EVALUATION OF PHYTOCHEMICAL ANALYSIS OF CALLUS TISSUE FROM INTERNODE EXPLANTS OF TRADITIONAL MEDICINAL PLANTS OF JATROPHA GOSSYPIIFOLIA LINN.HTML Full Text
EVALUATION OF PHYTOCHEMICAL ANALYSIS OF CALLUS TISSUE FROM INTERNODE EXPLANTS OF TRADITIONAL MEDICINAL PLANTS OF JATROPHA GOSSYPIIFOLIA LINN.
Kalaiyarasan and N. Hariram *
Department of Biotechnology, Kalasalingam Academy of Research Education, Krishnankoil, Tamil Nadu, India.
ABSTRACT: Jatropha gossypiifolia Linn. (Euphorbiaceae) is an ornamental shrub extensively used in traditional medicine for various ailments. In view of its potential medicinal properties, an attempt has been undertaken to devise an efficient protocol for in-vitro propagation from the internodes explants of J. gossypiifolia for a rapid and large-scale production as well as to study the phytochemical composition by gas chromatography and mass spectrometry (GC-MS). Further, the petroleum ether and ethyl acetate extracts of callus tissue were subjected to thin-layer chromatography (TLC) as well as for synthesizing silver and gold nanoparticles. The five bands resolved by TLC chromatogram were characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and screened for antimicrobial and antioxidant activities. Internode region of the one-year-old plant was used as explants. Internodal explants are cultured on Murashige and Skoog (MS) medium containing Naphthalene acetic acid (NAA) and 2,4-Dichlorophenoxy acetic acid (2,4-D) both at a concentration of 1.0 mg/L-1 induced the formation of callus between 14-18 days. This combination produced mature cream-colored calli by day 35. Explants growing on MS medium fortified with 2,4-D and benzyl amino purine (BAP), both a concentration of 2.0 mg/L-1 for 35 days, revealed compact light green calli due to greenish pigmentation. Silver and gold nanoparticles as well as the 3rd and 4th bands of TLC by using the ethyl acetate extracts of callus tissue, revealed antibacterial activity. The 3rd and 4th bands of TLC bands obtained by using petroleum ether and ethyl acetate extracts of callus tissue revealed antioxidant activity. A total of 31 compounds were obtained in GC-MS analysis, which belongs to different chemical classes. Among them, phytol, hexadecanoic acid, squalene, 9,12,15-Octadecatrienoic acid, 2-(acetyloxy)-1-[(acetyloxy)methyl]ethyl ester, (Z,Z,Z)-, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, 9-Octadecenoic acid (Z)-, 2,3-bis(acetyloxy)propyl ester and phthalic acid were some of the major compounds. The presence of these compounds in the callus tissue indicates that they are promising candidates for therapeutic use.
Keywords: MS medium, BAP, Jatropha, NAA, Antibacterial, GC-MS
INTRODUCTION: Jatropha gossypiifolia Linn. (Euphorbiaceae) is widely distributed all over the tropics and subtropics of Asia, Africa, Central and South America 1. J. gossypiifolia is popularly known as “bellyache bush”.
It is so named as it causes bellyache bush poisoning and eventually death in humans and animals, when the poisonous fruits which contain the toxic substance, tox albumin are eaten.
The roots, stem, and leaves of J. gossypiifolia have been used to treat various ailments, including intermittent fevers, stomachache, toothache, swollen mammae, eczema, cancer, carbuncles, dysentery, dyspepsia, venereal disease, and for bites of venomous animals 1, 2, 3, 4. The decoction of the leaf is used for itches, boils, burns, sprains, sores, swelling, eczema, and wound healing 5.
The leaves are also used as blood purifiers 2. Fresh leaf extract, as well as stem sap, are used to stop bleeding from the nose and to treat skin diseases 6. Pharmacological studies with different parts of J. gossypiifolia are shown to possess antibiotic, antioxidant, anti-inflammatory, antihypertensive, antimicrobial, anticholinesterase, hepatoprotective, insecticidal, pesticide properties, and antidote for poisoning 7, 8, 9.
Anti-inflammatory activity was manifested by the inhibition of acute carrageenan-induced paw edema and cotton pellet-induced granuloma formation in rats administered orally with the ethanolic extract of leaves of J. gossypiifolia 10. Diterpenoids (atrogossones) from J. gossypiifolia exhibited antiproliferative activity by inducing G2/M arrest and apoptosis in RKO colon cancer cells 11.
Other diterpenoids like falodone and jatrophone isolated from the roots of J. gossypiifolia also revealed antiproliferative activity in A549 (lung cancer cell line) and Hep G2 1886 (colon cancer cell line), respectively 12, 13. Extracts obtained from different parts of J. gossypiifolia and the diterpene jatrophenone have shown antimicrobial activity against various bacterial and fungal strains, including Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, Aspergillus fumigatus, A. flavus, Salmonella typhi, and Streptomyces pyogene 14, 15. The antioxidant properties of J. gossypiifolia extracts were reflected from the significant hydroxyl and superoxide radical scavenging activities and lipoxygenase inhibitory activity 16.
Phytochemical composition of the plant revealed the presence of flavonoids such as apigenin, vitexin, and isovitexin 17, lignins such as gossypifan, gossypilin, gossypidien, jatroiden, jatrodien etc. 18, The plant also contains saponins, tannins and phenolic acids 19. Plant growth regulators play a significant role in callus formation, each having a well-defined effect on callus induction, growth, and development 20, 21, 22. Cytokinins such as 2,4-Dichlorophenoxy acetic acid (2,4-D), benzyl amino purine (BAP), kinetin and auxins like Naphthalene acetic acid (NAA), Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA) have been used for regeneration. Among them, BAP at a concentration of 2.0 mg/L-1 was found to be more effective in shoot bud induction.
Besides, certain media additives like adenine sulphate, glutamine, and activated charcoal promoted a high frequency of multiple shoot proliferation (90%) within 15-20 days. Regeneration of hypocotyls, petiole, and leaf explants was observed when cytokinins like zeatin, kinetin, and N6 benzyladenine either singly or in combination with IBA were used 23.
In spite of the vast medicinal potentialities of J. gossypiifolia, efforts have not been taken to conserve this species from genetic extinction due to overexploitation. This continuous exploitation necessitates to employ in-vitro regeneration to produce this species for extraction of secondary metabolites for commercial purposes. Further, even though a number of reports are available to delineate in-vitro propagation of callus from other Jatropha species, J. gossypiifolia has been scarcely studied for callus formation. Therefore, an attempt has been made to provide a rapid regeneration protocol of this potent medicinal plant from internodal cuttings. Studies were also undertaken to evaluate the phytochemicals present in the callus tissue by gas chromatography and mass spectrometry (GC-MS) and thin layer chromato-graphy (TLC) and examine the anti-oxidant and antibacterial activities of gold and silver nanoparticles prepared using the callus of J. gossypiifolia.
MATERIALS AND METHODS:
Source and Sterilization of J. gossypiifolia Explants: Jatropha gossypiifolia plants grown in the Kalasalingam University Campus were used as a source of explants. The young internodal region of 3rd and 4th node from the apex were collected from one-year-old plants. The explants were washed under running water and then with a soap solution. The explants were surface sterilized with mercuric chloride (0.1%) for 5 min, and rinsed with double distilled water five times.
Culture Conditions of J. gossypiifolia Explants: The present study is focused on examining the influence of BAP and NAA on callus induction and growth of J. gossypiifolia explants. Tissue culture media were prepared according to the method of Murashige and Skoog (1962). The media contained sucrose (3%) and agar (0.8%) as solidifying agents. Internodes were excised aseptically into small pieces of 1 × 1 cm and inoculated on 24 basal medium for callus initiation. After a week, these explants were transferred to media containing phytohormones such as BAP (2 mg L-1) and NAA (1 mg L-1), 2,4-D (2 mg L-1) for regeneration. The pH of the media was set 5.6 ± 0.1. Media was steam-sterilized in an autoclave under 15 psi at 121 ºC for 20 min. Growth conditions were maintained at 27 °C with 16/8 h light-dark photoperiod with lux intensity of 3000 by cool white fluorescent tubes (Philips, India).
Preparation of Callus Tissue Extracts: Thirty-five-day-old mature callus tissue was extracted with petroleum ether and ethyl acetate for 8 h and concentrated using a rotary evaporator, and stored in a desiccator until use.
Synthesis of silver and Gold Nanoparticles using Callus Tissue: Silver and gold nanoparticles were synthesized by mixing 10 ml of callus tissue extract with 100 ml of 1 mM aqueous solution of silver and gold nitrates separately with constant stirring at room temperature.
The mixtures were heated at 60 °C and stored in the dark for 24 h. The change of colour of the solution was inspected visually. The reduction of Ag+ to Ag0 was confirmed by the colour change of solution from colourless to brown.
Characterization of Silver and Gold Nano-particles: Silver and gold nanoparticles were characterized by SEM analysis. In order to carry out SEM analysis, the solution containing silver nanoparticles was centrifuged at 10,000 rpm for 20 min and a drop of the sample was loaded on a stub and SEM analysis was then performed using the Scanning Electron Microscope (JSM-6360, JEOL). Compositional analysis and the confirmation of the presence of elemental silver was carried out through Energy-dispersive X-ray Spectroscopy (EDS) using the SEM equipped with an EDX attachment (Carl Zeiss, Germany).
TLC Method: TLC is an analytical technique used to separate a mixture of crude substances into its individual compounds. In the present study, an aliquot of petroleum ether and ethyl acetate extracts were spotted on TLC silica gel plates (10 × 15 cm) separately. The plates were developed using hexane and ethyl acetate (8:2) as the mobile phase.
After the run was over, the plates were visualized under visible and UV light (240 and 300 nm). In the present study, the chromatogram revealed five distinct bands. The separated bands were marked and their retention factor (Rf) values were calculated and recorded. The chromatogram was then photographed.
The five bands obtained using petroleum ether and ethyl acetate extracts were scratched off separately, dissolved in alcohol, filtered, concentrated, and then used for the antioxidant assay. For antibacterial assay, the TLC products obtained from ethyl acetate extract alone were used.
FTIR Analysis: FTIR spectral analysis was performed by TLC bioautographic method using the recovered concentrates of five TLC bands obtained from the ethyl acetate extracts of 35-day old callus tissue. FTIR spectral analysis was performed in FTIR instrument (IRTRACER-100, Shimadzu, Japan) with PC-based software and data processing. As mentioned earlier, each band of TLC was removed separately, mixed with absolute ethanol, filtered, and concentrated. The recovered concentrates of each band were mixed with KBr (100 mg) and made into pellets by applying pressure.
Antibacterial Activity: Antibacterial activity was screened by two ways viz., TLC bioautographic method as well as by silver and gold nanoparticles obtained from the ethyl acetate extract of 35-day old callus tissue. All bacterial strains Staphylococcus aureus (MTCC 96) (Gram-positive bacteria) Escherichia coli (MTCC 1652), and Pseudomonas aeruginosa (MTCC 2453) (Gram-negative bacteria) were provided by the Microbiology laboratory of the Meenakshi Mission Hospital. All bacterial strains were subcultured on nutrient agar for 24 h favorable to their growth prior to testing.
In the present study, the TLC chromatogram obtained by using ethyl acetate fraction of callus tissue showed five bands. Each of the five bands of TLC was scratched off separately, mixed with ethanol, filtered, and concentrated. The recovered concentrates of each band were then tested for antibacterial activity by the agar well diffusion method. at 37 °C.
Muller Hinton agar (MHA) plates were prepared by pouring 15 ml of molten media into sterile petriplates and then solidified. Wells of 7 mm diameter were made on the agar with cork borer. Silver and gold nanoparticles (20 μl) prepared using ethyl acetate callus extract were added into wells made in agar plates and allowed to diffuse for 5 min. DMSO alone added to the wells, served as test control, while wells loaded with ampicillin (20 μg/ml) served as a positive control. The plates were kept inverted in an incubator set at 37 ºC for 24 h. The growth inhibitory zones around the wells were measured using a ruler in centimeters and taken as an indication of antimicrobial activity. The assays were carried out in triplicates.
Determination of Total Antioxidant Activity: Antioxidant activity was evaluated by TLC bioautographic method. Each of the five bands of TLC chromatogram obtained by using petroleum ether and ethyl acetate extract was removed separately, mixed with absolute ethanol, filtered, and concentrated. The recovered solutions of each band was then tested for total antioxidant activity
The total antioxidant assay is based on the reduction of molybdenum (VI) to green phosphate/molybdenum (V) complex at an acidic pH by the sample analyte 25. An aliquot from the recovered solutions (0.3 ml) of each TLC band was added to 1 ml of reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate, heated to 95 °C for 90 min and then cooled to room temperature. The intensity of the green colour developed was read at 695 nm against a reagent blank using a double beam spectrophotometer (UV-160 A, Shimadzu Corporation, Kyoto, Japan). The total antioxidant activity is expressed as g equivalent of ascorbic acid.
Phytochemical Screening of Callus Tissue by GC-MS: The phytochemical analysis in the ethyl acetate extract of 35 day old callus tissue was carried out on GC-MS model HP 6890 (Agilent Technologies Ltd) fitted with an HP-5 MS (5% phenylmethyl siloxane) capillary column 30 m × 250 μm × 0.25 μm. The sample (1 µl) was injected to the injected port. The injector temperature was adjusted at 250 °C, while the detector temperature was fixed to 280 °C. The column temperature was raised from 40 °C to 220 ºC at a rate of 6 ºC/min. The flow rate of the carrier gas, helium was set 1 ml/min, and the split ratio was 1:50. The sample was vaporized, and the various components in the sample were separated and analyzed. The Mass spectra were acquired in scan mode (70 eV) in the range of 50–550 m/z. The spectral peak produced by each component was recorded. The retention time of the components was determined by matching the spectra with that of reference compounds.
Identification of Components: In the MS Program, National Institute Standard and Technology (NIST), Version 14.0, Wiley 8.0 library database of NIST having more than 62000 patterns were used for identifying the chemical components. The unknown phytochemicals were identified by comparing the spectra of both known and unknown compounds stored in the NIST database.
Statistical Analysis: The data were analyzed by one-way analysis of variance (ANOVA) using Statistical Software. The measurements are expressed as mean ± standard errors of the mean (SEM). A significance level of 0.05 was used for all statistical tests.
RESULTS AND DISCUSSION:
Callus Formation and Growth: The explants selected from mature plant internodes of J. gossypiifolia were cultured in callus induction medium at 26°C in the dark for 55 days. The callus formation was induced by a combination of cytokinin and auxin, which act synergistically to promote cell division, growth, development, differentiation, and the formation of plant organs 20, 22. The auxin 2, 4-D plays a key role in the induction of callus 26. Later, it was found that the addition of cytokinins acts in concert with auxins to enhance callus regeneration 27. MS media supplemented with only NAA has no significant effect on shoot proliferation 21. However, a number of studies have shown that NAA was the best plant growth hormone, and the optimal concentration was 1 mg/L-1 for producing green, compact, and fast-growing Jatropha callus (Rajore and Batra, 2007). In the present investigation, callus induction was achieved when explants were inoculated on MS media fortified with 2,4-D and NAA. Callusing was initiated between 14 and 18 days of explants transformation Fig. 1A-C. This is consistent with earlier studies of Rajore and Batra (2007), who have shown the induction of callus between 16-20 days from shoot tip explants of J. curcus. The callus growth reached 100% by 35 days Fig. 1.
FIG. 1: DIFFERENT STAGES OF CALLUS DEVELOPMENT ON MS MEDIA SUPPLEMENTED WITH NAA (1 MG/L-1) AND 2,4-D (1 MG/L-1)
Thirty-five days old explants growing on media containing 2,4-D (2 mg/L-1) with NAA (1 mg/L-1) produced white, cream-coloured calli. On the other hand, explants growing on MS medium fortified with 2,4-D (2 mg/L-1) and BAP (2 mg/L-1) for 35 days revealed compact and light green calli due to greenish pigmentation Fig. 2A and B. This finding gets further support from the study of de Oliveira et al. (2017), who had shown increased frequency of compact and light green calli, when MS media contained BAP (2 mg L-1). The grren calli have features opposite to that of cream-colored and friable ones, which have lower chances to differentiate. These observations infer that BAP plays a significant role in the induction and growth of green callus of J. gossypifolia, which may also imply BAP has an effect to preserve greenish pigmentation as well as to prevent senescence in callus tissue.
FIG. 2: CALLUS DEVELOPMENT ON MS MEDIA SUPPLEMENTED WITH NAA (1 MG/L-1) + 2,4-D (1 MG/L-1) WITH AND WITHOUT BAP ( MG/L-1)
Characterization of Silver and Gold Nanoparticles: Silver and gold nanoparticles were purified and scanned by SEM analysis Fig. 3, 4, and 5. SEM is the most widely used technique for characterizing nanoparticles in terms of the physical morphology of the particles. In the present study, SEM images suggest that biosynthesized silver nanoparticles are almost spherical in structure Fig. 4A-C.
The EDS profiles of the nanoparticles confirmed the presence of a characteristic silver signal at approximately 3 keV Fig 4C, which is typical for the absorption of silver nanoparticles due to surface plasmon resonance confirming that silver nanoparticles were biosynthesized successfully using J. gossififolia. Similarly, the presence of gold elements in the EDX analysis confirmed the formation of gold nanoparticles Fig 5A-C.
FIG. 3: SEM IMAGES WITH EDX ANALYSIS OF SILVER NANOPARTICLES
FIG. 4: SEM IMAGES WITH EDX ANALYSIS OF GOLD NANOPARTICLES
Legend for Fig. 4: Scanning electron microscopy images (a and b) represents gold nanoparticles synthesised using ethyl acetate extract of callus of J. gossypiifolia. Fig. 4C represents the Energy-Dispersive X-ray microanalysis of gold nano-particles.
FIG. 5: TLC CHROMATOGRAM SHOWING BANDS A – PETROLEUM ETHER EXTRACT; B – ETHYL ACETATE EXTRACT
Antibacterial Assays of Silver and Gold Nanoparticles and TLC Chromatogram Products: Silver and gold nanoparticles obtained by using 35-day old callus tissue were tested for antibacterial activity. The gold nanoparticles showed the highest antimicrobial activity with an inhibition zone of 2.1 cm against Gram-negative P. aeroginosa compared to silver nanoparticles, which showed an inhibition zone of 1.0 cm. Gold nano-particles also inhibited gram-positive S. aureus with inhibition zone of 1.9 cm, while silver nano-particles showed no antibacterial activity Fig. 6.
FIG. 6: ANTIBACTERIAL ACTIVITY OF SILVER AND GOLD NANOPARTICLES FROM CALLUS TISSUE AGAINST GRAM-NEGATIVE P. AEROGINOASA AND GRAM-POSITIVE S. AUREAS
Antibacterial activity was also evaluated by TLC bioautographic method. The recovered concentrates of each of the five TLC bands obtained by using ethyl acetate extract of 35-day old callus tissue Fig. 7A. were tested against P. aeroginosa (Gram-negative) and S. aureus (Gram-positive bacteria) by well diffusion method. Of the five bands of TLC tested, both 3rd and 4th bands alone have shown pronounced antibacterial activity against the test pathogens with zones of inhibition varying between 0.9-1.0 cm against Gram-positive bacteria S. aureus. In Gram negative bacteria P. aeroginosa, 3rd and 4th bands of TLC inhibited bacterial growth with zones of inhibition 1.2 and 1.7 cm, respectively Fig. 7B and C, Table 1.
The positive effects observed with the concentrates of TLC bands-3 and 4 indicate that the J. gossypiifolia callus synthesizes compounds responsible for antibacterial activity. This is consistent with a number of earlier studies, which have shown good antibacterial activity in the leaf, shoot and root extracts of J. gossypiifolia 28, 29, 15.
FIG. 7: ANTIBACTERIAL ACTIVITY AGAINST (A) GRAM-NEGATIVE P. AEROGINOSA AND GRAM-POSITIVE (B) S. AUREAS BY TLC BIOACTOGRAPHIC METHOD
TABLE 1: ANTIBACTERIAL ACTIVITY OF RECOVERED CONCENTRATES OF TLC BANDS AND SILVER AND GOLD NANOPARTICLES FROM CALLUS TISSUE OF J. GOSSYPIIFOLIA
|S. no.||Name of the micro-organism||TLC bands||Nanoparticles|
|In cm||In cm|
|+ve control (Ampicillin 20 µg/ disc)||
|TLC band 1||TLC band 2||TLC band 3||TLC band 4||TLC band 5||+ve control (Ampicillin 20 µg/ disc)||Control||Silver nanoparticls||Gold nanoparticls|
|1||P. aeroginosa (MTCC 2453||1.6||-||-||-||1.2||1.7||-||1.2||-||-||1.9|
|2||S. aureus (MTCC 96)||1.4||-||-||-||1.0||0.9||1.1||-||1||2.1|
Antioxidant Activity: The total antioxidant activity of recovered concentrates of five TLC bands obtained using petroleum ether and ethyl acetate extracts of 35-day old callus tissue were evaluated by comparing with that of ascorbic acid. Of the 5 TLC compounds tested, 3rd and 4th bands obtained using both petroleum ether and ethyl acetate extracts expressed the highest antioxidant activity, which has surpassed the reference compound, ascorbic acid Fig. 8. This finding gets support from a number of earlier studies, which have shown significant hydroxyl and superoxide radical scavenging activities of aqueous leaf crude extract of J. gossypiifolia 16, 4. These authors have claimed that the high content of phenols, tannins, and flavonoids present in the leaves are responsible for the antioxidant activity of this plant.
FIG. 8: ANTIOXIDANT ACTIVITY BY TLC BIOAUTOGRAPHIC METHOD USING ETHYL ACETATE EXTRACT OF CALLUS TISSUE OF J. GOSSYPIIFOLIA
FTIR Analysis: In the present study, FTIR spectra were obtained Fig. 9 from the recovered concentrates of five TLC bands resolved by using ethyl acetate extracts of 35-day old callus tissue. The 5 bands obtained on the TLC using ethyl acetate extract of J. gossypiifolia callus tissue are presented in Fig. 3B. The five bands of TLC were chaacterixed by FTIR in the spectral region of 500 to 4000 cm-1. A characteristic absorption maxima at 1096 cm-1, indicating the presence of C-O) for a hydroxyl (-OH), which was observed in TLC bands -2, -3 and -5 except band-3. Apart from this characteristic band, the five TLC bands showed the absorption maxima at 3000-4000 cm-1, which corresponds the hydroxyl groups. The absorption maxima at 2882 cm-1 and 2380 cm-1 (for C-H stretching), at 1641 cm-1 (C=C stretching), 1553-1565 cm-1 (C=O aromatic stretch), 974 cm-1 (C-H bending of aromatic hydrocarbons) and 798 cm-1 (aromatic carbons) were also observed Table 2.
FIG. 9: CHARACTERISATION OF FIVE TLC BANDS BY FTIR
TABLE 2: SHOWS THE CHARACTERISATION OF FIVE TLC BANDS BY FTIR AND FUNCTIONAL GROUPS
|TLC band 1||TLC band 2||TLC band 3||TLC band 4||TLC band 5||Functional groups|
|Wave numbers cm-1|
|3466.08||3485.01||3458.37||3462.22||3450.65||O-H||Hydrogen bonded alcohols, phenols|
|1552.70||--||1554.63||--||1552.70||C=O aromatic strecthing||Alkene|
Phytochemical Analysis of Callus Tissue Using GC-MS: Phytochemical screening of callus is required to identify the nature of bioactive components in order to discover novel therapeutic agents with better efficacy. In the present study,
The chromatogram peaks were identified by comparing with the spectra of known and unknown components stored in the NIST library.
GC-MS profiling of ethyl acetate extract of J. gossypiifolia callus revealed the presence of thirty-one phytocomponents. A distinct chromatogram of J. gossypiifolia callus is shown in Fig. 10.
The bioactive compounds with their retention time (RT), molecular formula, molecular weight, peak height and area are represented in Table 3. The phytocomponents in the callus of J. gossypiifolia showed a chromatogram with a retention time ranging from 3.94 to 23.61.
FIG. 10: GC-MS CHROMATOGRAM OF ETHYL ACETATE EXTRACT OF CALLUS TISSUE OF J. GOSSYPIIFOLIA
TABLE 3: COMPOUNDS IDENTIFIED BY GC-MS ANALYSIS FROM THE ETHYL ACETATE EXTRACT OF J. GOSSYPIIFOLIA
Among the identified 31 phytocomponents, some of them possess promising bioactivities. Phytol is a diterpene that possesses antimicrobial, antioxidant, anticancer, anti-inflammatory, anti-diuretic, immune-stimulatory, and anti-diabetic activities 30, 31. Hexadecanoic acid is known to exhibit strong antimicrobial and anti-inflammatory, antioxidant and cancer preventive activities. It is useful for the management of eye infections as well as bruises and erupted skins 32, 33, 34, 35. Squalene is a triterpene that act as natural antioxidants and possesses various pharmacological activities like antibacterial, antioxidant, anti-tumor, cancer preventive, chemopreventive and diuretic properties.
It is also a immunostimulant and a lipoxygenase-inhibitor 36, 37. 9,12,15-Octadecatrienoic acid, 2-(acetyloxy)-1-[(acetyloxy)methyl]ethyl ester, (Z, Z, Z)- is a fatty acid ester, which possesses anti-inflammatory, cancer preventive, antihistaminic, antiarthritic, anticoronary, antieczemic, antiacne, hepatoprotective, antiandrogenic and hypocho-lesterolemic properties 38, 39. 9-Octadecenoic acid (Z)-, 2,3-bis(acetyloxy)propyl ester is also a fatty acid ester, which possesses antifungal. anti-bacterial, anti-inflammatory, anti-alopecic, anemia-genic, antitumour, choleretic, dermatitigenic, antiandrogenic, allergenic, and hypocholesterol-emic properties 34. It is also a immunostimulant as well as lipoxygenase and 5α reductase inhibitor. 3, 7, 11, 15-Tetramethyl-2-hexadecen-1-ol is used in the treatment of asthma, Further, it is cancer-preventive and possesses anti-inflammatory and antimicrobial activities 40, 41. Based on the spectral data, it was found that the callus of J. gossypiifolia contained a large number of bioactive compounds. The analysis separated and identified various phytochemicals belonging to different chemical classes. The presence of various bioactive compounds justifies the propagation and use of this callus tissue for phytopharmaceutical purposes.
CONCLUSION: Taken together, the present study provided a rapid protocol for callus initiation and growth derived from the internode region of year old plant. The petroleum ether and ethyl acetate extracts of thirty-five-day-old callus tissue were resolved into 5 bands by TLC. Of the five bands, the third and fourth bands of TLC revealed antimicrobial and antioxidant activities. GC-MS analysis revealed 31 compounds of different classes. The presence of various bioactive compounds justifies it uses for various ailments by traditional practitioners.
ACKNOWLEDGEMENT: We thank the Vice President, Chancellor, and Vice-Chancellor, Kalasalingam University, Krishnankoil, Viruthunagar District, Srivilliputhur - 626126 Tamil Nadu, India, for their constant encouragement to carry out this work.
CONFLICTS OF INTEREST: The authors do not have any conflicts of interest.
- Puth LM and Post DM: Studying invasion: have we missed the boat? Ecol Lett 2005; 8: 715--21.
- Balee W: Footprints of the forest, kaapor ethnobotany- the historical ecology of plant utilization by an Amazonian people. Columbia University Press. New York 1994.
- Dabur R, Gupta A, Mandal TK, Singh DD, Bajpai V, Guray AM and Lavekar GS: Antimicrobial activity of some medicinal plants. Afri J Trad Comp Altern Med 2007; 4, 313-18.
- Félix-Silva J, Giordani RB, da Silva-Jr AA, Zucolotto SM and de Freitas FPM: Jatropha gossypifolia (Euphorbiaceae): A review of traditional uses, phytochemistry, pharmacology, and toxicology of this medicinal plant. Evid. Based Comp Altern Med 2014; 1–32.
- Lans C, Harper T, Georges K and Bridgewater E: Medicinal and ethnoveterinary remedies of hunters in Trinidad. BMC Comp. Altern. Med 2001; 1: 1–10.
- Oduola T, Adeosun OG, Oduola TA, Avwioro OG and Oyeniyi MA: Mechanisms of action of Jatropha gossypifolia stem latex as a haemostatic agent. Euro J Gen Med 2005; 2: 140-43.
- Panda BB, Gaur K, Kori ML, Tyagi LK, Nema RK, Sharma CS and Jain AK: Anti-inflammatory and analgesic activity of Jatropha gossypiifolia in experimental animal models. Glob J Pharmacol 2009; 3: 1-5.
- Purohit MC and Purohit R: Evaluation of antimicrobial and anti-inflammatory activities of bark of Jatropha gossypiifolia. World J Sci Technol 2011; 1: 1–5.
- Wu Q, Patocka J, Nepovimova E and Kuca K: Jatropha gossypiifolia and its biologically active metabolites: A mini review. J. Ethnopharmacol 2019; 234: 197-03
- Bhagat R, Ambavade SD, Misar AV and Kulkarni DK: Anti-inflammatory activity of Jatropha gossypiifolia leaves in albino mice and Wistar rat. J Sci Indust Res 2011; 70: 289-92.
- Zhang CY, Zhang LJ, Lu ZC, Ma CY, Ye Y, Rahman K, Zhang H and Zhu JY: Antitumor activity of diterpenoids from Jatropha gossypiifolia: cell cycle arrest and apoptosis-inducing activity in RKO colon cancer cells. J Nat Prod 2018; 81: 1701-10.
- Falodun A, Kragl U, Touem SMT, Villinger A, Fahrenwaldt T and Langer P: A novel anticancer diterpenoid from Jatropha gos Nat. Prod. Commun 2012; 7: 151-52.
- Asep S, Hening H, Gema SP, Gigih S, Widya MC and Sahhidin S: Anticancer activity of Jatrophone, an isolated compound from Jatropha gossypiifolia plant against hepatocellular cancer cell HEP G2 1886. Biomed Pharmacol J 2017; 10: 667–73.
- Gaikwad RS, Kakde RB, Kulkarni A.U and Gaikwad DR: Panchal VH: In-vitro antimicrobial activity of crude extracts of Jatropha species. Curr Bot 2017; 3: 09-15.
- Singh DD: Assessment of antimicrobial activity of hundreds extract of twenty Indian medicinal plants. Biomed Res 2018; 29: 1797-14.
- Kharat AR, Dolui AK and Das S: Free radical scavenging potential of Jatropha gossypiifolia. Asian J Chem 2011; 23: 799-01.
- Granados S, Balcázar N, Guillén A and Echeverri F: Evaluation of the hypoglycemic effects of flavonoids and extracts from Jatropha gossypiifolia Molecules 2015; 20: 6181-93.
- Saishri R, Ravichandran N, Vadivel V and Brindha P: Pharmacognostic studies on leaf of Jatropha gossypiifolia Int J Pharmaceut Sci Res 2016; 7: 163.
- Nwokocha AB, Agbagwa IO and Okoli BE: Comparative phytochemical screening of Jatropha L. species in the Niger Delta. Res. J Phytochem 2011; 5: 107-14.
- Rajore S and Batra A: Efficient plant regeneration via shoot tip explants in Jatropha curcas. J. Plant Biochem Biotechnol 2005; 14: 73–75.
- Biradar S, Waghmare V and Pandhure N: In-vitro callus and shoot induction in Jatropha curcas (Linn.). Trends Life Sci. 2012; 1: 38–41.
- Kumar S, Kumar V, Sharma MK, Kumar N, Kumar A, Tomar KPS, Sharma SK, Singh MK, Sengar RS and Jaiswal N: Effects of different plant growth regulators on in-vitro callus induction in physic nut (Jatropha curcas). J Appl Nat Sci 2015; 7: 30–37.
- Sujatha M and Mukta N: Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas. Plant Cell Tissue and Oran culture 1996; 44: 135-41.
- Murashige T and Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiolog. Plantar 1962; 15: 473-97.
- Prieto P, Pineda M and Aguilar M: Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem 1999; 269: 337-41.
- Chaudhury A and Qu R: Somatic embryogenesis and plant regeneration of truf-type bermudagrass: effect of 6-benzaldehyde in callus induction medium. Plant Cell Tiss Org Cult 2000; 60: 113-20.
- Bradley DE, Bruneau AH, Qu R: Effect of cultivar, explant treatment and medium supplements on callus induction and plantlet regeneration in perennial ryegrass. Int. Turfgrass Soc Res J 2001; 9: 152–56.
- Seth R and Sarin R: Analysis of the phytochemical content and anti- microbial activity of Jatropha gossypifolia Arch App Sci Res 2010; 2: 285-91.
- Sharma V, Kumawat TK, Seth R and Sharma A: Bioefficacy of crude extracts from Jatropha gossypifolia against human pathogens. Int J Biotech Bioengineer Res 2013; 4: 401-06.
- Wei LS, Wee W, Siong JYF and Syamsumir DF: Characterization of anticancer, antimicrobial, antioxidant properties and chemical compositions of Peperomia pellucida leaf extract. Acta Med Irani. 2011; 49: 670–74.
- Song Y and Cho SK: Phytol induces apoptosis and ROS-mediated protective autophagy in human gastric adenocarcinoma AGS cells. Biochem Anal Biochem 2015; 4: 211.
- Dilika F, Bremner PD and Meyer JJ: Antibacterial activity of linoleic and oleic acids isolated from Helichrysum pedunculatum: a plant used during circumcision rites. Fitoterapia 2000; 71: 450-52.
- McGraw LJ, Jager AK and Van staden J: Isolation of antibacterial fatty acids from Schotia brachypetala. Fitoterapia 2002; 73: 431-33.
- Swamy MK and Sinniah UR: A comprehensive review on the phytochemical constituents and pharmacological activities of Pogostemon cablin: an aromatic medicinal plant of industrial importance. Molecules 2015; 20: 8521-47,
- Thakora P, Subramaniana RB, Thakkarb SS, Rayb A, Thakkara VR: Phytol induces ROS mediated apoptosis by induction of caspase 9 and 3 through activation of TRAIL, FAS and TNF receptors and inhibits tumor progression factor Glucose-6-phosphate dehydrogenase in lung carcinoma cell line (A549). Biomed Pharmacother 2017; 92: 491–00.
- Amarowicz R: Squalene: a natural antioxidant?. European Journal of Lipid Science and Technology 2009; 111: 411-12.
- Kim SK and Karadeniz F: Biological importance and applications of squalene and squalane. Adv Food Nutr Res 2012; 65: 223-33.
- Sermakkani M and Thangapandian V: GC-MS analysis of Cassia italica leaf methanol extract. Asian J Pharm Clin Res 2012; 5: 90–94.
- Srinivasan K, Sivasubramanian S and Kumaravel S: Phytochemical profiling and GCMS study of Adhatoda vasica Int J Pharma BioSci 2014; 5: 714–20.
- Ogunlesi M, Okiei W, Ofor E and Osibote AE: Analysis of the essential oil from the dried leaves of Euphorbia hirta Linn (Euphorbiaceae), a potential medication for asthma. Afri J Biotech 2009; 8: 7042-50.
- Yu X, Zhao M, Liu F, Zeng S and Hu, J: Identification of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4- one as a strong antioxidant in glucose–histidine Maillard reaction products. Food Research International 2013; 51: 397-03.
- de Oliveira AB, Vendrame WA, Londe LCN and Sanaey M: Induction of different types of callus in Jatropha curcas L. hybrid accession at in-vitro Aust J Crop Sci 2017; 11: 874-79.
How to cite this article:
Kalaiyarasan A and Hariram N: Evaluation of phytochemical analysis of callus tissue from internode explants of traditional medicinal plants of Jatropha gossypiifolia Linn. Int J Pharm Sci & Res 2021; 12(12): 6403-15. doi: 10.13040/IJPSR.0975-8232.12(12).6403-15.
All © 2021 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
A. Kalaiyarasan and N. Hariram *
Department of Biotechnology, Kalasalingam Academy of Research Education, Krishnankoil, Tamil Nadu, India.
03 January 2021
23 May 2021
13 June 2021
01 December 2021