Research Article

Screening of Phytochemical Compounds and Antimicrobial Activity of Caralluma tuberculate by In-Silico Study

Khansa Jamil1, Muhammad Ramzan Khan1, Asad Jan2 and Ghulam Muhammad Ali1*

1Department of Plant Genomics and Biotechnology (NIGAB), National Agriculture Research Centre (NARC), Islamabad 44000, Pakistan; 2Institute of Biotechnology Genetic Engineering, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan.

Received | March 31, 2023; Accepted | October 27, 2023; Published | December 12, 2023

*Correspondence | Ghulam Muhammad Ali, Department of Plant Genomics and Biotechnology (NIGAB), National Agriculture Research Centre (NARC), Islamabad 44000, Pakistan; Email: drgmali5@gmail.com

Citation |Jamil, K., M.R. Khan, A. Jan and G.M. Ali. 2023. Screening of phytochemical compounds and antimicrobial activity of Caralluma tuberculate by In-Silico study. Sarhad Journal of Agriculture, 39(4): 983-989.

DOI | https://dx.doi.org/10.17582/journal.sja/2023/39.4.983.989

Keywords | Bioactive compounds, Antibacterial analysis, TLC, GC-MS, In Silico study

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Caralluma tuberculata is a perennial succulent herb, wild-growing in Pakistan, India (Andra Pradesh), the United Arab Emirates, Saudia Arabia, Iran, and Nigeria (Zarei et al., 2020; Bodoprost and Rosemeyer, 2007). The plant is widely distributed in Waziristan, Punjab, K.P., Dir, and Baluchistan (Fernie et al., 2004). Caralluma tuberculate locally known as Chung, marmot, pamanghi, aputag, and pamanky in Pashto (Khan et al., 2019; Zarei et al., 2020; Hamburger and Hostettmann, 1991). The genus Caralluma has a hundred species that are found in dry regions of the world. The genus belongs to the subfamily Asclepiadoideae and the family Apocynaceae (Harborne, 1986).

C. tuberculata is an upright, succulent herb without leaves. Fresh juice of C. tuberculata is used for the treatment of diabetes due to having some hypoglycemic activities.

Caralluma species have been used in folk medicine for hundreds of years in the treatment of various ailments. Its traditional application is reported in curing of simple disorders such as cuts, colds, coughs, and some complicated diseases including malaria, diabetes, kidney stones blood disorders, leprosy, diabetes, and rheumatism. It is also used for blood disorders, rheumatism, and leprosy (Baig et al., 2021; Khan et al., 2019). Both the urban and rural populations of Pakistan have employed the plant as a traditional anti-bacterial, antifungal, and anti-diabetic therapeutic agent (Khan et al., 2019; Rauf et al., 2013). Microorganisms have developed resistance to many antibiotics. Resistant bacteria are responsible for infections that are more difficult to treat, requiring the use of drugs that are more toxic and more expensive. In some cases, bacteria have become resistant to all known antibiotics, so there is a need to develop alternative antibiotic drugs from plants (Serwecinsk, 2020). One method is to identify plants that possess high antimicrobial activity. Current studies are based on the identification of antimicrobial activity and screening of medicinal bio-active compounds by using qualitative techniques and drug designing.

Materials and Methods

Collection of samples

The plants were collected from a glass house at the National Institute for Genomics and Advance Biotechnology, National Agriculture Research Center, Islamabad.

Sample preparation

To get rid of undesired foreign objects like soil and dust, fresh Caralluma tuberculata stems were washed with distilled water and then bleached with Clorox. Following washing, the plant material was air dried at room temperature in the shade, and shielded from the sun. It was then, finely pulverized into powder by using liquid nitrogen in piston and mortar. For extraction in a sonicator, set the frequency to 35 kHz, 4 g of dried stem powder was combined with 40 mL of methanol in a solvent to sample ratio of 1: 10 at 45°C and for 60 minutes, respectively. Extracts were micro-filtered through 0.45 m cellulose membrane filters, centrifuged, and solvent evaporated by vacuum evaporator (Roberts and Xia, 1995).

Chromatographic instrument and conditions

The screening of phytochemical compounds of Caralluma tuberculata was done by using TLC and GC-MS.

Thin layer chromatography (TLC)

Based on molecular weight and mobility, bioactive substances can be identified using a technique called TLC. Different solvents, methanol, ethanol, ethyl acetate, and chloroform were used to elute a maximum number of active compounds from the crude extract of the plant. The targeted sample was loaded on a silica plate. After loading the sample, the plate was dried and put into the TLC tanks having different solvents (methanol, ethanol, ethyl acetate, and chloroform) in each tank and the baseline sample did not directly touch the tank surface. The spots and colored bands were visualized under the short and long UV (254 and 365 nm respectively). Sprayed the dragendorffs and sulphuric acid reagent to evaluate the alkaloids and terpenoids compounds (Javed et al., 2018).

Gas chromatography and mass spectrum analysis

The column used for the GC program was DB-5 (0.32mm x 30m x 0.25µm) length of 30m. The equipment was Agilent 6890 6ºC, version N. 04.12, MS-5973. The carrier gas used in the GC-MS program was helium ml/min, and the software was chemstation. The oven’s temperature was increased from 200 °C to 700 °C at a pace of 6 °C per minute without holding. The temperature of the injector was 250 °C. The GC ran for 37 minutes in total. The MS’s library was NIST version-year 2011. The MS program’s source temperature was 280°C, and the inletline temperature was 230°C. The MS ran for 37 minutes in total (Aunsha and Sathish, 2021). The components of the test materials’ names, molecular weights, and structures were determined (Adnan et al., 2014).

Antibacterial screensing

Different microbial strains E. coli, Bacillus, Pseudomonas, and Staphylococcus were used to evaluate the antimicrobial effect of plant extract. The strains were ATTC strains obtained from the Food Sciences Research Institute (FSRI) NARC, Pakistan (Noreen, 2017; Baig et al., 2021). The disc diffusion method was carried out for antibacterial susceptibility testing according to the standard method (Paskialakshmi and Naziya, 2014).

Molecular docking

The Auto Dock Vina software has been used in the molecular docking study (De-Fatima et al., 2006). Four bioactive compounds 3,4-di-fluroanisole, d-fructose, acetyldimethy l, and 2-propionic acid of Caralluma plant were used as ligands to increase the expression of calprotectin anti-microbial protein in plants that inhibit the growth of pathogens by tightly binding transition metals e.g., Mg, Zn thereby preventing their uptake and utilization by invading microbes. The targeted ligands were docked against the active site of the calprotectin protein in plants (Falodu et al., 2009). The co-crystallized ligand (Parathion) of the enzyme was used to identify the binding location. The grid boxes had the following coordinates (x = −9.682; y = 4.274; z = −23.145 and x = 45.424; y = 92.375; z = 34.811). The grid’s box dimensions were set to 20 Å. Each docking experiment generated ten poses. Pymol software was used to analyze and display docking poses (Bourhia et al., 2020).

Results and Discussion

Thin layer chromatography

Bioactive compounds present in plants were detected by thin-layer chromatography scanning procedure. Elution and separation of bioactive compounds from crude extract vary with different solvents based on the nature of the solvent. Precise separation of compounds was done in a methanolic solvent. The spots and bands were visualized under short and long UV (254 and 365) respectively shown in Figure 1. Dragendorff reagent was used to detect alkaloids and 15% sulphuric acid was used to detect terpenoids. A red color band was shown on the top far away from the baseline that indicated the highest retention factor as compared to the terpenoid compound.

According to recent results, the Caralluma plant possesses many phytochemical compounds with highly medicinal properties. TLC results depicted that tannins and alkaloids bioactive compounds have highly anti-parasitic, anti-inflammatory, antifungal, antidiabetic, anticancer, and antibacterial activity (Albalawi et al., 2015). TLC showed that as compared to the other solvents, methanol eluted many spots that were determined after spraying of Dragendorff and 15% sulphuric acid under the wavelength of (254 and 365 nm) the little bit results were similar to a previous study (Ahmad et al., 2014). Plant extract contains many phytochemical compounds that possess highly medicinal properties.

 

 

Phyto-components by GC-MS report

Phytochemicals present in methanolic extracts of Caralluma tuberculata are tabulated and represented by a graphical method. 70 compounds were identified in the targeted plant by GC-MS analysis which are shown in Figure 2. The Prevailing compound was, 3, 4-di-fluroanisole, D-fructose, Acetyldimethyl, and 2-propionic acid identified by the GC-MS analysis.

The compounds identified in methanolic solvents by GC-MS analysis are medicinally valuable (Hussain et al., 2018; Khan et al., 2008) because Methanol was the best solvent for the extraction among the tested solvents (Salih et al., 2021).

Antibacterial analysis

In the current study, the antibacterial results revealed that the methanolic extract of the medicinal plant Caralluma possesses high antimicrobial activity against E. coli. and Bacillus causing 16mm±0.53 and 12.6±0.43 while ethyl acetate solvent of plant extract against E. coli. and Bacillus inhibited 08±0.0.03 and 9.6±0.32, respectively. Ethyl acetate extract showed the best inhibition against Pseudomonas and Staphylococcus at 15.6±0.54 and 9.8±0.41 respectively and the same extract exhibited the zone inhibition 0.68±0.14 and 0.53±0.51 against B. subtilis and P. aerugonosa represented in Table 1. In the current study, the antibacterial activity was indicated due to the presence of secondary metabolites phenolic, saponins, alkaloids, and tannins (Vanitha et al., 2019). These compounds have the potential to restrain the growth of many pathogenic bacteria. Our results are also to those of (Makeen et al., 2020). The high antibacterial activity of the Caralluma plant depicts that some natural compounds present in plants may lead to isolating the antibiotics.

 

Table 1: Antibacterial activity of methanolic and ethylacetate crude extract of Caralluma tuberculata.

Bacterial strain	Methanolic extract	Ethanolic extract
E. coli	16mm±0.53	08±0.0.03
B. subtilis	12.6±0.43	9.6±0.32
P. aerugonosa	0.68±0.14	15.6±0.54
S. aureus	0.53±0.51	9.8±0.41

 

In the present study, the methanolic extract obtained from Caralluma tuberculate extract exhibited strong antimicrobial activities against (E. coli and Bacillus) while ethyl acetate extract showed strong antimicrobial activity against (Pseudomonas, Staphylococcus, B. subtilis, and P. aeruginous) (Al-Snafi, 2017; ).

Molecular docking

The four bioactive compounds were subjected to molecular docking to enhance the expression of the antimicrobial protein calprotectin as shown in (Table 2 and Figure 3). All targeted compounds received scores ranging from (−3.5 to −5.9) kcal/mol with targeted protein. The ligand 1st received scores -5.9 kcal/mol, the ligand 2nd received -5.9 kcal/mol, the 3rd ligand secured -3.8 kcal/mol, and the 4th received -3.5 kcal/mol respectively. The compounds that achieved the best score (−5.9 kcal/mol) with calprotectin protein while the standard compound Parathion received a score of -5.6 which is slightly lower than the targeted ligand and hence −5.9 kcal/mol was selected as the best compound to enhance the expression of protectin antimicrobial protein in Caralluma plant.

 

Table 2: Docking sores and estimated absolute binding free energies (in kcal/mol) of the targeted ligands and protein, along with standard Parathion.

Ligand name	Protein name	Docking scores
3,4-Di-fluroanisole	Calprotectin	-5.9
D-fructose	Calprotectin	-5.9
Acetyldimethyl	Calprotectin	-3.5
2-propeonoic acid	Calprotectin	-3.8
Parathion	Calprotectin	-5.6

 

Image of docking analysis

The present study was conducted to isolate and design the antimicrobial drug by In-silico study. The identified phyto-components need further research on toxicological parameters to develop safe drugs.

Conclusions and Recommendations

The present study highlights the screening of bioactive compounds by chromatographic techniques and detects the antimicrobial activities of targeted phytochemicals by In-silico study. However, the identified phyto-components studies need further research on toxicological parameters to synthesize safe drugs.

Acknowledgments

Department of National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agriculture Research Center (NARC) Islamabad, Department of Food Sciences Research Institute (FSRI), National Agriculture Research Center (NARC) Islamabad, Institute of Biotechnology and Genetic Engineering (IBGE) and Higher Education Commission (HEC) Pakistan.

Novelty of Statement

According to this research findings Caralluma tuberculata possesses a novel antibacterial compound that can increase the efficiency of calprotectin antibacterial protein which can be used to synthesize novel antibacterial drugs.

Author’s Contribution

Khansa Jamil: Principal author, conducted research, analysis and wrote draft of the manuscript.

Muhammad Ramzan Khan: Helped and guidance in the research.

Asad Jan: Provided in technical assistance

Ghulam Muhammad Ali: Supervised the research and helped in proofreading.

Conflict of interest

The authors stated that there were no conflicts of interest present throughout the conduct of the current study. It is a part of my Ph.D. thesis and is submitted to the HEC repository directory.

References

Abdel, S.E., F.M. Harraz, S.A. Ghareib, A.A. Elberry, S. Gabr and M.I. Suliaman. 2011. Antihyperglycaemic and hypolipidaemic effects of the methanolic extract of Caralluma tuberculata in streptozotocin-induced diabetic rats. Nat. Prod. 25: 1171–1179. https://doi.org/10.1080/14786419.2010.490782

Abdel-Sattar, E.A., H.M. Abdallah, A. Khedr, A.B. Abdel-Naim and I.A. Shehata. 2013. Antihyperglycemic activity of Caralluma tuberculata in streptozotocin-induced diabetic rats. Food Chem. Toxicol., 59: 111–117. https://doi.org/10.1016/j.fct.2013.05.060

Abdel-Sattar, E., M.R. Meselhy and M.A. Al-Yahya. 2002. New oxypregnane glycosides from Caralluma penicillata. Planta Med., 68(5): 430-434. https://doi.org/10.1055/s-2002-32078

Adnan, M., S. Jan, S. Mussarat, A. Tariq, S. Begum, A. Afroz and Z.K. Shinwari. 2014. A review on ethnobotany, phytochemistry and pharmacology of plant genus Caralluma R. Br. J. Pharm. Pharmacol., 66: 1351–1368. https://doi.org/10.1111/jphp.12265

Ahmad, B.A., I.R. Abbas, S.J. Hussain, F. Bashir and D. Ahmad. 2014. Study on Caralluma tuberculata nutritional composition and its importance as medicinal plant. Pak. J. Bot., 46(5): 1677-1684.

Ahmad, V.U. and A. Basha. 2007. Spectroscopic data of steroid glycosides. Springer Sci. Bus. Media, 5: 142-189. https://doi.org/10.1007/978-0-387-39576-0

Albalawi, M.A., N.A. Bashir and A. Tawfik. 2015. Anticancer and antifolate activities of extracts of six Saudi Arabian wild plants used in folk medicine. J. Life Sci., 9: 34-40.

Al-Snafi, A.E., 2017. The pharmacology of Equisetum arvense. A review. IOSR J. Pharma., 7(2): 31-42.

Al-Yahya, M., E. Abdel-Sattar and E. Guittet. 2000. Pregnane glycosides from Caralluma russeliana. J. Natl. Prod., 27, 63(10): 14-51. https://doi.org/10.1021/np990530c

Anusha, S. and S. Sathish. 2021. UV, FT-IR, and GC-MS analysis of Caralluma fimbriata. J. Pharmacog. Phytochemist., 10(5): 288-291.

Baig, M.W., Ahmed, M., Akhtar, N., Okla, M.K., Nasir, B., Haq, I.U., Al-Ghamdi, J., Al-Qahtani, W.H. and AbdElgawad, H., 2021. Caralluma tuberculata NE Br manifests extraction medium reliant disparity in phytochemical and pharmacological analysis. Molecules, 26(24): 7530. https://doi.org/10.3390/molecules26247530

Bibi, Y., S. Tabassum, K. Zahara, T. Bashir, S. Haider. 2015. Ethnomedicinal and pharmacological properties of Caralluma tuberculata NE Brown. A review. Pure Appl. Biol., 4: 503. https://doi.org/10.19045/bspab.2015.44008

Bodoprost, J. and H. Rosemeyer. 2007. Analysis of phenacylester derivatives of fatty acids from human skin surface by reversed-phase HPTLC: Chromatography mobility as a function of physicochemical properties. Int. J. Mol. Sci., 8: 1111-1124. https://doi.org/10.3390/i8111111

Bourhia, M., M. Slighoua, S. Ibnemoussa, A. Bari, R. Ullah, A. Amaghnouje and D. Bousta. 2020. Phytochemical study on antioxidant and antiproliferative activities of Moroccan Caralluma europaea extract and its bioactive compound classes. Evid. Based Complement. Altern. Med., 2: 12-34. https://doi.org/10.1155/2020/8409718

Bruyns, P.V., A. Farsi and T. Hedderson. 2010. Phylogenetic relationships of Caralluma R. Br. (Apocynaceae). Taxon, 59(4): 1031-1043. https://doi.org/10.1002/tax.594004

De-Fatima, A., L.V. Modolo, L.S. Conegero, R.A. Pilli, C.V. Ferreira and L.K. Kohn. 2006. Lactones and their derivatives: Biological activities, mechanisms of action and potential leads for drug design. Curr. Med. Chem., 13: 3371-3384. https://doi.org/10.2174/092986706779010298

Falodun, A., R. Siraj and M.I. Choudary. 2009. GC- MS analysis of insecticidal stem essential oil of Pyrenacantha staudtii Hutch and Dalz (Icacinaceae). Trop. J. Pharm. Res., 8: 139-143. https://doi.org/10.4314/tjpr.v8i2.44522

Fayyaz, M., U. Sarwar, F. Nurjis, J.S. Ali, S. Tabassum and A. Raza. 2023. In vitro anti-cancer effect of polymeric nanoparticles encapsulating Caralluma tuberculata in cancer cells. Int. J. Adv. Nano Comput. Anal., 2(1): 22-38.

Fernie, A.R., R.N. Trethewey, A.J. Krotzky and K. Willmitzer. 2004. Metabolite profiling: From diagnostics to systems biology. Nat. Rev. Mol. Cell Biol., 5: 763-769. https://doi.org/10.1038/nrm1451

Haider, S.I., A. Asif, H.M.F. Rasheed, A. Akram and Q. Jabeen. 2022. Caralluma tuberculata exhibits analgesic and anti-arthritic potential by downregulating pro-inflammatory cytokines and attenuating oxidative stress. Inflammopharmacology, 30: 621–638. https://doi.org/10.1007/s10787-022-00949-5

Hamburger, M. and K. Hostettmann. 1991. Bioactivity in plants: the link between phytochemistry and medicine. Phytochemistry, 30: 3864-3874. https://doi.org/10.1016/0031-9422(91)83425-K

Harborne, J.B., 1986. Plant flavonoids in biology and medicine: Biochemical pharmacological, and structure–activity relationships. pp. 15-24.

Hussain, W., M. Ullah, G. Dastagir and L. Badshah. 2018. Quantitative ethnobotanical appraisal of medicinal plants used by inhabitants of lower Kurram, Kurram agency, Pakistan. Avicenna J. Phytomed. 8: 313. 

Iftikhar, N., A. Saleem, M.F. Akhtar, G. Abbas, S. Shah, S. Bibi, G.M. Ashraf, B.S. Alghamdi and T.S. Abujamel. 2022. In vitro and in vivo anti-arthritic potential of Caralluma tuberculata NE brown and its chemical characterization. Molecules, 27(19): 6323. https://doi.org/10.3390/molecules27196323

Ishaq, H., K. Rajendran and K. Nisar. 2023. A comprehensive review of medicinal uses and phytochemicals isolated from Caralluma tuberculata. Rheumatism, 176(96): 98. https://doi.org/10.25081/ia.2023-06

Javed, H., S. Tabassum, S. Erum, I. Murtaza, A. Muhammad, F. Amin and M.F. Nisar. 2018. Screening and characterization of selected drugs having antibacterial potential. Pak J. Pharmaceutical Sci., 31(3). http://142.54.178.187:9060/xmlui/handle/123456789/16359

Khan, M.A., K.H. Maqsood and O.S. Uslu. 2008. Caralluma tuberculata-An important medicinal plant to beconserved. Pak. J. Bot. 41(2): 1271-1582.

Khan, M.A., K. Maqsood and O.S. Uslu. 2019. Caralluma tuberculate- An important medicinal plant to be conserved. Biol. Divers. Conserv. 12: 189–196. https://doi.org/10.5505/biodicon.2019.33043

Makeen, H.A., S.J. Menachery, S.S. Moni, S.S. Alqahtani, Z. ur Rehman, M.S. Alam, S. Mohan and M. Albratty. 2020. Documentation of bioactive principles of the exudate gel (EG) from the stem of Carallumaretrospiciens (Ehrenb) and in vitro antibacterial activity–Part A. Arabian J. Chem., 13(8): 6672-6681.

Mathekaga, A.D. and J.J.M. Meyer. 1998. Antibacterial activity of South African Helichrysum species. South Afr. J. Bot., 64: 293-295. https://doi.org/10.1016/S0254-6299(15)30903-0

Noreen, S. 2017. A mini review on a Caralluma tuberculata NE Br. uncommon and wild succulents but having exciting pharmacological attributes. Pure Appl. Biol., 6(2): 748-761. https://doi.org/10.19045/bspab.2017.60080

Packialakshmi, N. and S. Naziya. 2014. Screening of antibacterial and phytochemical analysis of Caralluma fimbriata. Pharma Innovat., 3(6, Part B): 65.

Rauf, A., M. Jan, W. Rehman, and Muhammad, N. 2013. Phytochemical, phytotoxic and antioxidant profile of Carallumatuberculata NE Brown. Wudpecker J. Pharm. Pharmacol., 2(2): 21-25.

Rehman, R., M. Chaudhary, K. Khawar, G. Mannan and M. Zia. 2014. In vitro propagation of Caralluma tuberculata and evaluation of antioxidant potential. Biologia, 69(3): 341-349. https://doi.org/10.2478/s11756-013-0322-z

Roberts, J.K.M. and J.H. Xia. 1995. High-resolution NMR methods for study of higher plants Methods. Cell Biol., 49: 245-258. https://doi.org/10.1016/S0091-679X(08)61458-2

Ronald, H.A., 1997. Gas chromatography mass spectroscopy: Handbook of instrumental techniques for analytical chemistry. pp. 609-611.

Rahman, S., M. Zahid, A.A. Khan, T. Aziz, Z. Iqbal, W. Ali, F.F. Khan, S. Jamil, M. Shahzad, M. Alharbi and A. Alshammari. 2022. Hepatoprotective effects of walnut oil and Caralluma tuberculata against paracetamol in experimentally induced liver toxicity in mice. Acta Biochim. Polonica, 69(4): 871-878.

Salih, A.M., F. Al-Qurainy, M. Nadeem, M. Tarroum, S. Khan, H.O. Shaikhaldein, A. Al-Hashimi, A. Alfagham and J. Alkahtani. 2021. Optimization method for phenolic compounds extraction from medicinal plant (Juniperus procera) and phytochemicals screening. Molecules, 26(24):7454.

Serwecińska, L., 2020. Antimicrobials and antibiotic-resistant bacteria: A risk to the environment and to public health. Water, 12(12): 3313.

Vanitha, A., K. Kalimuthu, V. Chinnadurai and K.J. Nisha. 2019. Phytochemical screening, FTIR and GC-MS analysis of aqueous extract of Caralluma bicolor–An endangered plant. Asian J. Pharm. Pharmacol., 5(6):1122-1130.

Waheed, A., J. Barker, S.J. Barton, G.M. Khan, Q. Najm-us-Saqib, M. Hussain, S. Ahmed, C. Owen and M.A. Carew. 2011. Novel acylated steroidal glycosides from Caralluma tuberculata induce caspase-dependent apoptosis in cancer cells. J. Ethnopharmacol., 137: 1189–1196. https://doi.org/10.1016/j.jep.2011.07.049

Zarei, Z., D. Razmjoue and J. Karimi. 2020. Green synthesis of silver nanoparticles from Caralluma tuberculata extract and its antibacterial activity. J. Inorgan. Organ. Polymers Mater., 30: 4606-4614. https://doi.org/10.1007/s10904-020-01586-7