Phytochemical Assessment of Mimusops elengi Linn. Unripe fruits Methanol extracts using Modern Analytical Technique

 

Bishwanath Mishra*, Sujit Dash, Amaresh Chandra Sahoo, Prabhat Kumar Sahoo, Sazia Tabasum

Institute of Pharmacy and Technology, Salipur, Cuttack, Odisha- 754202, India.

 

ABSTRACT:

Mimusops elengi Linn. is a plant with high medicinal and commercial value. Although several secondary metabolites have been reported from different species of this plant, there has been not much information available on the complete profile of phytochemical constituents in Mimusops elengi Linn. The HPTLC fingerprinting of methanol extract showed 6 peaks having maximum R_f values 0.10, 0.27, 0.34, 0.51, 0.72, and 0.80 at 254nm. The same extract showed 3 peaks having maximum Rf value 0.25, 0.29 and 0.49 at 366 nm. This study applies Gas chromatography-Mass spectrometry technique to determine the possible chemical components in the methanol extract of Mimusops elengi Linn. Unripened fruits reports for the first time most extensive profile of the plant. The determination and identification of bioactive chemical compounds is established and based on the peak area, retention time molecular weight, and molecular formula. GC-MS analysis of Mimusops elengi Linn. revealed the existence of the 2,2’ Methylenebis [3,4,6-trichloroanisole] which was found to be major component followed by Z-1,9-Hexadecadiene, Cyclopentaneundecanoic-acid, 4-Methyloctanoic acid and E-10-Dodecen-1-ol propionate. The results obtained may be helpful to the further study of pharmacological action for their promising utilization as therapeutic agents.

 

 

 

INTRODUCTION:

Mimusops elengi Linn. tree is the indigenous and natural of the western peninsula. The tree is found in south India in dry evergreen forests from the Krishna southwards and in ravines in the hills up to 20 meters along the western coast and lower ghats in moist evergreen forests. The fruits are sweet and sour, aphrodisiac or inducing sexual desire, diuretic, astringent to the bowels, and good in gonorrhoea. The ripe fruit pulp is sweetish and astringent and has been used in treating chronic dysentery^1,2.

 

In recent years, human pathogens have grown resistance in the antiphon to the indiscriminate utilization of commercial drugs frequently employed in the treatment of infectious diseases³.

 

This situation, the inadmissible side effect of certain antibiotics, and the emergence of previously uncommon infections, has forced scientists to look for new substances from various sources, such as medicinal plants^4,5. The screening of plant extracts and plant products for different pharmacological activity has shown that plants represent a potential source of medicines.^6,7 The main purpose of this research was the screening of the secondary metabolites products from Mimusops elengi Linn. by using the modern analytical techniques like HPTLC and GC-MS.

 

MATERIALS AND METHODS:

Preparation of extract:

The air-dried raw fruits were loaded into soxhlet apparatus and subjected, levied to extraction for about 72 hours with methanol, chloroform, and petroleum ether (60-80⁰C) successively. After extraction, the solvent was distilled off and the extract was evaporated and concentrated in a rotary evaporator under reduced pressure. The extracts were stored or reserved in a refrigerator until tested. The four extracts were then subjected to phytochemical analysis. The extracts which showed the maximum phytochemical constituents had been subjected to the modern analytical techniques like HPTLC and GC-MS.

 

High performance thin layer chromatography:

HPTLC method is a modern sophisticated and automated separation technique derived from TLC. Pre-coated HPTLC graded plates and auto sampler was used to achieve or to attain precision, sensitive, significant separation both qualitatively and quantitatively. High performance thin layer chromatography (HPTLC) is a valuable and helpful quality assessment tool for the evaluation of botanical materials efficiently, accurately, and cost effectively. HPTLC method offers high degree of selectivity, sensitivity, and rapidity combined with single-step sample preparation^8,9. In addition it is a reliable, decisive method for the quantization of nanogram levels of samples. Thus this method can be conveniently, easily adopted for routine quality control analysis and investigation. It provides a chromatographic fingerprint of phytochemicals which is suitable for confirming the identity and purity of medicinal plant raw materials^10,11,12,13.

 

Basic steps involved in HPTLC

Extracts used              :  Methanol

Application mode     :  CAMAG Linomat 5

Development mode  :  CAMAG Twin Trough chamber.

 

Sample application:

The samples were dissolved in same solvent and 10μl quantity of sample was applied on the HPTLC silica merk 60F 254 graded plate sized 6cm x 10cm as narrow bands using CAMAG Linomat 5 injector.

 

Chromatogram development:

It was carried out in CAMAG Twin Trough chambers. Sample elution was carried out according to the adsorption capability of the component to be analyzed. After elution, plates were taken out of the chamber and dried.

 

Gas chromatography – Mass Spectrum analysis:

The GC-MS analysis of the plant extract was contrived in a (789 Agilent) instrument under computer control at 70 eV. About 1 liter of the methanol extract was injected into the GC-MS using a micro syringe and the scanning was done for 45 minutes.^14,15,16 As the compounds were separated or apportioned, they are eluted and fluted from the column and entered a detector that was capable of creating an electronic signal whenever a compound was detected. The higher or larger the concentration in the sample, the bigger was the signal obtained which was then processed by a computer. The time from when the injection was made and contrived (Initial time) to when elution occurred is referred to as the retention time (RT). While the instrument was run, the computer bring about or develop a graph from the signal called Chromatogram. Each of the peaks in the chromatogram represented the signal created when a compound eluted from the Gas chromatography column into the detector. The x-axis showed the RT and the y-axis measured the intensity of the signal to quantify the component in the sample injected. As individual compounds eluted from the gas chromatographic column, they entered or filed the electron ionization (mass spectroscopy) detector, where they were bombarded or besiege with a stream of electrons causing them to break apart into fragments. The fragments obtained and collected were indeed charged ions with a certain mass. The M/Z (mass/charge) ratio obtained was calibrated, graded from the graph obtained, which was called the mass spectrum graph which is the fingerprint or identify of a molecule. Before analyzing and evaluating the extract using gas chromatography and Mass spectroscopy, the temperature of the oven, the flow rate of the gas used, and the electron gun were programmed initially. The temperature of the oven was maintained and controlled at 100°C. Helium gas was used as a carrier or bearer as well as eluent. The flow rate of helium was set to 1ml per minute. The electron gun of mass detector emancipated and liberated electrons having the energy of about 70eV.The column employed here for the separation of components or factor was Elite 1(100% dimethyl poly siloxane). The identity of the components in the extracts was assigned and accredited by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures. Compounds were identified and classified by comparing their spectra to those of the Wiley and NIST/EPA/NIH mass spectral libraries.

 

RESULTS AND DISCUSSION:

High-performance thin layer chromatography (HPTLC) technique is the most simple and fastest separation technique available today which gives better precision and accuracy with extreme flexibility for various steps. In this study the HPTLC fingerprinting exhibiting number of peaks, maximum R_f value of methanol extract are shown in (Figure 1 and 2) for 254nm and 366nmrespectively. The methanol extract showed 6 peaks having maximum R_{f value} 0.10, 0.27, 0.34, 0.51, 0.72, and 0.80 at 254nm. The same extract showed 3 peaks having maximum Rf value 0.25, 0.29 and 0.49 at 366 nm. This HPTLC technique may be useful for both the identification and the quality evaluation of preparations containing Mimusops elengi Linn. unripe fruit extract.

The GC-MS chromatogram (Figure-3) of methanolic extract of Mimusops elengi Linn. unripened fruits revealed five major compounds which belonged to various classes of chemicals. The result revealed that 2,2’-Methylenebis[3,4,6-trichloroanisole] (RT-4.30) was found to be major component followed by Z-1,9-Hexadecadiene (RT-24.00), Cyclopentaneundecanoic-acid (RT-24.09, Figure 3.6), 4-Methyloctanoic acid (RT-22.37). A very small quantity of E-10-Dodecen-1-ol propionate (RT-23.39) was also reported. The peak report of the chromatogram obtained with details of peak number, retention time, area percentage, name of the identified component, its molecular formula and molecular weight, are presented in Table1. The mass spectrum was obtained with a mass/charge ratio on the x-axis and relative intensity on the y-axis in the sample injected. As individual compounds eluted from the gas chromatographic column, they entered the electron ionization (mass spectroscopy) detector, where they were bombarded with a stream of electrons causing them to break apart into fragments. The fragments obtained were actually charged ions with a certain mass. The M/Z (mass/charge) ratio obtained was calibrated from the graph obtained, which was called as the Mass spectrum graph and which is the fingerprint of a molecule. GCMS is one of the techniques to identify the bioactive constituents of long, branched-chain hydrocarbons, acids, alcohols, esters, etc.

 

Figure 1: HPTLC chromatogram and spectral analysis of Mimusops elengi Linn. raw fruits extracts at 254 nm

 

Figure 2: HPTLC chromatogram and spectral analysis of Mimusops elengi Linn. raw fruits extracts at 366 nm

 

Figure 3: GC-MS chromatogram of methanol extract of Mimusops elengi Linn.raw fruits

 

Table 1: Compounds identified by GC-MS analysis of methanol extract of Mimusops elengi Linn.unripen fruits

 

CONCLUSION:

The Study on Mimusops elengi Linn. unripe fruits shows that the methanol extract may be the mixture of different components. Gas chromatography and mass spectroscopy analysis put on view the availability of the various compounds with variable molecular weight. This experiment showed that the stronger extraction capacity of methanol could have produced number of bioactive constituents. These various bioactive compounds might be utilized for the expansion for the drug development without any side effects, purely in traditional way.4-Methyloctanoic acid and E-10-Dodecen-1-ol propionate can be isolated and used as flavouring agents^17,18. At this end, it can be concluded that the in vivo studies on the crude extract will open up to new ways for natural drug that can be employed for clinical trials which may generate successful results in the future. The medicinal activities of some of the compounds are not known yet. It is quite possible that further research on these compounds could through light on the medicinal efficacy of Mimusops elengi Linn. This is a preliminary work towards understanding more about the medicinal and pharmaceutical aid efficacy of Mimusops elengi Linn.

 

REFERENCE:

1.      Kirthikar KR, Basu BD. Indian Medicinal Plants, 2nd ed.; Popular Publications: Dehradun, 1999.

2.      Satyapal U, Mahajan D, Tatke P, Naharwar V. Phytochemical Investigation and Assessment of Antioxidant and Antimicrobial Potential of Bark of Mimusops elengi. Research Journal of Pharmacy and Technology. 2014; 7(11): 1226-1230.

3.      Sharma H, Karnwal A. Impact of Herbal Extracts in Biocontroling of Four Human Pathogenic Bacteria- an in-vitro Study. Research Journal of Pharmacy and Technology. 2018; 11(7): 2895-2900.

4.      Marchese A, Shito GC. Resistance patterns of lower respiratory tract pathogens in Europe. International Journal of Antimicrobial Agents. 2001; 16(1): 25-29.

5.      Poole K. Overcoming antimicrobial resistance by targeting resistance mechanisms. The Journal of Pharmacy and Pharmacology. 2001; 53 (3): 283-94.

6.      Amani S et al. Antimicrobial activities in some argentine medicinal plants. Acta Horticulture 1998; 501: 115-122

7.      Salvat A et al. Screening of some plants from Northern Argentina for their antimicrobial activity. Letters in Applied Microbiology. 2001; 32(5): 293-97.

8.      Harborne JB. Phytochemical methods. Edn 3rd London, Chapman and Hall, 1998, pp. no-1-28.

9.      Wagner H, Baldt S, Zgnaisnki EM. Plant drug analysis, New York, Berlin, Springer, 1996; 355-357.

10.   Daharwal SJ, Shrivastava S. Preliminary Phytochemical Screening and HPTLC Fingerprinting of Extracts of Thuja occidentalis. Research Journal of Pharmacy and Technology. 2019; 12(10): 4782-4784.

11.   Anto Arockia Raj A, Vinnarasi J, Venkataraman R, Augustin M. HPTLC Fingerprinting Analysis of Tannin Profile on Canthium coromandelicum and Flueggea leucopyrus willd. Research Journal of Pharmacy and Technology. 2018; 11(12): 5355-5358.

12.   Patel GH, Prajapati ST, Patel CN. HPTLC Method Development and Validation for simultaneous Determination of Cinitapride and Pantoprazole in Capsule Dosage Form. Research Journal of Pharmacy and Technology. 2011; 4(9): 1428-1431.

13.   Patidar P, Singh S, Dubey D, Dashora K. Estimation of Rutin in Tephrosia purpurea by HPTLC Method. Research Journal of Pharmacy and Technology. 2013; 6(1): 58-60.

14.   Moualla N, Naser M. Using GC/MS to Study the Chemical Composition of Essential Oil of Thymus vulgaris L. at AL-Qadmous Area, Syria. Research Journal of Pharmacy and Technology. 2015; 8(4): 437-442.

15.   Priya S, Nethaji S, Sindhuja B. GC-MS Analysis of Some Bioactive Constituents of Diospyros virginian. Research Journal of Pharmacy and Technology. 2014; 7(4): 429-432.

16.   Aravind R, Bindu AR, Bindu K, Alexeyena V. GC-MS Analysis of the Bark Essential Oil of Cinnamomum malabatrum (Burman. F) Blume. Research Journal of Pharmacy and Technology. 2014; 7(7): 754-759.

17.   http://www.thegoodscentscompany.com/data/rw1552251.html

18.   Hussein J, Hameed IH, Hadi I, Mohammed. Using Gas Chromatography-Mass Spectrometry (GC-MS) Technique for Analysis of Bioactive Compounds of Methanolic Leaves extract of Lepidium sativum. Research Journal of Pharmacy and Technology. 2017; 3981-3989.

 

 

 

Received on 29.12.2019           Modified on 07.06.2020

Accepted on 19.09.2020         © RJPT All right reserved