Rapid Phytochemical Profiling of Moringa oleifera and Mucuna pruriens Leaf Extracts for their Pharmacological Activity by GC-MS

Introduction

Traditional knowledge systems have repeatedly proven their roots in healing multiple diseases/ disorders/ discomforts in the human mankind by using herbal alternatives instead of modern medicine. Preliminary screening of such herbal remedies in the form of a whole plant or in parts is hence an essential aspect of standardization. Such screenings are performed in multiple ways as per the guidelines of World Health Organization (WHO). Post this evaluation; assessment of bioactivities and proving those therapeutic values by using in-vivo or in-vitro techniques becomes crucial. Phytoconstituents or secondary metabolites are the medico-therapeutic agents of plants.

Mucuna pruriens and Moringa oleifera belong to the Fabaceae and Moringaceae family respectively from kingdom plantae. These two families are known for their distinguished medicinal properties. Anti-inflammatory, antidiabetic, Antibacterial, Antioxidant, etc. are some of the few prominently known activities. Herbal plants are the good source of medicinal ingredients having potency in developing new drugs which begins with identifying active ingredients from plant sources. The screening of plant extracts is a novel approach to evaluate therapeutically active phytoconstituents in the plant samples.¹

Variety of plant components with multiple pharmacological activity are screened in preliminary physical and chemical investigations. These molecules have fundamental functional groups such as hydroxyl, alcohols, aldehydes, benzene rings, steroids.²

Plant samples are to be prepared via suitable extraction techniques with optimized suitable solvent for getting maximum components for analysis. Extraction is a crucial first stage when the sample has to be analyzed by modern analytical techniques. Suitable extraction protocol is a key to precise separation, identification and analysis of active ingredients.³ Post extraction separation and simultaneous identification or characterization has to be done. Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), etc. are known to be the most used chromatographic techniques for preliminary screening of components.

In herbal research chromatographic fingerprints are the most often and prominently used for quality control purposes to unveil the phytochemical profile of the plant.⁴

GC-MS is another such tool which has brought revolution in the field of analytical chemistry. This amalgamation of chromatography and spectroscopy gives us new dimensions of information on our samples. Samples in volatile state are easily analyzed by GC-MS whereas the ones which are not volatile in the original state can be converted to volatile organics by derivatization techniques.

Materials and Methods

Collection of plant material

Fresh Moringa oleifera and Mucuna pruriens leaves are collected from Fort area, Mumbai, Maharashtra.

Preparation of extract

Both the plant leaves are sun dried and grounded in mixer   grinder. 1gm of these finely grounded plant leaves powder was weighed separately and following extracts were prepared using 10ml of the respective solvent in separate labelled flasks as per the conditions.

Extract I: Steady maceration Methanol MPL

Extract II: Accelerated Maceration Methanol MPL

Extract III: Accelerated Maceration Ethanol MPL

Extract IV: Soxhlet Methanol MPL

Extract V: Steady maceration Methanol MOL

Extract VI: Accelerated maceration Methanol MOL

Extract VII: Soxhlet Methanol MOL

The accelerated extract was exposed to ultrasonication at the temperature of 45ºC for          30 minutes and was kept for overnight extraction at steady state. After 24hrs of incubation the filtered extract was further diluted 1:100 times and 1ml volume is injected in the GC-MS system. The sound vibrations are generated in a sonicator the accelerated extraction process is expected to give good yield of the extract.

GC-MS analysis

Different extracts were prepared and filtered using 0.2micron syringe filters and injected in the GC-MS system with an injection volume of 1ml for all the samples. The temperature used was a programmed method for the present work. Sample injector was kept at 240ºC temperature. The Carrier gas flow was 1.0mL/minute. Rtx-5sil MS capillary column was used for the analysis. The initial column temperature was 70ºC and was kept constant for 2 minutes, it was then increased up to 280ºC at the rate of 10ºC/minute followed by a hold at the said temperature for 10 minutes. The total run time was 33 minutes. The split mode of injection was also applied and Interface was at 270ºC. MS was operated on scan mode for Retention time (R_t in minutes) determination of reference standards with a mass range of (m/z) 35-600. 

Results 

Table 1: Prominent molecules found in GC-MS screening and approximate % content

Table 2: Name of the component with the structure and their activity

 Table 3: Different components identified from Mass spectra of Extract 1

 Table 4: Different components identified from Mass spectra of Extract 1

 Table 5: Different components identified from Mass spectra of Extract 2

 Table 6: Different components identified from Mass spectra of Extract 3

 Table 7: Different components identified from Mass spectra of Extract 4

 Table 8: Different components identified from Mass spectra of Extract 5

 Table 9: Different components identified from Mass spectra of Extract 5

 Table 10: Different components identified from Mass spectra of Extract 6

 Table 11: Different components identified from Mass spectra of Extract 6

Table 12: Different components identified from Mass spectra of Extract 7

 Table 13: Different components identified from Mass spectra of Extract 7

 Discussion

The above tables are prepared from Total Ion Chromatograms (TICs) and Mass spectras by all the extracts run by GC-MS of the two aforementioned plants. Peak height and peak areas are also tabulated. Table 1 shows the approximate percentage content of distinguished components in the plants. As per the results, Octadecanoic acid, hexadecenoic acid, Amyrins, Sitosterol and Lupeol are found to be the prominent components in both the plants. Both the plants are widely distributed in nature and are effective to reveal many medicinal properties and other biological activities. Leaf extracts are considered for revealing other important phytochemicals which can be an asset for curing multiple other diseases or disorders.

Octadecanoic acid

Stearic acid is extracted in highest amount in moringa extract by accelerated maceration process. Soxhlet extract has worked best for extraction from MPL. Heating the extract at controlled rate increases the concentration of the components.

Hexadecenoic acid

Palmitic acid is found to be extracted in maximum concentration by Soxhlet extraction from both the plants. Repetitive continuous method of extraction with appropriate heat seems to have worked best for hexadecenoic acid.

Beta amyrin

Accelerated maceration with ultrasonication and heating gives more yield of beta amyrin. Mucuna pruriens is showing more concentration as compared to Moringa oleifera.

Alpha amyrin

Highest extracted analyte is alpha amyrin in both the plants. Alpha amyrin shows lowest solubility in ethanol extract. Velvet beans extracts are producing more amyrins considering their therapeutic importance.

Gamma sitosterol:

Gamma sitosterol is extracted more by acceleration of extraction. It is non polar molecule which is extracted better with acceleration of sound waves being introduced.

Lupeol

Lupeol is also a significant non polar molecule which is extracted better in just Mucuna pruriens with Soxhlet extraction. Soxhlet is a continuous extraction process used for better yield of non-polar analytes. 

Conclusion

Alcoholic accelerated maceration is proven to be the most suitable method for both the plants in-order to extract maximum number of phytoconstituents. Alcohol is an effective organic solvent having good extractive power and are stable agents for preserving the extracts for longer duration of time. GC-MS or any other instrumental techniques used for analysis are compatible with such organic solvents preferably alcoholic solvents as they are neither extremely non-polar nor they are highly polar, hence alcoholic extracts are most preferred. Soxhlet is also showing satisfactory results but considering the sustainable and greener approach; usage of low volumes of solvents is possible in maceration. Both the techniques of extraction have their pros and cons. Methanol is considered to be most useful solvent of choice. GC-MS is also considered as a greener technique considering its negligible consumption of solvents and ease of analysis. GC-MS uses gas as the mobile phase and does not involve any liquid for its use as it is not an environment friendly approach. Since, the injection volume is negligible the sample consumption required is also minimal. GC-MS is a tool showing high sensitivity and selectivity having the ability to provide qualitative and quantitative analysis of the data. It is known to be the most versatile tool which can analyze wide range of samples including volatile organic compounds, drugs, biological samples, pesticides. GC analyses volatile samples & MS works in vacuum hence, hyphenating these two techniques for identification of plant components is a much quicker and suitable approach in herbal chemistry. The NIST library reveals majority of components in the form of ions by GC-MS in both the plants. All the above components are predominantly best antioxidants and hence have crucial role in showing action against multiple diseases.

Acknowledgements

The authors thank Department of Bioanalytical Sciences, Ramnarain Ruia Autonomous College, Mumbai, Department of Chemistry, St. Xavier’s College (Autonomous), Mumbai and P.S. Ramanathan Advanced Instrumentation Centre, Ruia College for their unconditional support throughout the progression of the work. 

Conflict of Interest

Neither the authors nor the institutions have any conflict of interest. 

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Abbreviations List

MPL extract– Mucuna pruriens leaf extract

MOL extract– Moringa oleifera leaf extract