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Research Article - International Research Journal of Plant Science ( 2022) Volume 13, Issue 3

Gc-Ms analysis, in-vitro neuroprotective and antioxidant studies on ethanolic and acetone extract of oledenlandia corymbosa

Gomathi V1, Babitha M.C2*, C. Umamaheswara Reddy3, M.Kumar4 and Venkateswarlu B.S5
 
1Professor, Department of Pharmacology, Vinayaka Mission�s College of Pharmacy, Vinayaka Mission Research Foundation (Deemed to be University), Ariyannur, Salem-636308, India
2Research Scholar, Vinayaka Mission�s college of Pharmacy, Vinayaka mission research foundation (Deemed to be university), Ariyannur, Salem 636308, India
3Deputy Registrar (Invigilation), Sri Ramachandra institute of Higher Education and Research (D.U), Porur, Chennai-600116, India
4Professor and Head, Department of Pharmaceutical chemistry, Vinayaka Mission�s College of Pharmacy, Vinayaka mission research foundation (Deemed to be University), Ariyannur, Salem-636308, India
5Principal, Vinayaka Mission�s College of Pharmacy, Vinayaka Mission Research Foundation (Deemed to be University), Ariyannur, Salem-636308, India
 
*Corresponding Author:
Babitha M.C, Research Scholar, Vinayaka Mission�s college of Pharmacy, Vinayaka mission research foundation (Deemed to be university), Ariyannur, Salem 636308, India, Email: mcbabith@gmail.com

Received: 13-Apr-2022, Manuscript No. IRJPS-22-60606; Editor assigned: 15-Apr-2022, Pre QC No. IRJPS-22-60606(PQ); Reviewed: 29-Apr-2022, QC No. IRJPS-22-60606; Revised: 20-Jun-2022, Manuscript No. IRJPS-22-60606(R); Published: 27-Jun-2022, DOI: http:/dx.doi.org/10.14303/irjps.2022.018

Abstract

The study was aimed to explore the phytochemicals present in the ethanol and acetone extracts of Oldenlandia corymbosa by GC-MS analysis and to determine the neuroprotective and antioxidant potential of both of the extracts. The GC-MS analysis of the ethanol and acetone indicated the presence of carotenoids, polyphenols, diterpenes, and fatty acids. Neuroprotective effect of ethanolic (OCWE) and acetone extract of Oldenlandia Corymbosa (OCWA) on IMR-32 Neuroblastoma cell lines were evaluated by MTT assay and the results showed that ethanol extract of the plant got more neuroprotective activity on cell lines. The antioxidant results showed that both of the extracts had almost similar DPPH and ABTS radical scavenging. However, the ethanol extract had more potent antioxidant power than the acetone extract. Study data suggest that whole plant of Oldenlandia corymbosa possesses neuroprotective and anti-oxidative activities and the best neuroprotective and anti-oxidant activity being exhibited by the ethanolic extract of the plant.

Keywords

Oldenlandia Corymbosa, GC-MS analysis, MTT assay, DPPH assay, ABTS assay antioxidant, Neuroprotective

Introduction

Oldenlandia corymbosa Linn.is a flowering plant belonging to the Rubiaceae family and is commonly found in India, tropical East Asia, Java Island and in Srilanka. It is commonly known as Parppatakapullu and is one of the chief ingredients in various ayurvedic preparations like Amritarishtam, Mahatiktaghrtam, Candanasavam, Jatyadi tailam (Das et al., 2019). Oldenlandia corymbosa reported to have immunoprotective activity and used in many traditional medicines to treat ulcers, bronchitis, pelvic and uterine inflammation. Aqueous extract of the plant contains arabinose, rhamnose, mannose, zylose glucose and galactose (Sivapraksam et al., 2014).

Materials and Methods

Plant Material

Whole plant of Oldenlandia corymbosa was collected from Kottayam, Kerala. The plant was identified and authenticated by Dr. M.U. Sharief, The Scientist ‘E’ & Head of office, Botanical survey of India, Southern regional center, Coimbatore. Identification No.: BSI/SRC/5/23/2020/ Tech/63.

Preparation of ethanol and acetone extract

Oldenlandia corymbosa plants (Whole plant) were washed several times with distilled water to remove complete impurities present in it. Then it is dried at room temperature and coarsely powdered and the powder was extracted with ethanol and acetone for 50 hours. Alcohol and acetone are completely removed under reduced pressure, and semisolid extracts were obtained (Redfern et al., 2014).

GC-MS analysis

Shimadzu GC-MS (Model Number: QP2010S) with GC –MS solutions software was used to carry out GC-MS profiling of Oldenlandia corymbosa extract (ethanol and acetone). Chromatographic conditions: Elite-5 MS column( fused silica) of 30 mm length,0.25mm internal diameter and 0.25μ thickness was used. The carrier gas used was Helium at a flow rate of 1 ml /min and the injection volume of the sample was 1.0 microlitre. The oven temperature is maintained at 280°C. The total time taken for GC running was 50 min. By comparing the average peak area to the total area relative percentage amount of each component was calculated.

Invitro Neuroprotective Effect

MTT assay method: In-vitro Neuroprotective potential of selected extract of Oldenlandia Corymbosa (ethanol& acetone) was assessed using IMR-32 Neuroblastoma cells (purchased from NCCS Pune was maintained in Dulbecco’s modified eagles media). After attaining sufficient growth of the cell line Lipopolysaccharide (1 μg/ml) was added to induce neuroinflammation and incubated for one hour, prepared extracts were added to the respective wells. The sample content in the well after 24 hours of incubation period were removed and MTT solution was added to all tests and cell control wells again incubated for 4 hours. After removing the supernatant MTT solubilization solution (DMSO) was added and absorbances were measured by microplate reader at a wavelength of 540 nm (Chang et al., 2001).

In-vitro antioxidant Assay

DPPH Radical Scavenging assays and ABTS assay were used to assess antioxidant potential of the extracts.

DPPH Radical Scavenging assay

Antioxidant activities of ethanol and acetone extracts were determined by DPPH assay. Samples of different concentrations (12.5 μg/mL to 200 μg/mL) mixed with DPPH and this reaction mixture incubated at room temperature in dark condition for 20 minutes, a control without test compound is also prepared. Absorbance was measured at 517 nm and as the positive control Ascorbic acid was used (Rajurkar & Hande, 2011).

ABTS assay

Antioxidant activities of ethanol and acetone were determined by ABTS assay. Extracts of different concentrations mixed with ABTS solution. Generated radical monocation was reduced in the presence of antioxidants present in the extract. Absorbance was measured at 734 nm. Ascorbic acid was used as standard (Massada, 1976).

Results and Discussion

Identification of Components

Detection of phytoconstituents was done using National Institute Standard and Technology (NISTII) and WILEY 8 library. For characterizing phytoconstituents, parameters such as comparison of retention time and peak, computer matching, and the characteristic fragmentation patterns of the mass spectra were used (Adams, 2007; Cazes, 2004; Bhardwaj et al., 2020).

Phytochemicals detected from the chromatogram are shown in Table 1 (ethanol extract) and (Tables 2 and 3) (acetone extract).The chromatograms (Figures 1 and 2) exhibited the presence of numerous biologically active compounds (Lozano et al., 2018).

Peak Retention Time Area Area% Name Base m/z
1 16.784 1736537 1.53 4-ALLYL-1,2-DIMETHYOXY BENZENE 178.10
2 21.463 2473631 2.17 HEPTADECANE 57.05
3 26.712 7619606 6.69 NEOPHYTADIENE 68.05
4 26.858 3992898 3.51 HEXAHYDFARNESYLACETONE 58.05
5 27.586 2216814 1.95 3,7,11,15-TETRAMETHYL-2-HEXADECEN-1-OL 82.05
6 28.399 3106963 2.73 1-AMINODECANE, N-TRIflUROACETYL 184.10
7 28.481 19021396 16.71 METHYLPALMITATE 74.05
8 29.254 4233864 3.72 HEXADECANOIC ACID 73.05
9 31.709 32147298 28.25 9,12-OCTADECADIENOIC ACID, METHYL ESTER 67.05
10 32.045 21380475 18.79 PHYTOL 71.05
11 32.264 4600472 4.04 METHYL STEARATE 74.05
12 35.738 1803139 1.58 METHYL MELISSICATE 74.05
13 36.241 2380057 2.09 4,8,12,16- TETRAMETHYLHEPTADECAN 4-OLIDE 99.05
14 38.691 1790455 1.57 GLYCEROL .BETA.
PALMITATE
57.05
15 43.317 1704582 1.50 SQUALENE 69.05
16 45.557 3605707 3.17 9(11)-DEHYDROERGOSTERYL BENZOATE 251.15

Table 1. List of Chemical fractions identified in the Ethanol Extract of Oldenlandia Corymbosa.

Peak Retention Time Area Area% Name Base m/z
1 20.721 9,939,510.0 3.552 DECANE, 1-BROMO-2-METHYL 57.3163
2 21.306 9,573,032.0 3.421 TETRATETRACONTANE 57.3163
3 21.891 11,929,057.0 4.263 HEXATRIACONTANE 57.2465
4 22.467 16,370,365.0 5.850 TETRATETRACONTANE 43.2645
5 24.127 10,850,997.0 3.878 HEPTACOSANE 43.1946
6 25.008 6,647,038.0 2.375 ETHYL ISO-ALLOCHOLATE 43.1946
7 25.133 14,378,199.0 5.138 STIGMASTEROL 55.2897
8 25.288 6,993,453.0 2.499 1-HEPTATRIACOTANOL 55.2198
9 25.398 33,330,096.0 11.911 LANOSTEROL 55.2198
10 25.918 23,234,928.0 8.303 4,22-STIGMASTADIENE-3-ONE 55.2198
11 26.153 20,960,502.0 7.491 2,2-DIBROMOCHOLESTANONE 55.2198
12 26.748 11,069,512.0 3.956 THUNBERGOL 43.1946

Table 2. List of Chemical fractions identified in the Acetone Extract of Oldenlandia Corymbosa.

Compound name Nature of compound Activity established
Neophytadiene Diterpene Antiinflammatory (Lozano-Grande et al., 2018)
Squalene Triterpene Antioxidant (Kaur et al., 2011)
Stigmasterol Sterol Antipyrectic, Antiinflammatory (Ka et al., 2005)
Thunbergol Diterpene Natural antioxidant

Table 3. Phytocompounds were proven for their antioxidant and anti-inflammatory activity.

In vitro neuroprotective Effects

The viability of cells was evaluated by direct observation of cells by Inverted phase contrast microscope followed by MTT assay. The data obtained are given in the table 4. Percentage viability against concentration graphically represented in figure 3.

Sample
Concentration (µg/mL
Viable
Cells (%)
CONTROL
Viable
Cells (%)
LPS
Viable
Cells (%)
OCWA
Viable
Cells (%)
OCWE
CONTROL 100 ----- ----- -----
LPS 54.61±0.01 ----- -----
6.25 ----- ----- 61.11±2.179 55.36±1.605
12.5 ----- ----- 61.31 ± 2.271 59.52±1.297
25 ----- - ---- 66.31±2.232 66.10±2.118
50 ----- ----- 69.69±2.535 76.06±1.757
100 ----- ----- 77.80±2.323 85.91±2.499

Table 4. PercentageViability of cells by MTT assay.

DPPH Radical Scavenging Assay

It is one of most popularly used assay method to detect antioxidant potential of herbal extracts. Table 5 and figure 4 exhibited ethanol extract had got more antioxidant activity compared to ethanol extract of the plant. IC 50 Value from DPPH assay for ethanol extract is 130.56 μg/mL and acetone extract is 189.33 μg/mL.

Concentrations
 (µg/mL)
Scavenging of DPPH free radical (% activity of ethanol extract) Scavenging of DPPH free radical (% activity of acetone extract)
12.5 12.57 18.84
25 30.40 29.34
50 41.33 32.65
100 49.20 40.36
200 60.21 49.43

Table 5. DPPH Assay of extracts of Oldenlandia Corymbosa.

ABTS Assay Method

From the ABTS study also it is clear that ethanol extract has got more antioxidant potential compared to acetone extract. Table 6 and Figure 5. IC 50 Value from ABTS assay for ethanol extract is 1328.38 μg/mL and acetone extract is 1462.17 μg/Ml.

Concentrations
(µg/mL)
Scavenging of ABTS assay (% activity of ethanol extract) Scavenging of ABTS assay (% activity of acetone extract)
12.5 11.72 8.29
25 21.68 18.12
50 35.78 30.33
100 51.42 49.88
200 61.01 57.34

Table 6.  ABTS Assay of extracts of Oldenlandia Corymbosa.

Conclusion

In the present work attempts were made to scientifically validate and standardize the antioxidant and neuroprotective potential of Oldenlandia corymbosa. GC-MS analysis revealed that the plant is rich in phytoconstituents having antioxidant and anti-inflammatory activity. The antioxidant activity against DPPH and ABTS free radicals showed ethanol extract had got more antioxidant activity. Invitro neuroprotective activity against IMR-32 cell lines exhibited by both plant extract this may be due to the presence of antiinflammatory fractions such as neophytadiene, squalene, stigmasterol and thunbergol. Further studies on these plants can result in the development of newer drug entities that can be most efficiently used in neuroinflammation mediated neurodegenerative diseases.

Acknowledgement

The authors thank Department of pharmacology, Vinayaka mission’s college of pharmacy, Yercad main road, Salem for providing the necessary facility to accomplish the work.

Conflict of Interest

The authors declare that there is no conflict of interests regarding the publication of this article.

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Citation: Babitha et al., (2022). Gc-Ms analysis, in-vitro neuroprotective and antioxidant studies on ethanolic and acetone extract of oledenlandia corymbosa. IRJPS. 13: 018.