussion
3.1. Molecular docking studies
Compounds 3, 9, 12, 22, 32 and 37 have very good binding energy with the particular targets. The comparative molecular docking results of compounds 3, 9, 12, 22, 32 and 37 of Cassia fistula have more binding energy (kcal/mol) with target protein are tabulated in Table 1 and interaction tabulated in Figures 3, 4, and 5.
Table 1.
Molecular Docking Interaction of Abundant Medicinal Phytochemicals of C. fistula with the L. donovani Drug Target Proteins and Pancreatic lipase colipase.
| S.No. | Proteins | Ligands | Binding energy kcal/mol | Interacting residues |
|---|---|---|---|---|
| 1 | Sterol 24-C-methyltransferase (PDB ID 5WP4) | S-Adenosyl-L-Homocysteine | 10.1 | Ala 106, Arg 66, Asn 126, Asp 82, Asp 107, Gly 61, Leu 128, Phe 83, Trp 16, Tyr 131, Val 108 |
| 3 | 10.6 | Arg 9, Asp 82, Leu 128, Phe 83, Val 87, Trp 16 | ||
| 9 | 13.1 | Asp 82, Met 28, Phe 83, Trp 16 | ||
| 12 | 11.5 | Ala 106, Gly 61, Leu 128, Met 28, Phe 83, Trp 16, Trp 127, Tyr 131, Val 108 | ||
| 22 | 10.7 | Arg 9, Asp 82, Leu 128, Phe 83, Trp 16, Trp 127 | ||
| 32 | 11.2 | Asp 82, Leu 128, Phe 83, Trp 16, Val 108 | ||
| 37 | 11.4 | Asp 82, Leu 128, Met 28, Phe 83, Trp 16 | ||
| 2 | Nucleoside hydrolase (PDB ID 5TSQ) | β-D-ribofuranose | 6.4 | Asn 39, Asn 168, Asp 10, Asp 14, His 240, Tyr 225 |
| 3 | 9.3 | Ala 78, Ala 161, Arg 233, Asn 168, Asp 14, Asp 241, Gln 80, His 240, Met 152, Phe 167, Tyr 225 | ||
| 9 | 16.8 | Ala 78, Asp 10, Asp 14, Gln 80, His 82, His 240, Phe 167 | ||
| 12 | 16.0 | Ala 78, Gln 80, His 82, His 240, Ile 81, Phe 167, Tyr 225 | ||
| 22 | 8.9 | Ala 78, Ala 161, Arg 233, Asn 39, Asn 168, Ile 81, His 82, His240, Phe 167, Tyr 225 | ||
| 32 | 8.5 | Ala 78, Ala 161, Arg 233, Asn39, Asn 168, Ile 81, Phe 167, His 82, His 240, Tyr 225 | ||
| 37 | 11.5 | Ala 78, Gln 80, Asp 241, Arg 233, Glu 166 | ||
| 3 | Pancreatic Lipase Colipase (PDB ID 1LPB) | Methoxyundecylphosphinic Acid | 6.2 | Ala 178, Arg 256, Asp 79, His 263, Phe 215, Phe 77, Ile 209, Pro 180, Tyr 114 |
| 3 | 9.0 | Asp 79, His 263, Ile 78, Phe 215, Pro 180, Ser 152, Tyr 114 | ||
| 9 | 9.4 | Phe 215 | ||
| 12 | 8.9 | Phe 215, Tyr 114 | ||
| 22 | 9.4 | Ala 260, Asp 79, Arg 256, Leu 264, His 263, Phe 77, Ser 152, Tyr 114 | ||
| 32 | 9.5 | Asp 79, His 151, His 263, Phe 215, Tyr 114 | ||
| 37 | 16.8 | Ile 209, Pro 180, Tyr 114 |
Figure 3.
Binding pattern of C. fistula major chemical constituents with the Pancreatic lipase colipase. Two-dimensional (2D) and its significant interactions with (A) Butein, (B) Lup-20(29)-en-28-al, 3beta-hydroxy, (C) Betulin (D) (±) Naringenin, (E) Kaempferol, (F) Lupa-12,20(29)-dien-3-one.
Figure 4.
Binding pattern of C. fistula major chemical constituents with the Sterol 24-C-methyltransferase (SMT). Two-dimensional (2D) and its significant interactions with (A) Butein, (B) Lup-20(29)-en-28-al, 3beta-hydroxy, (C) Betulin (D) (±) Naringenin, (E) Kaempferol, (F) Lupa-12,20(29)-dien-3-one.
Figure 5.
Binding pattern of C. fistula major chemical constituents with the Nucleoside hydrolase (NH). Two-dimensional (2D) and its significant interactions with (A) Butein, (B) Lup-20(29)-en-28-al, 3beta-hydroxy, (C) Betulin (D) (±) Naringenin, (E) Kaempferol, (F) Lupa-12,20(29)-dien-3-one.
3.2. Physicochemical parameters
The comparative analysis results of physicochemical parameters for Cassia fistula stem bark and small branches are tabulated in Table 2. The results of all the parameters of stem bark comply with the Ayurvedic Pharmacopeia of India (API) standards.
Table 2.
Physicochemical parameters of Cassia fistula stem bark and small branches.
| S. No. | Test parameter | Results |
|
|---|---|---|---|
| Stem bark | Small branches | ||
| 1. | pH (10% w/v aqueous solution) | 6.90 | 7.60 |
| 2. | Total ash content (% w/w) | 7.46 | 2.69 |
| 3. | Acid insoluble ash (% w/w) | 0.26 | 0.01 |
| 4. | Water soluble extractive (% w/w) | 19.29 | 5.68 |
| 5. | Alcohol soluble extractive (% w/w) | 20.14 | 1.55 |
| 6. | Loss on drying at 105 °C (% w/w) | 8.56 | 8.14 |
pH of the small branches was found slightly higher as compared to stem bark and the percentage of other parameters like total ash content, acid insoluble ash, loss on drying at 105 °C, water soluble extractive and alcohol soluble extractive values were found fewer in the small branches as compare to stem bark of the Cassia fistula plant [24, 25].
3.2.1. Preliminary phytochemicals screening
The comparative preliminary phytochemicals screening results of aqueous and ethanol extracts of Cassia fistula stem bark and small branches are tabulated in Table 3. The results reveal the presence of similar phytochemicals in stem bark and small branches except flavonoids which were present in both extracts of stem bark and absent in both extracts of small branches [21, 22, 23].
Table 3.
Preliminary phytochemicals screening tests of Cassia fistula stem bark and small branches.
| S. No. | Phytochemical constituents | Results |
|||
|---|---|---|---|---|---|
| Stem bark |
Small branches |
||||
| Aqueous extract | Alcohol extract | Aqueous extract | Alcohol extract | ||
| 1. | Alkaloids | + | - | + | - |
| 2. | Coumarins | - | ++ | - | + |
| 3. | Flavonoids | + | +++ | - | - |
| 4. | Furanoids | +++ | +++ | - | + |
| 5. | Phenols | ++ | +++ | + | + |
| 6. | Quinones | +++ | +++ | + | ++ |
| 7. | Reducing sugars | ++ | +++ | + | - |
| 8. | Saponins | +++ | - | +++ | - |
| 9. | Sugars (Carbohydrate) | - | + | +++ | ++ |
| 10. | Tannins | ++ | ++ | ++ | + |
| 11. | Triterpenoids | - | +++ | - | +++ |
3.2.2. HPLC chromatographic profiling of Cassia fistula
The obtained residue weights and extractive values of extraction are given in Table 4.
Table 4.
Extractive values of Cassia fistula stem bark and small branches.
| S. No | Name of the solvent for extraction | Stem bark |
Small branches |
|||||
|---|---|---|---|---|---|---|---|---|
| Weight of sample (g) | Weight of extract (g) | Percentage of extract | Weight of sample (g) | Weight of extract (g) | Percentage of extract | |||
| 1. | Successive extraction | Hexane | 10.0108 | 0.0546 | 0.55 | 10.0128 | 0.0539 | 0.54 |
| Chloroform | 0.0469 | 0.47 | 0.0883 | 0.88 | ||||
| Ethyl acetate | 1.1537 | 11.52 | 0.0808 | 0.81 | ||||
| Ethanol | 1.2442 | 12.43 | 0.3595 | 3.59 | ||||
| 2. | Total ethanol | 10.0217 | 2.4911 | 24.86 | 10.0172 | 0.3387 | 3.38 | |
While comparing the HPLC chromatographic profiling of successive extracts of Cassia fistula, it was observed that 19 peaks in stem bark and 16 peaks in small branches of the samples were detected in hexane extracts, 10 peaks in stem bark and 10 peaks in small branches of the samples were detected in chloroform extracts, 06 peaks in stem bark and 10 peaks in small branches of the samples were detected in ethyl acetate extracts, 07 peaks in stem bark and 09 peaks in small branches of the samples were detected in ethanol extracts. In the comparison of total ethanol extracts, 07 peaks in stem bark and 09 peaks in small branches of the samples were detected.
It was observed that the number of peaks in stem bark and small branches of the plant sample were almost similar and the retention time of each peak in stem bark was coincide with the retention of small branches of the sample. Therefore, similarity was observed in stem bark and small branches of the Cassia fistula plant. The detailed peak identification and peak area results are shown in Figures 6, 7, 8, 9, and 10 and Tables 5, 6, 7, 8, and 9.
Figure 6.
HPLC profiling chromatogram of successive hexane extracts of Cassia fistula stem bark and small branches.
Figure 7.
HPLC profiling chromatogram of successive chloroform extracts of Cassia fistula stem bark and small branches.
Figure 8.
HPLC profiling chromatogram of successive ethyl acetate extracts of Cassia fistula stem bark and small branches.
Figure 9.
HPLC profiling chromatogram of successive ethanol extracts of Cassia fistula stem bark and small branches.
Figure 10.
HPLC profiling chromatogram of total ethanol extracts of Cassia fistula stem bark and small branches.
Table 5.
HPLC peaks details of successive hexane extracts of Cassia fistula stem bark and small branches.
| Stem bark |
Small branches |
||||||
|---|---|---|---|---|---|---|---|
| Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % | Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % |
| 1 | 1.589 | 149.50922 | 0.8897 | 1 | 1.580 | 103.48606 | 0.7698 |
| 2 | 1.718 | 139.68979 | 0.8313 | 2 | 1.708 | 90.54295 | 0.6735 |
| 3 | 1.882 | 200.25746 | 1.1917 | 3 | 1.847 | 184.42491 | 1.3718 |
| 4 | 2.247 | 1444.48193 | 8.5960 | - | - | - | - |
| 5 | 2.291 | 420.46875 | 2.5022 | - | - | - | - |
| 6 | 2.349 | 1856.65857 | 11.0488 | 4 | 2.352 | 3193.80786 | 23.7569 |
| 7 | 2.784 | 457.82062 | 2.7244 | 5 | 2.776 | 420.07623 | 3.1247 |
| 8 | 2.938 | 333.25076 | 1.9831 | - | - | - | - |
| - | - | - | - | 6 | 3.129 | 406.59952 | 3.0245 |
| 9 | 3.204 | 2033.92200 | 12.1037 | 7 | 3.191 | 1360.34729 | 10.1188 |
| - | - | - | - | 8 | 4.652 | 1159.55225 | 8.6252 |
| 10 | 4.936 | 2209.57007 | 13.1489 | 9 | 4.984 | 863.45996 | 6.4228 |
| 11 | 5.973 | 1354.29077 | 8.0593 | 10 | 5.963 | 3289.10571 | 24.4657 |
| 12 | 6.334 | 2420.41626 | 14.4037 | - | - | - | - |
| 13 | 7.053 | 1200.93823 | 7.1467 | - | - | - | - |
| 14 | 7.942 | 340.91782 | 2.0288 | 11 | 8.076 | 167.28749 | 1.2444 |
| 15 | 8.561 | 227.86253 | 1.3560 | 12 | 8.522 | 115.64956 | 0.8602 |
| 16 | 8.965 | 558.20044 | 3.3218 | 13 | 8.946 | 1330.86389 | 9.8995 |
| 17 | 9.968 | 173.10760 | 1.0301 | 14 | 10.051 | 290.90503 | 2.1639 |
| 18 | 10.759 | 749.83698 | 4.4622 | 15 | 10.713 | 233.54915 | 1.7372 |
| 19 | 11.473 | 532.97852 | 3.1717 | 16 | 11.424 | 234.07089 | 1.7411 |
| Total | 1.680424 | 100.0000 | Total | 1.344374 | 100.0000 | ||
Table 6.
HPLC peaks details of successive chloroform extracts of Cassia fistula stem bark and small branches.
| Stem bark |
Small branches |
||||||
|---|---|---|---|---|---|---|---|
| Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % | Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % |
| 1 | 2.686 | 1413.94092 | 17.5177 | 1 | 2.604 | 2169.96729 | 15.5223 |
| 2 | 2.870 | 2256.06689 | 27.9510 | 2 | 2.873 | 4167.65039 | 29.8122 |
| 3 | 3.068 | 3059.15527 | 37.9006 | 3 | 3.095 | 3431.93970 | 24.5495 |
| - | - | - | - | 4 | 3.246 | 2771.26514 | 19.8235 |
| 4 | 4.203 | 606.60406 | 7.5154 | 5 | 4.128 | 445.39230 | 3.1860 |
| - | - | - | - | 6 | 4.300 | 518.91888 | 3.7120 |
| - | - | - | - | 7 | 5.401 | 78.72145 | 0.5631 |
| 5 | 6.099 | 104.24109 | 1.2915 | 8 | 6.072 | 60.75570 | 0.4346 |
| 6 | 6.822 | 225.29892 | 2.7913 | 9 | 6.787 | 200.31689 | 1.4329 |
| 7 | 7.181 | 167.93848 | 2.0806 | 10 | 7.147 | 134.73520 | 0.9638 |
| 8 | 8.284 | 82.74257 | 1.0251 | - | - | - | - |
| 9 | 9.296 | 92.94195 | 1.1515 | - | - | - | - |
| 10 | 9.871 | 62.58374 | 0.7754 | - | - | - | - |
| Total | 8071.51389 | 100.0000 | Total | 1.39797e4 | 100.0000 | ||
Table 7.
HPLC peaks details of successive ethyl acetate extracts of Cassia fistula stem bark and small branches.
| Stem bark |
Small branches |
||||||
|---|---|---|---|---|---|---|---|
| Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % | Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % |
| 1 | 1.247 | 7609.18701 | 25.0800 | 1 | 1.252 | 1533.26746 | 14.2287 |
| 2 | 1.331 | 5945.04541 | 19.5949 | 2 | 1.355 | 1782.12549 | 16.5381 |
| 3 | 1.419 | 4751.75391 | 15.6618 | 3 | 1.425 | 1718.84949 | 15.9509 |
| 4 | 1.587 | 7001.36768 | 23.0766 | 4 | 1.631 | 1884.67175 | 17.4897 |
| 5 | 1.728 | 5003.45557 | 16.4914 | 5 | 1.734 | 2621.03247 | 24.3232 |
| - | - | - | - | 6 | 2.166 | 774.16815 | 7.1843 |
| 6 | 2.622 | 28.91096 | 0.0953 | 7 | 2.696 | 197.91785 | 1.8367 |
| - | - | - | - | 8 | 3.033 | 183.10434 | 1.6992 |
| - | - | - | - | 9 | 3.471 | 32.90925 | 0.3054 |
| - | - | - | - | 10 | 3.644 | 47.82133 | 0.4438 |
| Total | 3.033974 | 100.0000 | Total | 1.077594 | 100.0000 | ||
Table 8.
HPLC peaks details of successive ethanol extracts of Cassia fistula stem bark and small branches.
| Stem bark |
Small branches |
||||||
|---|---|---|---|---|---|---|---|
| Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % | Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % |
| 1 | 1.181 | 6042.50293 | 16.8212 | 1 | 1.199 | 1168.21667 | 12.4974 |
| 2 | 1.244 | 6150.87988 | 17.1229 | 2 | 1.255 | 920.84351 | 9.8510 |
| 3 | 1.344 | 6271.53662 | 17.4587 | 3 | 1.348 | 1409.40515 | 15.0775 |
| 4 | 1.637 | 1.72378e4 | 47.9868 | 4 | 1.642 | 2877.07544 | 30.7784 |
| - | - | - | - | 5 | 1.794 | 1562.69836 | 16.7174 |
| - | - | - | - | 6 | 2.014 | 1131.03308 | 12.0996 |
| 5 | 2.750 | 163.38998 | 0.4548 | 7 | 2.882 | 136.38690 | 1.4590 |
| 6 | 3.571 | 28.72318 | 0.0800 | - | - | - | - |
| 7 | 4.580 | 27.19285 | 0.0757 | 8 | 4.579 | 31.10106 | 0.3327 |
| - | - | - | - | 9 | 4.849 | 110.95047 | 1.1869 |
| Total | 3.592204 | 100.0000 | Total | 9347.71065 | 100.0000 | ||
Table 9.
HPLC peaks details of total ethanol extracts of Cassia fistula stem bark and small branches.
| Stem bark |
Small branches |
||||||
|---|---|---|---|---|---|---|---|
| Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % | Peak No. | Ret. Time [min] | Area [mAU∗s] | Area % |
| 1 | 1.150 | 1.41661e4 | 34.8629 | 1 | 1.162 | 9786.41602 | 19.8888 |
| 2 | 1.250 | 8181.31152 | 20.1343 | 2 | 1.265 | 2775.98022 | 5.6416 |
| - | - | - | - | 3 | 1.360 | 6830.32959 | 13.8812 |
| 3 | 1.536 | 1.09232e4 | 26.8823 | - | - | - | - |
| 4 | 1.665 | 6782.29297 | 16.6913 | 4 | 1.638 | 1.38504e4 | 28.1480 |
| - | - | - | - | 5 | 1.792 | 1.41270e4 | 28.7100 |
| 5 | 2.104 | 345.49762 | 0.8503 | - | - | - | - |
| 6 | 2.462 | 151.43039 | 0.3727 | - | - | - | - |
| 7 | 2.929 | 83.77605 | 0.2062 | 6 | 2.922 | 741.33044 | 1.5066 |
| - | - | - | - | 7 | 3.416 | 149.57335 | 0.3040 |
| - | - | - | - | 8 | 3.674 | 637.74231 | 1.2961 |
| - | - | - | - | 9 | 4.823 | 306.90524 | 0.6237 |
| Total | 4.063364 | 100.0000 | Total | 4.920564 | 100.0000 | ||
3.2.3. LC-MS chromatographic profiling of Cassia fistula
The LC-MS profiling chromatograms of Cassia fistula stem bark and small branches are given in Figure 11 and retention time, name of compound, molecular formula, molecular weight and maximum peak area are given in Table 10 (see chemical structure in Figure 2). From the report of LC-MS, it was observed that, the LC-MS analysis of active compounds showed similarity in the both extracts of stem bark and small branches of the plant [26, 27].
Figure 11.
LC-MS Chromatogram of ethanol extracts of Cassia fistula stem bark and small branches.
Table 10.
LC-MS Peak details of ethanol extracts of Cassia fistula stem bark and small branches.
| Stem bark | |||||
|---|---|---|---|---|---|
| Peak No. | Ret. Time | Name of the compound | Molecular Formula | Molecular Weight | Area Maximum |
| 1 | 1.072 | Betaine | C5H11NO2 | 117.0787 | 15260766.1 |
| 2 | 1.191 | Nicotinic acid | C6H5NO2 | 123.0318 | 393452.2136 |
| 3 | 2.509 | (-)-Epigallocatechin | C15H14O7 | 306.073 | 464997.9638 |
| 4 | 7.388 | Epicatechin | C15H14O6 | 290.0781 | 2571077.704 |
| 5 | 11.094 | Butein | C15H12O5 | 272.0676 | 411811.0544 |
| 6 | 13.95 | 4-Methoxycinnamic acid | C10H10O3 | 178.0627 | 722746.2581 |
| 7 | 14.321 | Aloin A | C21H22O9 | 418.1255 | 969916.0451 |
| 8 | 14.432 | Quercetin | C15H10O7 | 302.0421 | 265656.2337 |
| 9 | 14.855 | Luteolin | C15H10O6 | 286.047 | 745514.2985 |
| 10 | 15.004 | Rhamnetin | C16H12O7 | 316.0576 | 299669.8381 |
| 11 | 20.072 | 4-Hydroxycoumarin | C9H6O3 | 162.0313 | 860891.3235 |
| 12 | 20.11 | Caffeic acid | C9H8O4 | 180.0418 | 2391754.538 |
| 13 | 20.925 | (E)-parinaric acid | C18H28O2 | 276.2083 | 250512.54 |
| 14 | 21.633 | Abietic acid | C20H30O2 | 302.2238 | 194720.7413 |
| 15 | 22.935 | Oleanolic acid | C30H48O3 | 438.3488 | 231825.8614 |
| 16 | 23.856 | Lup-20(29)-en-28-al, 3beta-hydroxy- | C30H48O2 | 440.3647 | 194223.063 |
| 17 | 24.326 | Nervonic acid | C24H46O2 | 366.3489 | 155724.8206 |
| 18 | 24.488 | Erucamide | C22H43NO | 337.3335 | 15176586.26 |
| 19 | 26.046 | Betulin | C30H50O2 | 442.3803 | 399168.5256 |
| 20 |
26.662 |
(22E)-Stigmasta-5,22-dien3-ol |
C29H48O |
412.3692 |
26940935.26 |
|
Small branches | |||||
|
Peak No. |
Ret. Time |
Name of the compound |
Molecular Formula |
Molecular Weight |
Area Maximum |
| 1 | 1.01 | 3-Hydroxypyridine | C5H5NO | 95.03695 | 301739.2746 |
| 2 | 1.036 | Betaine | C5H11NO2 | 117.07878 | 72844002.46 |
| 3 | 1.19 | Nicotinic acid | C6H5NO2 | 123.03192 | 2574509.917 |
| 4 | 2.504 | 4-Piperidone | C5H9NO | 99.06827 | 2786020.512 |
| 5 | 6.456 | 8-Hydroxyquinoline | C9H7NO | 145.05255 | 845239.8029 |
| 6 | 7.575 | Adipic acid | C6H10O4 | 146.05771 | 673583.4342 |
| 7 | 9.276 | Vanillin | C8H8O3 | 152.04697 | 1048031.784 |
| 8 | 10.336 | Quinine | C20H24N2O2 | 324.18294 | 401047.9444 |
| 9 | 10.473 | β-Asarone | C12H16O3 | 208.10939 | 740911.8038 |
| 10 | 10.893 | (E)-Ferulic acid | C10H10O4 | 194.0576 | 836774.0259 |
| 11 | 11.431 | Butein | C15H12O5 | 272.06804 | 660531.6233 |
| 12 | 12.455 | (±)-Naringenin | C15H12O5 | 272.06804 | 136131.3751 |
| 13 | 13.517 | 7-Ethoxycoumarin | C11H10O3 | 190.06275 | 4987298.85 |
| 14 | 13.605 | Apigenin | C15H10O5 | 270.05223 | 266768.5365 |
| 15 | 13.945 | 4-Methoxycinnamic acid | C10H10O3 | 178.06282 | 1101857.784 |
| 16 | 14.678 | (E)-4-Methoxycinnamic acid | C10H10O3 | 178.06282 | 106125.6885 |
| 17 | 15.588 | Kaempferol | C15H10O6 | 286.04717 | 320138.5085 |
| 18 | 16.847 | Aloe-emodin | C15H10O5 | 270.05223 | 181000.967 |
| 19 | 18.293 | (+)-[6]-Gingerol | C17H26O4 | 294.18236 | 274825.6524 |
| 20 | 19.738 | (+)-ar-Turmerone | C15H20O | 216.15097 | 168543.0028 |
| 21 | 19.947 | 6-Gingerol | C17H26O4 | 294.18236 | 451219.7348 |
| 22 | 20.028 | 10-Gingerol | C21H34O4 | 350.24452 | 212893.1035 |
| 23 | 20.152 | Caffeic acid | C9H8O4 | 180.04178 | 1885849.78 |
| 24 | 20.153 | 4-Hydroxycoumarin | C9H6O3 | 162.03131 | 998683.3751 |
| 25 | 20.572 | (E)-parinaric acid | C18H28O2 | 276.20818 | 7882595.386 |
| 26 | 22.208 | (+/-)-Methoprene | C19H34O3 | 310.24982 | 3161195.753 |
| 27 | 23.708 | Oleanolic acid | C30H48O3 | 438.34877 | 1783708.336 |
| 28 | 23.812 | Lup-20(29)-en-28-al, 3beta-hydroxy- | C30H48O2 | 440.36464 | 802911.4302 |
| 29 | 23.833 | Asiatic acid | C30H48O5 | 488.34922 | 335493.9399 |
| 30 | 23.851 | Lupa-12,20(29)-dien-3-one | C30H46O | 422.35384 | 874681.9728 |
| 31 | 24.482 | Erucamide | C22H43NO | 337.33344 | 23537542.84 |
| 32 | 26.037 | Betulin | C30H50O2 | 442.37984 | 1024698.489 |
| 33 | 26.663 | (22E)-Stigmasta-5,22-dien3-ol | C29H48O | 412.36937 | 130035124.4 |
3.2.4. GC-MS chromatographic profiling of Cassia fistula
The detailed peak identification shown in Figure 12 and retention time, compound name, molecular weight and maximum peak area, are given in Table 11. The significant similarities have been observed in the GC-MS chromatographic profiling of volatile content of the Cassia fistula stem bark and small branches [26, 27].
Figure 12.
GC-MS Chromatogram of volatile content of Cassia fistula stem bark and small branches.
Table 11.
GC-MS Peak details of volatile content of Cassia fistula stem bark and small branches.
| Stem bark | |||||
|---|---|---|---|---|---|
| Peak No. | Ret. Time | Name of the compound | Molecular Formula | Molecular Weight | Area Maximum |
| 1 | 14.683 | Azulene | C10H8 | 128 | 1,084,225.0 |
| 2 | 14.753 | Naphthalene | C10H8 | 128 | 521,754.2 |
| 3 | 14.853 | Azulene | C10H8 | 128 | 738,852.2 |
| 4 | 14.913 | 1h-Indene, 1-Methylene | C10H8 | 128 | 688,024.4 |
| 5 | 26.393 | 2-Nitro-5-Aminobenzoic acid | C7H6O4N2 | 182 | 580,434.5 |
| 6 | 26.923 | 6-Methylquinolinic acid diamide | C8H9O2N3 | 179 | 260,495.9 |
| 7 |
26.968 |
Benzoic acid, 4-(acetylamino)-2-nitro |
C9H8O5N2 |
224 |
304,433.7 |
|
Small branches | |||||
|
Peak No. |
Ret. Time |
Name of the compound |
Molecular Formula |
Molecular Weight |
Area Maximum |
| 1 | 15.103 | Naphthalene | C10H8 | 128 | 464,276.4 |
| 2 | 15.203 | Naphthalene | C10H8 | 128 | 1,027,875.1 |
| 3 | 15.313 | Naphthalene | C10H8 | 128 | 378,814.3 |
| 4 | 15.363 | Azulene | C10H8 | 128 | 339,622.5 |
| 5 | 23.066 | 1-Butanol, 4-Butoxy | C8H18O2 | 146 | 617,018.8 |
| 6 | 23.111 | Sulfurous acid, 2-propyl tridecyl ester | C16H34O3S | 306 | 272,625.6 |
| 7 | 23.162 | (2s,3s)-(-)-3-Propyloxiranemethanol | C6H12O2 | 116 | 261,251.5 |
| 8 | 23.592 | 1-Butanol, 4-Butoxy | C8H18O2 | 146 | 421,100.4 |
| 9 | 24.242 | Di-N-Decylsulfone | C20H42O2S | 346 | 1,948,454.4 |
3.2.5. Quantitative estimation of rhein biomarker compound in Cassia fistula stem bark and small branches by HPLC
(i) Test solution:
The residues obtained from ethanol extracts of stem bark and small branches of Cassia fistula were accurately weighed in triplicate and dissolved in HPLC grade methanol using 5 ml volumetric standard flasks, filtered through 0.22 μ membrane filters and used for HPLC analysis.
(ii) Standard solution:
1.0 mg of rhein reference standard was accurately weighed and dissolved in HPLC grade methanol and the volume was made up to 5 ml to obtain 0.20 mg/ml rhein stock solution.
(iii) Chromatographic conditions:
| Column | - | ZORBAX Eclipse XBD- C18 |
| (4.6 mm × 150 mm), 5μm particle size | ||
| Detection | - | VWD Detector at 247 nm |
| Mobile phase | - | Acetonitrile: Phosphate Buffer (55:45) |
| Flow rate | - | 1.2 ml/min |
| Injection volume | - | 10 μl |
| Retention time | - | 3.076 |
| Mode of operation | - | Isocratic elution |
(iv) Calibration curve:
0.20 mg/ml rhein stock solution was appropriately diluted further to obtained a concentration of 0.05, 0.025, 0.0125, 0.00625 mg/ml of rhein. Each of the standard solution was run through HPLC system and recorded the respective peak areas. Calibration curve was established for peak area vs concentration of rhein applied shown in Figure 13.
Figure 13.
HPLC Chromatogram of Rhein Standard and Calibration curve.
(v) Estimation of Rhein:
Injected 10 μl each of the test solution to HPLC system. Record the chromatogram and determine the area of the peak of the test solution corresponding to that rhein as described above from the calibration curve. Calculated the amount of rhein present in the residues extracted in ethanol for each test sample of Cassia fistula stem bark and small branches is given in Figure 14 and Table 12.
Figure 14.
Estimation of Rhein in Ethanol extracts of Cassia fistula stem bark and small branches.
Table 12.
Estimation of Rhein in ethanol extracts of Cassia fistula stem bark and small branches.
| S. No. | Name of extract | Rhein (% w/w) |
|||
|---|---|---|---|---|---|
| Stem bark |
Small branches |
||||
| Results | Mean | Result | Mean | ||
| 1. | Ethanol extract | 0.0096 | 0.0084% | 0.0252 | 0.0257% |
| 0.0077 | 0.0259 | ||||
| 0.0080 | 0.0261 | ||||
∗Percentage of results was given from the means of triplicates for stem bark and small branches samples of optimized extracts of ethanol.
The results obtained from HPLC analysis shows that stem bark contains 0.0084% and small branches having 0.0257% of rhein in Cassia fistula.
4. Conclusion
The results obtained from HPLC analysis shows that stem bark contains 0.0084% and small branches having 0.0257% of rhein in Cassia fistula.
Compounds 3, 9, 12, 22, 32 and 37 gave excellent interaction with 5WP4, 5TSQ and 1LPB target protein in molecular docking. Compounds 3, 9 and 12 obtained in stem bark and small branches of the plant while compounds 22, 32 and 37 were present in small branches only. So small branches are more efficiently work as Antileishmanial drug as well as Pancreatic lipase inhibitor than stem bark on the basis of molecular docking. Similarities in different chromatographic profiles, phytochemical analysis of various extracts of stem bark and small branches and quantitative estimation of rhein suggests that, the small branches may have almost similar active chemical constituents like stem bark. Hence, the study provides the base for further study to recommend small branches in place of stem bark and vice-versa after comparison and confirmation of the same for pharmacological activities.
Declarations
Author contribution statement
Ajay Kumar Meena: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
R. Ilavarasan; Ravindra Singh; N. Srikanth; K. S. Dhiman: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.
Vikas Ojha: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Ayyam Perumal: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data availability statement
Data will be made available on request.
Declaration of interest's statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
The authors are very grateful to Director General and Deputy Director General, CCRAS New Delhi for providing encouragement and facilities for carrying out this work.
References
- 1.Pawar A.V., Patil S.J., Killedar S.G. Uses of Cassia fistula Linn as a medicinal plant. Int. J. Adv. Res. Dev. 2017;2(3):85–91. [Google Scholar]
- 2.Gupta R.K. first ed. CBS Publishers & Distributors; 2010. Medicinal & Aromatic Plants; pp. 116–117. [Google Scholar]
- 3.Gupta M., Mazumder U.K., Rath N., Mukhopadhyay D.K. Antitumor activity of methanolic extract of Cassia Fistula L seed against Ehrlich Ascites carcinoma. J. Ethanopharmacol. 2000;72:151–156. doi: 10.1016/s0378-8741(00)00227-0. [DOI] [PubMed] [Google Scholar]
- 4.Anonymous . Volume 5. Government of India Publication; New Delhi: 2001. pp. 8–9. (Ayurvedic Pharmacopoeia of India). Part 1. [Google Scholar]
- 5.Mahmoud, et al. Facile synthesis of reduced graphene oxide by Tecoma stans extracts for efficient removal of Ni (II) from water: batch experiments and response surface methodology. Sustain. Environ. Res. 2022;32:22. [Google Scholar]
- 6.Trease G.E., Evans W.C. Vol. 13. Alden Press Oxford; 1989. pp. 268–298. (Text Book of Pharmacognosy). [Google Scholar]
- 7.Ching Kuo Lee, Ping Hung Lee, et al. The chemical constituents from the aril of Cassia Fistula L. J. Chin. Chem. Soc. 2001;48:1053–1058. [Google Scholar]
- 8.Theesan B., Vidushi S.N., Okezie I.A. Phytochemical constituent of Cassia fistula. Afr. J. Biotechnol. 2005;4:1530–1540. [Google Scholar]
- 9.Kuo Yueh-Hsiung, Lee Ping-Hung, Wein Yung-Shun. Four new compounds from the seeds of Cassia fistula. J. Nat. Prod. 2002;65:1165–1167. doi: 10.1021/np020003k. [DOI] [PubMed] [Google Scholar]
- 10.Barthakur N.N., Arnold N.P., Alli I. The Indian Labernum (Cassia fistula L.) fruit: an analysis of its chemical constituents. Plant Foods Hum. Nutr. 1995;47:55–62. doi: 10.1007/BF01088167. [DOI] [PubMed] [Google Scholar]
- 11.Sujogya K.P., Padhi L.P., Mohanty G. Antibacterial activities and phytochemical analysis of Cassia fistula (linn.) Leaf. J. Adv. Pharm. Technol. Research. 2011;2(1):62–67. doi: 10.4103/2231-4040.79814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Verma S. Pharmacological review on Cassia fistula Linn (amaltas) Int. J. Pharmaceut. Chem. Biol. Sci. 2016;6(3):332–335. [Google Scholar]
- 13.Kulkarni A., Govindappa M., Channabasava Chandrappa C.P., Ramachandra Y.L., Prasad S.K. Phytochemical analysis of Cassia fistula and it’s in vitro antimicrobial, antioxidant and anti-inflammatory activities. Adv. Med. Plant Res. 2015;3(1):8–17. [Google Scholar]
- 14.Biji C.V. Plant drug analysis - a comparative analysis of Cassia fistula. Int. J. Appl. Res. Technol. 2017;2(1):60–72. [Google Scholar]
- 15.Dallakyan S., Olson A.J. Small-molecule library screening by docking with PyRx. Methods Mol. Biol. 2015;1263:243–250. doi: 10.1007/978-1-4939-2269-7_19. [DOI] [PubMed] [Google Scholar]
- 16.Trott O., Olson A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010;31:455–461. doi: 10.1002/jcc.21334. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Shamsi A., Mohammad T., Anwar S., AlAjmi M.F., Hussain A., Rehman M.T., Islam A., Hassan M.I. Glecaprevir and Maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy. Biosci. 2020;40:450. doi: 10.1042/BSR20201256. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Tabrez S., Rahman F., Ali R., Abdulaziz S.A., Mohammed A.B., Ayed A.F., Alaidarous M.A., Banawas S., Aziz B.D.A., Rub A. Assessment of the Antileishmanial potential of Cassia fistula leaf extract. ACS Omega. 2021;6:2318–2327. doi: 10.1021/acsomega.0c05629. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Wilson I. 2011. Genome Biology12; p. I18. [Google Scholar]
- 20.Kashif M., Tabrez S., Husein A., Arish M., Kalaiarasan P., Manna P.P., Subbarao N., Akhter Y., Rub A. Identification of novel inhibitors against UDP-galactopyranose mutase to combat leishmaniasis. J. Cell. Biochem. 2018;119:2653–2665. doi: 10.1002/jcb.26433. [DOI] [PubMed] [Google Scholar]
- 21.Kokate C.K., Purohit A.R., Gokhale S.B. 42nd edition. Nirali Prakashan Pune; 2008. Pathway to Screen Phytochemical Nature of Natural Drugs. Pharmacognosy; pp. 56–61. [Google Scholar]
- 22.Parekh J., Chanda S. Antibacterial and phytochemical studies on twelve species of Indian medicinal plants. Afr. J. Biomed. Res. 2007;10:175–181. [Google Scholar]
- 23.Trease G.E., Evans W.C. eleventh ed. Bailliere Tindall; London: 1989. Pharmacognosy; pp. 45–50. [Google Scholar]
- 24.Ayurvedic Pharmacopoeia India. first ed. Volume VII. Department of AYUSH, Ministry of Health and Family Welfare, Government of India; New Delhi (India): 2013. Part - 1. [Google Scholar]
- 25.World Health Organization . 2002. Quality Control Methods for Medicinal Plants, Geneva; pp. 28–31. [Google Scholar]
- 26.Gopalakrishnan S., Vadivel E. GC-MS analysis of some bioactive constituents of Mussaenda frondosa Linn. Int. J. Pharma Bio Sci. 2011;2(1):313–320. [Google Scholar]
- 27.Murty V.K., Rao T.V.P., Venkataeswaralan V. Vol. 23. 1967. Chemical Examination of cassia Fistula; p. 515. [Google Scholar]
Associated Data
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Data Availability Statement
Data will be made available on request.