INTRODUCTION:

In the literature, polyphenolic compounds are reported from plant metabolites and have shown their effectiveness against infectious diseases, including cardiovascular diseases. Various studies were conducted clinically and epidemiologically and showed a prominent linkage between dietary consumption of flavonoids and a decline in the rate of cardiovascular diseases^1,2.

These flavonoids have immunopharmacological activity, and this activity is mainly due to their molecular structure or the identification of some active flavonoid moieties that provoke immunological activity against infectious diseases. This is one of the most considerable areas of interest in the field of drug discovery. To understand the structure-activity relationship in plant metabolites, particularly crude flavonoids, several subclasses of flavonoids (natural ones) may be involved or reported. Inspite of these crude flavonoids being isolated from medicinal plant products, they are a potential candidate for drug design, which may be able to understand the structure-activity relationship of crude flavonoids and compare fractionation of crude flavonoids isolated from medicinal plants with different solvent systems^3,4. In this study, we worked on crude flavonoids isolated from Gymnosporia montana and compared their antioxidant and antimicrobial activity with fractionated flavonoids using different solvent systems. In addition, FTIR analysis was also performed in order to assess the molecule that is responsible for various immunobiological activities.

Gymnosporia montana (family Celastraceae; called Vikro), a medicinal plant, has been reported in several states in India, including Gujarat and Maharashtra, and is used to treat a variety of ailments. One familiar example is seen in the Saurashtra region of Gujarat, India, where leaf juice is widely used for curing the diseases, especially jaundice (consumption of leaves mixed with a proportionate amount of cow milk is taken in the morning)^5,6. In the present communication, we determined the flavonoid content of hydroethanolic leaf and fruit extracts from Gymnosporia montana and fractionated them with three solvent systems, determining their antioxidant and anti-inflammatory potential.

MATERIALS AND METHODS:

Collection and extraction:

Gymnosporia montana (leaves and fruit) were obtained from Victoria Park, Bhavnagar City, Gujarat, India. This plant's leaves were thoroughly washed with tap water to remove dirt before being air dried. These plant materials were homogenised into a fine powder.

The leaves and fruit powders of Gymnosporia montana were extricate with hydroethanolic extracts to get crude flavonoids. In order to extricate the flavonoid (hydroethanolic extracts and their fractions) from the leaves and fruits of Gymnosporia montana. On the basis of thin layer chromatography (TLC), the results may reveal the existence of spots more in the case of hydroethanolic extract as compared to methanolic extract. Further fractionation of hydroethanolic extracts using three different solvent systems (i.e., acetone, chloroform, and petroleum ether). In each set of experiments, crude flavonoids were fractionated with three different solvent systems (i.e., chloroform, acetone, and petroleum ether) and these samples were placed separately in centrifuge tubes. These tubes were opened for a period of time, and this filtrate (i.e., solvent) was evaporated. Finally, dried extracts (chloroform, acetone, and petroleum ether) of hydroethanolic extracts of leaves and fruits containing flavonoids were tested for the determination of antioxidant and anti-inflammatory activity.

Preparation of extracts:

A dried extract of hydroethanolic leaves containing flavonoids (4g) of Gymnosporia montana was extracted with acetone (40ml) on a shaking machine for 5 minutes. Centrifuging (4000rpm, 10min) the samples and decanting the supernatant was done three times, and finally the extracts were combined. Similarly, dried material was soaked for 72hours in chloroform (1:20 w/v ratio) at room temperature, and its concoction was percolated applying cotton wool and then filter paper to collect the supernatant. Collect the material and then proceed with the extirpation and filtration processes, repeated two times. The pooled supernatant ones were collected and then subjected to the evaporation process to bring down the pressure at 40°C to achieve the dried extract. In contrast, dried powder was extracted by the Soxhlet apparatus at an exalted temperature (65°C) using petroleum ether. The filtrates were procured after extraction, and these were dried at room temperature, ultimately forming a crude extract (gummy concentrate) ^7,8. Finally, the extracts were stored in a suitable container with proper marking and maintained in proper conditions.

Identification and isolation of flavonoids by TLC:

The presence of flavonoids in hydroethanolic leaf and fruit extracts of Gymnosporia montana was identified through TLC using a silica gel TLC plate (15 x 5cm; 3 mm thickness). Samples were applied as spots in capillary tubes using this mobile phase in the ratio of 1:0.5:0.4 (chloroform: methanol: water). After completion of the solvent run, spots were identified using Dragondorff’s reagent (spraying agent). For the identification of spots on a TLC plate using vanillin hydrochloric acid reagent. Finally, under ultraviolet light (254 and 365nm), plates were examined for fluorescent spots to identify the colour of flavonoids content and its retardation factor (Rf) using the formula Rf = distance travelled by the respective solvent extract/distance travelled by the solvent.

On this plate, solvent moved some distance, dried the plate, and was finally observed under ultraviolet light for examining fluorescent spots. Flavonoids were detected in these spots (yellow in colour with a Rf of 0.57). Finally, these spots were marked and scraped off with silica gel. Finally, the powder was collected and dissolved in phosphate buffered saline (pH 7), followed by centrifugation (10,000rpm; 5minutes). This step was repeated twice to ensure the complete removal of the adsorbent. The powder was then stored in tubes under refrigeration.

FT-IR analysis:

Leaves and fruit extracts of hydroethanolic extract were taken in a mortar and pestle, mixed with KBr salt, and compressed into a thin pellet⁷. For spectroscopy measurements of the samples, a Perkin Elmer Spectrum 2 FTIR spectrometer was applied, and the scan range selected for these studies was between 4000 and 500 cm−1.

Stickability of flavonoid and phenolic content:

The flavonoid content of Gymnosporia montana leaves and fruit extract fractions (acetone, chloroform, and petroleum ether) at various concentrations (10-1000 µg/ml) was determined by mixing them with an AlCl3 (2%; 0.5ml) ethanolic solution. This experiment was performed in triplicate and kept in the dark. Firstly, quercetin was used as a standard (0-250µg) and dissolved in ethanol, from which serial dilutions were formulated. In this test, samples (0.5ml) were mixed in the dark with an AlCl3 (2%; 0.5mL) ethanolic solution. After incubation for 45 minutes, the solutions were analysed in the UV-Vis spectrophotometer at a wavelength of 415nm⁸.

In leaves and fruits, the phenolic compounds in Gymnosporia montana were quantified using gallic acid as a standard, plotted on the standard curve (0–250 µg/ml) and analysed at 765nm using a UV-Vis spectrometer. An aliquot of solution (gallic acid dilution, 0.1ml) was jumbled with Folin-Ciocalteu (0.5ml, Sigma-Aldrich) for 5minutes. In the dark, these solutions were jumbled with a 2% Na2CO3 (0.4ml) solution and incubated for 2hours before analysis⁹.

Antioxidant activity:

This activity was evaluated in a hydroethanolic extract containing flavonoids using a slightly modified spectrophotometric method¹⁰. This technique is totally based on the DPPH radical, which is scavenged by plant derived antioxidants through proton donation and forms reduced DPPH. So, pairing of electrons will occur, and solutions of extract may lose their colour depending on the electrons' consumption. Finally, the colour of the extract after reduction may change from purple to yellow, and its antioxidant activity is also demarcated by a reduction in absorbance at 517nm.

The extracts (leaves and fruit) were prepared at different concentrations (10–100μg/ml) and these tubes were made up to 2ml with aqueous ethanol. A fresh solution of DPPH in aqueous ethanol (0.6mM) was prepared. DPPH (0.5ml) solution was added up to each tube, and the tubes were thoroughly mixed at room temperature before being incubated in the dark, with absorbance measured at 517nm. As controls, an aqueous-ethanol (2 mL) solution was mixed with DPPH (0.5mL). Aqueous ethanol (2ml) was taken as a blank. The percentage inhibition of the radicals was premeditated to make use of the antioxidant properties of these extracts: Percent inhibition= [(A control-A sample)/A control] * 100.

Anti-Inflammatory Activity In vitro:

For assessing the anti-inflammatory effect using different concentrations of Gymnosporia montana. Different concentrations were prepared, and samples (100µl) were taken in centrifuged tubes. Add typhoid vaccine (25µg/ml; 1:1000 dilution; 0.5ml) to all tubes, and samples in the form of a mixture were incubated for 20 minutes at 37^◦C. These samples were kept in a water bath for 10 minutes at 37^◦C. The tubes (Eppendorf) were cooled down, and their absorbance value was calculated in an ELISA reader at 630nm. The percentage of inhibition was calculated as mentioned below. Similarly, typhoid vaccine (0.025mg/ml; 1:100 dilutions; 0.5ml) was used for these studies instead of bovine serum albumin (BSA).

% Typhoid vaccine inhibition = (Abs Control − Abs sample/Abs control) × 100

Statistical examination:

Values were obtained in the form of Mean ± S.E. and their statistical examination was implemented using a one-way ANOVA test.

RESULTS:

FT-IR analysis:

In this study, we extracted the flavonoids from a hydroethanolic extract of Gymnosporia montana using three solvent systems (chloroform, acetone, and petroleum ether). For confirmation of flavonoids through FTIR, which mainly gives information or confirmation on the basis of functional groups. The results of FTIR, as shown in Fig. 1, give information in the form of a spectra with different structural arrangements on the basis of intensities, shapes, widths, and peaks. The results of FTIR are totally different in hydroethanolic extract and its fractions (chloroform, acetone, and petroleum ether), but these extracts have the same functional groups but differ in certain wavelength ranges.

Estimation of phenolic and flavonoid content:

Total phenolic content was estimated in the hydroethanolic extract containing flavonoids and its fractions using the Folin-Ciocalteu reagent. The results were presented in Table 1 and showed that total phenolic content was reported in chloroform and acetone extracts of the leaf fraction (87.21±4.22 and 59.44± 3.84) as compared to petroleum ether extract (43.65± 1.86). In contrast, fruit hydroethanolic extract and its fractions have less phenolic content as compared to the control. The total phenolic content of chloroform and acetone extracts was solvent-dependent and intimate as GAE. Similarly, the content of flavonoids expressed as quercetin equivalents, varied from 24.9±1.36 to 62.07± 3.18mg quercetin equivalent/g extract. The chloroform extract showed the highest range of flavonoid content, followed by acetone and petroleum ether extracts (Fig.2).

Table 1. Estimation of total phenolic content in fractionated leaves and fruit content containing flavonoids of Gymnosporia montana

S. No.	Samples	Fractionation Extract	Total Phenolic content (mg GAE/100 g)
1	Leaves (flavonoids, hydroethanolic extract)	Acetone	59.44 ± 3.84
		Chloroform	87.21 ± 4.22
		Petroleum ether	43.65 ± 1.86
2	Fruit (flavonoids, hydroethanolic extract)	Acetone	24.24 ± 2.04
		Chloroform	27.2 ± 2.18
		Petroleum ether	21.73 ± 1.92

Total phenolic content results were expressed in Mean ± S.E. as GAE/100g

Fig.2. Estimation of flavonoid content from leaves and fruit fractionation.

Values were expressed in the form of Mean ± S.E. Readings were expressed in the form of µg using Quercetin as standard for these studies.

Antioxidant activity:

The results showed the scavenging effects of hydroethanolic extract and its fractions of Gymnosporia montana on DPPH radicals, and the results were expressed in the following sequence: Chloroform extract > acetone extract > petroleum ether extract. The effective concentration (EC50) values of scavenging DPPH radicals for chloroform extract and acetone extract were 64.2±1.86 and 48.6±1.76μg/ml, respectively. In addition, the antioxidant prospective of fruit fractions was found to be moderate when compared with the standard, i.e., ascorbic acid. This study may have revealed that chloroform extract and acetone extract have noteworthy antioxidant activity; the occupancy of phenolic compounds is reported and could be determinable by the pronounced exorbitant antiradical properties (Fig.3).

Fig.3. Antioxidant activities of fractionated extracts.

Values were expressed in the form of Mean ± S.E. in the form of µg/ml using ascorbic acid as standard for these studies.

Anti-Inflammatory Activity:

The results demonstrated the anti-inflammatory activity of a hydroethanolic extract fractions of Gymnosporia montana on the typhoid vaccine. In this study, it will be revealed that the leaves and fruit fraction showed an anti-inflammatory effect at higher doses as compared to standard. The typhoid vaccine was used as a standard and showed an antigen-specific immune response as compared to the control. In short, chloroform and acetone leaf extract showed better anti-inflammatory effects as compared to the fruit fraction, which had less anti-inflammatory effects (Fig.4).

Fig.4. Anti-inflammatory activities of fractionated extracts of Gymnosporia montana by different solvents at various concentrations using Typhoid vaccine as antigen.

Values were expressed in the form of Mean±S.E. using Typhoid vaccine as the standard for these studies.

DISCUSSION:

In literature, the medicinal properties of plants are mainly determined on the basis of the metabolites (primary and secondary) present in them. Several metabolites (e.g., tannin, flavonoids, etc.) contribute significantly to medicinal quality. Identification and isolation of flavonoids from the hydroethanolic leaf extract using a solvent system, i.e., the mobile phase [CHCl3: CH3OH: H2O in the ratio of 1:0.5:0.3]. These TLC studies of hydroethanolic leaf extract confirmed the existence of flavonoids, which may help to support the knowledge of medicinal plants. Further confirmation is required for the existence of flavonoids, and it may only happen on the basis of functional groups, which are mainly determined through FT-IR^11,12. This technique is widely used in the food and pharmaceutical industries for the identification of active ingredients in plants, vegetables, fruits, and microorganisms. The spectra of these extracts are related to the presence of aromatic compounds in plants. In addition, phenolic and flavonoid content was also estimated in these extracts, which is directly related to the human health benefits of having good antioxidant properties.

The qualitative analysis of hydroethanolic leaf extracts containing flavonoids that were fractionated with acetone, chloroform, and petroleum ether was conducted using FT-IR, which gives fast and accurate measurements. In FTIR, fruit extract revealed the existence of alcohols, phenols, esters, and aromatic compounds. Leaf extract and its fraction showed the same trend as fruit extract. The FTIR technique was applied in order to examine and analysed the functional groups as mentioned in Figures 1. In these figures, the FTIR peaks of fractionated chloroform, acetone, and petroleum ether in the FTIR spectrum of Gymnosporia montana hydroethanolic leaf extract containing flavonoids are reported. In order to cross-examine the absorption spectra in the assortment from 2000 to 3000 cm⁻¹ to confirm the presence of flavonoids. In chloroform extract, a peak at 3597.08 revealed the presence of amide groups. The peaks at 2051.61 cm^-1, 1665.60 cm^-1 and 1470.56 cm^-1 refer to the presence of alkynes, diketones and nitrosamine in the ring. The peak at 802.10 cm^-1 corresponds to aromatic compounds. In the acetone extract, peaks at 3913.91, 3774.35, 3681.46, and 3661.76 revealed the presence of amide group (–C (=O) N=). The peaks at 2050.00 cm^-1, 1649.70 cm-1 and 1375.44 cm^-1 refer to the presence of stretching bond of alkynes [C=C] molecule, diketones and isopropyl group. In petroleum ether extract, peaks at 3015.21 cm^-1 to 3925.11 cm^-1 revealed the presence of amide groups. The peaks at 2043.00 cm^-1, 1717.51 cm^-1, 1633.27 cm^-1and 1442.77 cm^-1 refer to the presence of alkynes (C=C), diketones and nitrosamine). The results of the chloroform extract using FT-IR spectroscopy confirm the presence of various chemical constituents such as alcohols, phenols, esters, and alkyl halides. In contrast, acetone extract confirmed the presence of primary and secondary amines, esters, and aromatic amines. Similarly, petroleum ether revealed the presence of alcohols, phenols, and aromatic compounds.

The main intention of this study was to examine the antioxidant and anti-inflammatory activity of Gymnosporia montana. Medicinal plants are chemico-biological molecules that generate active moieties in them and are also revealed in other organisms. Some chemico-biological molecules may enhance or exist for their own survival within the plant. In some cases, these chemical-biological molecules act as herbicides to inhibit the growth of competing plants, such as the salicylic acid produced by willows. So, phyto-compounds are investigated to play a major role in the plant's transformation into its environment, but they also constitute the principal origin of pharmaceuticals^13,14. The use of plant-derived formulations provides a foundation for modern prophylactic and therapeutic areas of the biological sciences. In this regard, plant species were reported on the basis of literature and their medicinal uses, so our major aim is to get the high flavonoid content from fractionated samples of plant products. Hence, these fractionated samples were isolated and screened for antioxidant and anti-inflammatory activity. A lot of efforts were made to get effective antioxidants from medicinal plant products as an alternative to synthetic antioxidants. These antioxidants may be able to down-regulate the free radicals responsible for causing diseases, which can have serious effects on the cardiovascular system^15,16. In view of this, the results of these studies may indicate that chloroform and acetone extracts of Gymnosporia montana showed higher antioxidant activity as compared to petroleum ether extract. These results may be supported by the fact that there is an enhancement in the phenolic and flavonoid content of these extracts at higher doses. In short, these polyphenolic compounds are plant secondary metabolites that have been reported in several plant species and play a key role in antioxidant activity. In literature, phytochemicals like flavonoids are extensively used and show their effectiveness against pathogenic microorganisms.

In literature, plant-derived metabolites are generally applied for medicinal purposes because they induce anti-inflammatory activity and are considered cheap, safe, and affordable. In view of this, work on a standardized fraction of hydroethanolic leaves containing flavonoids using chloroform and acetone extracts showed inhibition in antigen-specific immune response as compared to petroleum ether extract. In contrast, the fruit hydroethanolic fraction has less activity as compared to the leaf fraction. Our study may explore some possibilities pertaining to identifying the potential antioxidant and anti-inflammatory compounds in Gymnosporia montana.

CONCLUSION:

Hence, flavonoid compounds could apparently be accountable for the antioxidant and anti-inflammatory activity of Gymnosporia montana. However, additional research should be fetch out to understand the interrelationship between flavonoids and the antioxidant and anti-inflammatory activity of Gymnosporia montana. In short, these extracts might be a good source of phenolic compounds and a potent natural phenolic antioxidant. Further research is needed to determine the active molecules in the chloroform and acetone extracts of Gymnosporia montana for antioxidant and anti-inflammatory activity.

REFERENCES:

1. Mothana RA, Abdo SA, Hasson S, Althawab F, Alaghbari SA, Lindequist U. Antimicrobial, antioxidant and cytotoxic activities and phytochemical screening of some yemeni medicinal plants. Evidence-Based Complementary Altern Med. 2010; 7: 323-330.

2. Cushnie TT, Lamb AJ. Recent advances in understanding the antibacterial properties of flavonoids. Int J Antimicrob Agents. 2011; 38:99-107.

3. Vauzour D, Rodriguez-Mateos A, Corona G, Oruna-Concha MJ, Spencer JP. Polyphenols and human health: prevention of disease and mechanisms of action. Nutrients. 2010; 2: 1106-1131.

4. Baba SA, Malik SA. Determination of total phenolic and flavonoid content, antimicrobial and antioxidant activity of a root extract of Arisaema jacquemontii Blume. J Taibah Univ Sci. 2015; 9: 449-454.

5. Sutariya S, Singh Y, Bajpai AB, Gupta A. Assessment of antioxidant and anti-inflammatory activities of secondary metabolites from leaves and fruit extract of Gymnosporia montana. International Journal of Pharmaceutical Sciences and Research. 2022; 13 (10): 4221-4227.

6. Sutariya S, Shah AA, Bajpai AB, Sharma RJ, Pandhurnekar CP, Gupta A. Fourier transform infrared spectroscopy (FTIR) analysis, antioxidant and anti-inflammatory activities of leaf and fruit extracts of Gymnosporia montana. Materials Today: Proceedings. 2022; 73 (1): 134-141.

7. Kumar SS, Manoj P, Giridhar P. Fourier transform infrared spectroscopy (FTIR) analysis, chlorophyll content and antioxidant properties of native and defatted foliage of green leafy vegetables. J Food Sci Technol. 2015; 52(12): 8131–8139.

8. Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P, Vidal N. Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds. Food Chem. 2006; 97: 654–660.

9. Khan MI, Sri Harsha PSC, Giridhar P, Ravishankar GA. Pigment identification, nutritional composition, bioactivity, and in vitro cancer cell cytotoxicity of Rivina humilis L. berries, potential source of betalains. LWT Food Sci Technol. 2012; 47: 315–323.

10. Sadasivam S, Manickam A. Biochemical methods. 8th. New Delhi: New Age International Publisher. 2008.

11. Kumar RA, Ramaswamy M. Phytochemical screening by FTIR spectroscopic analysis of leaf extracts of selected Indian medicinal plants. Int J Curr Microbiol Appl Sci. 2014; 3: 395-406.

12. Hussain K, Ismail Z, Sadikun A, Ibrahim P. Evaluation of metabolic changes in fruit of piper armentosum in various seasons by metabolomics using fourier transform infrared (FTIR) spectroscopy. Int J Pharm Clin Res 2009; 1: 68–71.

13. Ioannone F, Di Mattia CD, De Gregorio M, Sergi M, Serafini M, Sacchetti G. Flavanols, proanthocyanidins and antioxidant activity changes during cocoa (Theobroma cacao L.) roasting as affected by temperature and time of processing. Food Chem. 2015; 174: 256–262.

14. Heo SJ, Cha SH, Lee KW, Jeon YJ. Antioxidant activities of red algae from Jeju Island. Algae. 2006; 21: 149–156.

15. Ananthi S, Raghavendran HRB, Sunil AG, Gayathri V, Ramakrishnan G, Vasanthi, HR. In vitro antioxidant and in vivo anti-inflammatory potential of crude polysaccharide from Turbinaria ornata (Marine Brown Alga). Food Chem Toxicol. 2010; 48: 187–192.

16. Fleita D, El-Sayed M, Rifaat, D. Evaluation of the antioxidant activity of enzymatically-hydrolyzed sulfated polysaccharides extracted from red algae: Pterocladia capillacea. LWT Food Sci Technol. 2015; 63: 1236–1244.

Received on 09.02.2024 Revised on 01.06.2024

Accepted on 07.08.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):81-88.

DOI: 10.52711/0974-360X.2025.00013

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.