GC-MS PROFILING OF BIOACTIVE COMPOUNDS IN ASPARAGUS RACEMOSUS: IMPLICATIONS FOR PHARMACOLOGICAL PROPERTIES

GC-MS PROFILING OF BIOACTIVE COMPOUNDS IN ASPARAGUS RACEMOSUS: IMPLICATIONS FOR PHARMACOLOGICAL PROPERTIES

Anup Hemant Eden and Vootla Shyam Kumar *

P. G. Department of Studies in Biotechnology and Microbiology, Karnatak University, Dharwad, Karnataka, India.

ABSTRACT: Background: Plants for remedial applications are well known, realizing the pharmacological potentials of plants through analytical techniques. Asparagus racemosus is a well-known medicinal plant analyzed using biological approach. The present study was carried out to evaluate the antioxidant, ani-inflammatory, antibacterial effects and the possible bioactive compounds present in the chloroform, methanol and aqueous extract of A. racemosus plant. Methods: The whole plant was subjected to serial solvent extraction using chloroform, methanol, and aqueous. Phytochemical screening, total phenolic and total flavonoid contents were determined using standard methods and was followed by GCMS and FTIR for biological activity of compounds. The plant extracts were assessed for antioxidant activity by DPPH and FRAP assays, anti-inflammatory activity by protein denaturation method, and antibacterial activity by agar well diffusion method. Results: The phytochemical tests revealed the presence of phytochemicals like alkaloids, flavonoids, glycosides, triterpenoids, and saponins. The GCMS of whole plant chloroform, methanol and aqueous extract revealed the compounds that are rich in free radical scavenging activity and proven to be the best source for antioxidant activity. The phenolics, flavonoids are linked with its antibacterial property. Conclusion: The present study helps to find out the novel bioactive compounds having pharmacological properties to formulate the safer drugs for the treatment of deadly diseases bothering mankind.

Keywords: Phytoconstituents, Asparagus racemosus, Antioxidant activity, Antibacterial activity, Anti-inflammatory activity

INTRODUCTION: Plants represent a prominent source for many pharmaceuticals as the phytochemical compounds or the secondary metabolites present in the plants have been used for treating a number of human ailments. Drugs obtained from medicinal plants comprise 25% of total drugs in developed countries and about 80% in developing countries ¹.

Plant products have been part of phytomedicines since time immemorial and can be derived from barks, leaves, flowers, roots, fruits, seeds. India has a rich cultural history as plants are the primary source of medicine in Ayurveda, Siddha, and Unani systems of medicine ².

The secondary metabolites or bioactive compounds derived from the plants are the primary components of phytomedicine. They have numerous applications in the treatment of various ailments including, chronic and infectious diseases ³. Despite this, oxidative stress is the primary cause for the development and progression of several diseases ⁴. Antioxidants reduce the harmful effects of oxidative damage caused by reactive oxygen species (ROS). These antioxidants are found in variety of plant species that can reduce the oxidative damage caused on by ROS ⁵, resulting in the prevention of cancer, cardiovascular diseases, and other neurological disorders ⁴. Therefore, the major focus in modern pharmacology is the exploration of medicinal plants for their bioactive metabolites, such as phenols, alkaloids, terpenoids etc., which have various pharmacological effects like antioxidant, anti-inflammatory, and antibacterial properties ⁴.

Asparagus racemosus (Family: Asparagaceae), is a tropical and subtropical medicinal plant of India. A. racemosus is a woody climber growing to 1-2 m in height. The unique characteristics of this plants are its small, uniform and pine needles like leaves with whitish coloured flowers ⁶. It is commonly known as ‘Shatavari’ and found to have immense pharmacological properties such as antioxidant, anti- HIV, hepato-protective, anti-diarrhoeal, antiulcer, and antibacterial effects ^7-9. A. racemosus has been used in Ayurveda as a galactagogue, aphrodisiac, anodyne, diuretic, antispasmodic and nervine tonic since time immemorial ¹⁰.

Even though the plant A. racemosus has many therapeutic applications, the systematic analysis of this plant is still undetermined in terms of their chemical constituents. The present study was carried out to identify the phytochemical compounds and their pharmacological functions as present in the chloroform, methanol and aqueous extracts of the plant through GCMS and FTIR analysis. The plant extracts were also evaluated for their antioxidant, antibacterial, and anti-inflammatory properties.

MATERIAL AND METHODS:

Collection of Sample: The disease-free plant sample was collected from the Botanical Garden of Karnataka University in Dharwad, Karnataka, India (15.4589ºN, 75.0078ºE). The plant sample was authenticated by the Botanical Survey of India at the Southern Regional Centre in Coimbatore and identified as Asparagus racemosus, belonging to the Asparagaceae family Fig. 1. The collected sample was wash thoroughly several times with tap aqueous, shade dried, grind to fine powder and stored in tight sealed containers. The nature of the powder is examined physically by the characteristics such as color, odour and texture.

FIG. 1: HABITAT OF THE PLANT ASPARAGUS RACEMOSUS

Solvent Extraction: The serial solvent extraction of the coarsely powdered sample was done using Soxhlet apparatus with (1:10) ratio with chloroform, methanol, and aqueous as a solvent. After extraction the solvent was evaporated and the residue was stored in airtight containers for further use.

Preliminary Phytochemical Screening: The presence of various phytochemicals including alkaloids, flavonoids, glycosides, phenols, tannins, steroids, saponins, terpenoids, carbohydrates and proteins were analysed in the crude extracts of Asparagus racemosus plant using standard procedures ^{11, 12, 13}.

Estimation of Total Phenolic Content (TPC): The total phenolic content of the A. racemosus plant extract was determined by Folin-Ciocalteu method. The plant extract was oxidized using 1.5 mL of Folin-Ciocalteu (FC) reagent and 7.5% Sodium carbonate (Na₂CO₃) solution. After 1 hour incubation at room temperature, the absorbance was read at 750nm. The amount was calculated using gallic acid calibration curve and expressed as Gallic acid equivalent (GAE) mg/g of sample ¹⁴. The experiment was performed in triplicates and results were expressed as mean ± standard deviation.

Estimation of Total Flavonoid Content (TFC): The total flavonoid content of A. racemosus plant extract was determined using a spectrophotometric method. The plant extract was mixed with 10% Aluminum chloride (AlCl₃) and 1M sodium acetate. The absorbance was measured at 415nm after the incubation in the dark for 45min.The experiment was performed in triplicates and results were expressed as mean ± standard deviation. The amount was calculated using Quarcetin calibration curve and the results were expressed as quercetin equivalent (QE) mg/g of sample ¹⁵.

GCMS Analysis: GC-MS analysis of the different extracts of A. racemosus was carried out by using the GC-MS instrument (Model GCMS-QP 2010 plus, Shimadzu). The instrument was operated in electron impact mode at ionization voltage (70 eV), injector temperature (250ºC) and detector temperature (280ºC). About 1µL of the sample was injected into mobile phase consisting of helium (99.9% purity) at a flow rate of 1mL/min. The oven temperature was initially programmed at 60ºC (isothermal for 2 min) before being raised to 100 ºC and finally to 280ºC at a rate of 5 ºC/min for 9 min.

The Gas Chromatogramran for 34 minutes in total and the relative percentage amount of each component was calculated by comparing its average peak area to the total area. The comparison was made between the spectra of unknown component and the spectrum of the known components with the help of the National institute of Standards and Technology-5 (NIST-5) library, and the compounds were identified including the compound’s name, their molecular formula, molecular weight and their structure.

FTIR Spectroscopic Analysis: FTIR analysis of A. racemosus plant was performed using Perkin Elmer Spectrum system (version 10.7.2), which was used to detect the characteristic peaks ranging from 500-4000 cm^-1 and their functional groups. The peak values of the FTIR were recorded.

Pharmacological Activity of Asparagus racemosus:

In-vitro Antioxidant Activity of A. racemosus Plant Extract:

2, 2-diphenyl-1-picryl-hydrazyl (DPPH) Radical Scavenging Assay: Free radical scavenging activity of the plant extracts of A. racemosus was evaluated according to Vardhini et al. method ¹⁶. 1mL of DPPH solution was mixed with different concentrations of the plant extracts (50, 100, 150, 200, and 250 µg). The mixture was incubated for 30 min in the dark at room temperature and the absorbance was measured at 517 nm. Ascorbic acid was used as standard reference. The capacity of radical scavenging activity was calculated using the following equation:

DPPH scavenging effect (%) = (A_C – A_S)/ A_C ×100

Where, A_C is the absorbance of the control reaction and A_S is the absorbance of the sample. The experiment was performed in triplicates and the IC₅₀ value was calculated for all the samples.

Ferric Ion Reducing Power Assay: Ferric ion reducing power (FRAP) assay was measured according to Asraoui method ¹⁷ with slight modifications. The different concentration of A. racemosus plant extracts (50, 100, 150, 200, and 250 µg) were added to the mixture containing 2.5 mL of 0.2 M phosphate buffer and 2.5mL of 1% potassium ferrocyanide. This mixture was incubated at 50ºC for 30 min. After cooling, 2.5 mL of 10% trichloroacetic acid and 0.5 mL of 0.1% ferric chloride was added. The mixture was left at room temperature for 10 min to form bluish green colour complex. The absorbance was measured at 700 nm and Ascorbic acid was used as a standard.

Anti-inflammatory Activity: Anti-inflammatory activity of different extracts of A. racemosus plant was evaluated by protein denaturation assay using Aspirin as a standard drug ¹⁸. To the different concentrations (50, 100, 150, 200, 250µg) of the plant extracts, 1 mL of phosphate buffered saline and 50 µL of bovine serum albumin (BSA) was added, and incubated for 15 min at room temperature followed by incubation at 70 ºC for 30 min. After cooling, the absorbance was read at 660 nm. The percent of inhibition of protein denaturation was calculated using the following formula:

% Protein denaturation activity = (A_C – A_T)/A_C × 100

Where, A_C is the absorbance of the control reaction and A_T is the absorbance of the sample, and IC₅₀ value was calculated for all the samples.

Antibacterial Activity: Antimicrobial activity of Asparagus racemosus plant extracts were studied by agar well diffusion method ¹⁹ using Pseudomonas aeruginosa, Escherichia coli, Xanthomonas cultures as the test organisms. 100µL of the saline suspension was swabbed uniformly over the sterile agar plates. The different concentrations of the plant extracts (30, 60, 90 and 120µg) along with standard drug Ciprofloxacin (30 µL) were added to the medium. The plates were incubated at 37 ºC for 24 hours and the diameter of zone of inhibition formed around the well after incubation was measured in millimetres.

Statistical Analysis: The experiments were performed in triplicates and the results were expressed as mean ± standard error. The significance was analyzed through two-way ANOVA analysis with significant p value (0.01 and 0.05).

RESULTS AND DISCUSSION:

Preliminary Phytochemical Screening: Plants are a substantial source of potentially useful bioactive components for the development of novel chemotherapeutic medicines due to their rich phytochemicals ²⁰. Medicinal plants are renewable source of drugs, offering safety and efficacy with minimal side effects compared to synthetic drugs, and have been used since time immemorial and their utility is increasing day by day in the present world ²¹. The medicinal plant A. racemosus is a boon to mankind as its rich phytochemicals are used to treat numerous diseases. The qualitative phytochemical screening of different extracts of A. racemosus showed the presence of alkaloids in methanol and aqueous extracts; sterols in chloroform and aqueous extracts; flavonoids, glycosides, phenols, tannins and saponins were detected in all the three extracts i.e., chloroform, methanol and aqueous extracts whereas terpenoids, carbohydrates and proteins were not detected in any of the extracts. The result of the phytochemical analysis is presented in Table 1.

Total Phenolic and Flavonoid Content: Several reports tend to show that secondary metabolites that are phenolic nature including flavonoids are responsible for the variety of pharmacological activities ^{22, 23}. Because of this, the total phenolic and total flavonoid contents of A. racemosus plant extracts were determined. In case of total phenolic content, the aqueous extract had the highest amount (73.79 ± 0.009 mg/g of GAE), followed by methanol extract (31.23 ± 0.003 mg/g of GAE), and chloroform extract being the least (10.43 ± 0.006 mg/g of GAE) respectively. While in case of total flavonoid content the aqueous extract of A. racemosus plant showed the maximum flavonoid content (97.43 ± 0.003 mg/g of QE), followed by methanol (84.54 ± 0.005 mg/g of QE) and then the chloroform extract (34.40 ± 0.004 mg/g of QE). A wide range of variation was observed in total phenolic and total flavonoid content in each extract Table 2.

TABLE 1: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF THE WHOLE PLANT EXTRACTS OF ASPARAGUS RACEMOSUS

‘+’ indicates Present; ‘-’ indicates absent

TABLE 2: TOTAL PHENOL AND TOTAL FLAVONOID CONTENT FROM ASPARAGUS RACEMOSUS WHOLE PLANT EXTRACTS

The results are expressed as mg/g equivalent ± standard deviation

FIG. 2: EXTRACTION OF THE WHOLE PLANT ASPARAGUS RACEMOSUS; A. SOXHLET EXTRACTION OF THE PLANT; B. EXTRACTED SOLVENT; C. CRUDE EXTRACT

GC-MS Analysis: The GCMS spectra of chloroform, methanol and aqueous extract of A. racemosus plant showed the presence of 19, 64 and 54 compounds, respectively. Based on the peak area, Phytol, Tetratetracontane, Eicosane and Octadecanal are the major compounds found in chloroform extract. Methanol and aqueous extracts had Sucrose as major compounds while 1,2-Dithiolane-3-carboxylic acid, and Dotriacontane major compounds found in aqueous extract. The compounds identified had a diverse pharmacological propertysuch as Stigmasterol was known to have anti-inflammatory, antioxidant, antimicrobial, anticancer, antiarthritic and anti-asthama activity ^{24, 25}. Phytol was known to have antimicrobial, anti-inflammatory, antiallergic, anticancer, diuretic, antidiabetic, cytotoxicity, antiproliferative, cancer preventive properties ^{24, 26}. The GCMS spectra, compound name, retention time, peak area, molecular weight, molecular formula and their uses are depicted Table 3-5 and Fig. 3-5).

FIG. 3: GC-MS CHROMATOGRAM OF CHLOROFORM EXTRACT OF A. RACEMOSUS PLANT

FIG. 4: GC-MS CHROMATOGRAM OF METHANOL EXTRACT OF A. RACEMOSUS PLANT

FIG. 5: GC-MS CHROMATOGRAM OF AQUEOUS EXTRACT OF A. RACEMOSUS PLANT

TABLE 3:  COMPOUND IDENTIFIED FROM CHLOROFORM EXTRACT OF A. RACEMOSUS AND ITS USES

TABLE 4:  COMPOUND IDENTIFIED FROM METHANOLIC EXTRACT OF A. RACEMOSUS AND ITS USES

TABLE 5: COMPOUND IDENTIFIED FROM AQUEOUS EXTRACT OF A. RACEMOSUSPLANT AND ITS USES

FTIR Analysis: FTIR analysis provide information about the functional groups of the compounds ²⁷ and the A. racemosus plant extracts revealed several functional groups based on the peak value ratio. The functional groups present in the chloroform, methanol, and aqueous extracts of A. racemosus plant were identified based on the peak values in the IR region. The results showed the presence of C=C, C-H, C=O, O-H, C-N, C-O, C-F, S=O, S-C≡N, and N-H groups. The peak values, their respective functional groups and their bond nature are represented in Table 6-8 and Fig. 6-8.

FIG. 6: FTIR SPECTRA OF THE WHOLE PLANT CHLOROFORM EXTRACT OF A. RACEMOSUS

TABLE 6: FTIR INTERPRETATION OF COMPOUNDS OF WHOLE PLANT CHLOROFORM EXTRACT OF A. RACEMOSUS

FIG. 7: FTIR SPECTRA OF THE WHOLE PLANT METHANOLIC EXTRACT OF A. RACEMOSUS

TABLE 7: FTIR INTERPRETATION OF THE WHOLE PLANT METHANOLIC EXTRACT OF A. RACEMOSUS

FIG. 8: FTIR SPECTRA OF WHOLE PLANT AQUEOUS EXTRACT OF A. RACEMOSUS

TABLE 8: FTIR INTERPRETATION OF WHOLE PLANT AQUEOUS EXTRACT OF A. RACEMOSUS

Antioxidant Activity: The antioxidative properties of flavonoids are due to several different mechanisms, such as scavenging of free radicals, chelation of metal ions, such as iron and copper and inhibition of enzymes responsible for free radical generation. Natural antioxidants are more popular these days because of their potential to improve health and fend off diseases ²⁸. The antioxidant activity of A. racemosus whole plant extract was measured and compared with ascorbic acid in different concentrations. DPPH radical scavenging ability of the A. racemosus whole plant extracts exhibited potent antioxidant activity. The result revealed that the aqueous extract exhibited highest percentage of inhibition (73.42 ± 0.531%) at 250 µg and lesser IC₅₀ value of 146.84and in comparison, the standard ascorbic acid had 89.01 ± 0.173% inhibition with IC₅₀ value of 84.61. The percentage inhibition of DPPH and IC₅₀ values are represented in Fig. 9 and Table 9 respectively.

TABLE 9: ANTIOXIDANT ACTIVITY OF A. RACEMOSUS WHOLE PLANT EXTRACT BY DPPH

Results are expressed as mean ± standard error

FIG. 9: ANTIOXIDANT ACTIVITY OF A. RACEMOSUS WHOLE PLANT EXTRACT BY DPPH

The FRAP assay was performed to determine the reducing power of A. racemosus plant extracts. Greater the absorbance of the extracts corresponds to their greater antioxidant activity. Among the extracts, aqueous extract exhibited the highest activity with varying absorbance between 0.331 ± 0.010 to 0.786 ± 0.004. The results are represented in Fig. 10 and Table 10 respectively.

TABLE 10: ANTIOXIDANT ACTIVITY OF A. RACEMOSUS WHOLE PLANT EXTRACT BY FRAP ASSAY

Results are expressed as mean ± standard error

FIG. 10: ANTIOXIDANT ACTIVITY OF A. RACEMOSUS WHOLE PLANT EXTRACT BY FRAP ASSAY

Anti-inflammatory Activity: The non-steroidal anti-inflammatory drugs function by protecting albumin protein denaturation in response to heat treatment ²⁹. Hence, the chloroform, methanol and aqueous extracts of A. racemosus plant was evaluated for their ability to inhibit the denaturation of albumin protein which was attributed to the presence of flavonoid and sterols present in the plant extract. In our study, the chloroform extract showed maximum anti-inflammatory activity (66.71 ± 0.384%at 250 µg) with an IC₅₀ value of 152.88whereas the standard Aspirin revealed an IC₅₀value of 51.63 with the protein inhibition percentage of 87.41 ± 0.262%. The methanolic, chloroform and aqueous extract of A. racemosus plant significantly inhibits the production of nitric oxide which shows a key role in inflammation. The results are represented in the Table 11 and Fig. 11.

TABLE 11: ANTI-INFLAMMATORY ACTIVITY OF A. RACEMOSUS WHOLE PLANT EXTRACT

Results are expressed as mean ± standard error

FIG. 11: ANTI-INFLAMMATORY ACTIVITY OF A. RACEMOSUS WHOLE PLANT EXTRACT

Antibacterial Activity: Plant extracts are a fantastic source of pathogen-fighting antibacterial compounds. They can therefore be utilised to treat a variety of infectious disorders brought on by virulent microorganisms ³⁰. The antibacterial activity of A. racemosus whole plant extract was studied against E. coli, P. aeruginosa and Xanthomonas sp. The methanolic extract exhibited the highest zone of inhibition against Xanthomonus sp. with maximum inhibitory zone of 14 mm followed by E. coli with 11 mm and P. aeruginosa with 9 mm zone of inhibition at 1200 µg. The aqueous extract was sensitive against P. aeruginosa (11 mm). Whereas, the chloroform extract was insensitive against the tested pathogens. Table 12 and Fig. 12-14 display the measured zone of inhibition for A. racemosus plant extracts.

TABLE 12: ZONE OF INHIBITION (IN MM) OF A. RACEMOSUS WHOLE PLANT EXTRACTS

FIG. 12: ANTIBACTERIAL ACTIVITY OF THE A. RACEMOSUSPLANT EXTRACTS AGAINST P. AERUGINOSA; A. AQUEOUS EXTRACT, B. METHANOL EXTRACT, C. STANDARD CIPROFLOXACIN

FIG. 13: ANTIBACTERIAL ACTIVITY OF THE A. RACEMOSUSPLANT EXTRACTS AGAINST E. COLI; A. AQUEOUS EXTRACT, B. METHANOL EXTRACT, C. STANDARD CIPROFLOXACIN

FIG. 14: ANTIBACTERIAL ACTIVITY OF THE A. RACEMOSUSPLANT EXTRACTS AGAINST XANTHOMONAS SP.; A. AQUEOUS EXTRACT, B. METHANOL EXTRACT, C. STANDARD CIPROFLOXACIN.

CONCLUSION: Several bioactive compounds found in the plants were thought to have medicinal qualities. These were considered as crucial components by modern pharmaceutical companies for manufacturing one-fourth of all medications. Screening medicinal plants for their therapeutic uses is therefore gaining importance in recent years. In the current study, the aqueous extract was found to be high in phenols and flavonoids and was also more effective in free radical scavenging and anti-inflammatory actions which is supported by the presence of various bioactive compounds identified through GC-MS analysis. Thus, the study suggests that the extracts could be utilized as an excellent source of natural antioxidants and a new molecule in the creation of anti-inflammatory drugs. This study adds a vital component to the pharmacological uses of A. racemosus plant and the identification of the novel plant metabolites from A. racemosus paved the way to discovery of new drugs. However, further research could be encouraged to isolate the bioactive compounds to determine the mechanism(s) behind their pharmacological effects.

ACKNOWLEDGEMENT: The authors are thankful to the Department of Biotechnology and Microbiology, Karnatak University, Dharwad for providing the facility to conduct the research experiments and also thankful to the USIC, Karnatak University, Dharwad for helping in the analytical instruments to conduct the experiments.

Ethical Approval: Not applicable because the present research work doesn’t involve any humans or animal study.

CONFLICT OF INTEREST: The authors hereby declare no conflict of interest.

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    Eden AH and Kumar VS: GC-MS profiling of bioactive compounds in Asparagus racemosus: implications for pharmacological properties. Int J Pharm Sci & Res 2025; 16(3): 791-09. doi: 10.13040/IJPSR.0975-8232.16(3).791-09.

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