INTRODUCTION:

In literature, pharmacological studies were conducted using medicinal plants against newly modified or synthesized new molecules which represents a major breakthrough for developing new drug entities against several pathogenic diseases^1,2. So, these medicinal plants having primary and secondary metabolites showed several health benefits for human. Most of the metabolites especially flavonoids and terpenoids are currently used and applied as drugs against several pathogenic diseases and number of studies are still is in progress. The present study of flavonoids and terpenoids (crude extract) from the leaves and fruit extract of Gymnosporia montana which can be worn as a basic study to investigate and survey the other possible mechanisms that may follow and come out in an anti-inflammatory and antimicrobial activity.

Gymnosporia montana is a branched, woody, spinescent, traditional herbaceous shrub. The Roth exists over various regions of India and it hails from family Celastraceous. The atmosphere of southeast Asia is most favourable for growth of this shrub.it bears young prickly stem having thick yellowish leaves measuring (3 X 5 X 3.7) cm in dimensions^3,4. The leaf is fastened to stem via a slender long petiole of about 4-9.8cm. The leaf blade is elliptic or obovate, tapering at the base, round at the apex, whole or crenulate, and elliptic or obovate. Axillary cymes form the inflorescences. The pedicel of flower is long and slender, with tiny, lanceolate bracts. The calyx lobes are extremely tiny, roughly elliptic-oblong, ciliate, and circular at the tip. The petals are elliptic-oblong and 2.84mm long. The stamen filaments are flattened and dilate at the base whereas nectary disc is succulent and has ten lobed edges. The ovary is orbicular and glabrous. The model is characterized by fertile blooms that are strongly 2-3 lobed. The fruits are 5mm long globose capsules that become purple as they develop. The seeds are glabrous and have an aril imbedded in them. This shrub of Discifloral group is of high utility is ayurvedic research. It is reported to be efficacious in the prevention of diseases e.g. diarrhoea, jaundice, inflammation etc.^5-7.

Flavonoids (hydroxylated phenolic compounds) and terpenoids that are present in medicinal plant products which showed a wide range of immunobiological activities. One of the most familiar examples of flavonoid (i.e. quercetin) which is currently applied in clinical trials against prostate cancer whereas terpenoid (e.g. artemisinin) isolated from Artemisia species which are highly effective against cancer and malaria^8-10. One of the largest class of natural products i.e. terpenoids which may be organized as C5 (hemiterpenes); C10 (monoterpenes); C15 (sesquiterpenes); C20 (diterpenes) etc. These terpenoids are found in various organisms, especially plants, fungi, and marine animals. Terpenoids are of significant and appreciable interest due to the extensive and wide range of immunobiological activities i.e. antifungal, antiviral, and antiparasitic activities^11-13.

In contrast, flavonoids considered as one of the major constituent of human diet called as non-nutrients. These flavonoids are reported in food materials i.e. consumption of fruits and vegetables. The absorption of these dietary based flavonoids which are liberated from the chewing food and it is totally dependent on several physicochemical properties (i.e. molecular size, configuration, lipophilicity, solubility etc.). In order to explore this activity of flavonoids and terpenoids from other medicinal plants which may be more effective against other diseases or disorders^10-13. In this study, we screened its metabolites (flavonoids and terpenoids) for determining its anti-inflammatory and antimicrobial activity of Gymnosporia montana leaves.

MATERIALS AND METHODS:

Procurement, authentication of plant material:

Gymnosporia montana leaves were collected from Victoria Park, Bhavnagar, Gujarat, India in the month of December, 2021 and were taxonomically identified by Department of Botany, M K Bhavnagar University, Bhavnagar, Gujarat, India.

Preparation of plant material:

Leaves (1kg) and fruit (purplish or black in colour when ripped; 1kg) of Gymnosporia montana were taken and washed under running tap water pertaining to remove unwanted impurities. The fruits along with leaves were cut into fine chips and shaded in a dried area under normal environmental condition and finally homogenized to prepared the coarse powder and store them in tight jars.

Extraction of secondary metabolites (flavonoids and terpenoids):

Leaves (5g) and fruits extract (3g) of Gymnosporia montana were taken and mashed in liquid nitrogen (-196 °C) to prepare powdered material. This material was then soaked in methanol (80% in distilled water) and then boiled at 80-90°C for 2 h. Cooled down the solution and extract was collected in the form of filtrate using whatman filter paper and further draw out using ethyl acetate (20ml) along with distilled water (40ml). Further, Gymnosporia montana leaves extract was shaken using vortex mixer only for 1-2 minutes and then further incubated for 24 h at room temperature. After incubation, we observe two different layers i.e. upper layer having ethyl acetate (further heated to evaporate it) and in lower layer having flavonoid content which may be settled at the bottom. In addition, this sample was again heated to vaporize the layer having ethyl acetate at the top and flavonoid content is in concentrated form which may settled at the bottom. For confirmation, using qualitative test was performed for confirming the flavonoid content. For these studies, quercetin used as a standard.

For extraction of crude terpenoids from leaves (5g) and fruits extract (3g) of Gymnosporia montana using methanol, chloroform and sulphuric acid. First and foremost, plant leaves powder was percolated and immersed in alcohol for 48 h. After incubation, supernatant (filtrate) was accumulated or collected and then extricate with petroleum ether using separating funnel. The ether extract was treated as total terpenoids. For authentication, using qualitative test was performed for confirming the terpenoid content.

High Performance Thin-layer chromatography (HPTLC):

For confirmation and verification of flavonoids and terpenoids content through HPTLC and this experiment was executed on silica gel HPTLC plates using mobile phase (ethyl acetate: methanol: formic acid: water) in the ratio of 20:2.5:0.5:2 (v/v). For these studies, quercetin (standard) and Gymnosporia montana leaves having crude flavonoids and terpenoids (5.0µL, 0.5 mg/ml) were taken to the plates (10 mm bands), sample of crude flavonoids and terpenoids with CAMAG-Linomat IV automated spray on band applicator equipped with a syringe (100µl) and operated through these settings: band length (10mm); application rate (10 sec/ µl); distance (1.5cm; plate side edge) and distance (2cm, plate bottom). For these studies, CAMAG TLC Scanner 3 was applied and computed and calibrated bands having software (Version 4 X, WIN CATS)¹⁴.

Antimicrobial activity:

For the antimicrobial activity, flavonoids and terpenoids stock solution (100mg/mL) extracted from leaves and fruits of Gymnosporia montana using the disc diffusion method. For these studies, we used bacterial strains i.e. Staphylococcus aureus and Pseudomonas aeruginosa. In this study, sterile blank discs (6.0mm diameter), were saturated with non-identical dilutions of leaves and fruits extracts ranging from 2mg/disc to 0.0312 mg/disc. Since these cultures were incubated overnight using a turbidometer to yield approximately 1 X 10⁸ CFU/ml. Extract (flavonoids and terpenoids)-saturated discs (20 μl) were maintained on agar plates and incubated (37°C, 24h) them. For these studies, DMSO (20μl) and vancomycin discs (30μl) was used as a negative and positive control for these studies. Antibacterial activities of flavonoids and terpenoids were then work out by evaluating the zone of inhibition in millimetres (mm)^15,
16.

In vitro, antimicrobial activity of flavonoids and terpenoids stock solution (100mg/mL) extracted from leaves and fruits of Gymnosporia montana was determined through broth microdilution method using 96-well microtitre plates. Firstly, two-fold dilution of flavonoids and terpenoids extract were diluted starting from 100mg/ml in distilled water. MIC values were evaluated using micro-dilution method (standard broth) which is endorsed and nominated by the NCCLS and CLSI methodology with some rectifications.

In this experiment, serial two fold dilutions of the flavonoids and terpenoids extracts (100mg/ml to 3.125 mg/ml) were formulated. In 96 well plate, test samples of flavonoids and terpenoids (50μl) and assembled bacterial suspension (1 × 10⁶ CFU/ml, 50μl) were added into each well. In this study, microbial suspension (50 μl) and broth (50μl) used as negative control. After addition of samples, plates were agitated pertaining to mix well and then incubated at 37ºC, 24 h. All these tests were performed and carried out in triplicate. The lowest range of concentration which showed OD at 620 nm less than control^15,16.

Antioxidant activity:

In this study, flavonoids and terpenoids is in crude from extracted from the leaves and fruit extract of Gymnosporia montana for determining its antioxidant activity against DPPH [2,2-diphenyl-1-picrylhydrazyl] radical. This activity was performed and evaluated through UV spectrophotometry (518nm). In this experiment, variable concentration of Gymnosporia montana (flavonoids and terpenoids; leaves and fruit extract) were prepared using analytical methanol and standard antioxidant (Vitamin C) was used for these studies. Briefly, extract (flavonoids/terpenoids, 1ml) and methanol (3ml) were taken and mixed with 1.0mM DPPH (0.5Ml) of in methanol. This reaction was kept at room temperature (30 minutes) whereas blank (methanol and DPPH) in equal amount used for this study. Flavonoids and terpenoids samples of leaves and fruit extract were prepared in triplicates and calculate the mean value of absorbance was obtained. The percentage inhibition of radical scavenging activity was calculated i.e. absorption of the blank sample subtracted with absorption of the extract and divided by the absorption of blank sample. In addition, leaves and fruit extract having 50% inhibition (IC50) was calculated from the plot of inhibition percentage against leaves and fruit extract concentration¹⁷.

In Vitro Anti-Inflammatory Assays (inhibition of Protein Denaturation):

To evaluate its anti-inflammatory effects of the flavonoids and terpenoids of leaves and fruit extracts were used with minor changes¹⁸. Briefly, extracts of leaves and fruits (having flavonoids or terpenoids) or diclofenac sodium using variable concentrations (0.5-500μg/ml) was homogenised with bovine serum albumin (5g/100ml; 1ml). Incubate these extracts along with BSA at 37°C for 30-45 minutes whereas control tube having mixture of distilled water and BSA. For this experiment, denaturation of the proteins along with or without extracts were caused by placing the samples having flavonoids or terpenoids in a water bath for 10 minutes at 70°C. The mixture was cooling inside the ambient room temperature, and the activity each mixture was measured at 660 nm. Each test was done three times. The following formula was used to calculated inhibition percentage:

Absorbance of control – Absorbance of sample

% inhibition = -----------------------------------------------

in protein Absorbance of Control

RESULTS:

HPTLC studies:

The results of these studies related to HPTLC analysis of flavonoids and terpenoids as shown in Fig.1. From these studies, it may have confirmed that Gymnosporia montana having content of flavonoids and terpenoids in leaves and fruit extract.

Antimicrobial activity:

This activity was performed using variable concentration of flavonoids and terpenoid (extracted from leaves and fruit extract) against the tested bacterial strains were evaluated through inhibition zones (presence or absence) using disk diffusion method. As per the studies, it may have reported the inhibition zone produced by leaves and fruit extracts having flavonoids and terpenoids on different bacterial strains as shown in Table 1. From this studies, it may have revealed that flavonoid extract of leaves and fruit extract showed inhibitory effects against both bacterial strains, while terpenoid content exhibited less inhibitory effects as shown in Table 1. Methanol used as negative control and also showed inhibition in the range of 6-8mm. In contrast, MIC values were carried out against 2 bacterial strains using leaves and fruit extract as shown in Table 2.

Track peaks of flavonoids

Track peaks of terpenoids

Flavonoid (Leaves extract)

Flavonoid (Fruit extract)

Terpenoid (Leaves extract)

Terpenoid (Fruit extract)

Fig.1: HPTLC analysis of flavonoids and terpenoids from leaves and fruit extract.

Table 1: Inhibition zone (mm) of crude extracts (flavonoids and terpenoids) from the leaves and fruits of Gymnosporia montana against bacterial strains

*Concentration of crude extracts of Gymnosporia montana* against bacterial strains (mg/disc)**	*Staphylococcus aureus*		*Pseudomonas aeruginosa*		Antibiotic (Gentamycin) (inhibition zone in mm)
	Leaves extract (inhibition zone in mm)		Fruit extract (inhibition zone in mm)
	Flavonoids	Terpenoids	Flavonoids	Terpenoids
4	16.4	12.5	13.6	6.5	-
2	14.2	11.6	11.4	3.6	-
1	13.5	10.2	10.5	0	19.2
0.5	10.5	6.2	7.2	0	16.4
0.25	7.4	0	0	0	12.2
0.125	0	0	0	0	-
0.0625	0	0	0	0	-
0.03125	0	0	0	0	-

Table 2. Minimum Inhibitory concentration (MIC) of Gymnosporia montana against bacterial strains

Concentration (µg/ml)	*Staphylococcus aureus*		*Pseudomonas aeruginosa*		Antibiotic (Gentamycin)
	Leaves extract		Fruit extract
	Flavonoids	Terpenoids	Flavonoids	Terpenoids
3.9	+	+	+	+	+
7.8	+	+	+	+	+
15.6	+	+	+	-	+
31.25	+	-	+	-	+
62.5	+	-	-	-	+
125	-	-	-	-	-
250	-	-	-	-	-
500	-	-	-	-	-

Table 3. Percentage inhibition of DPPH and IC50 for leaves and fruit extract of Gymnosporia montana compared with Vitamin C

Concentration (µg/ml)	Percentage inhibition (leaves extract)		Percentage inhibition (Fruit extract)		Percentage inhibition by Vitamin C
	Flavonoids	Terpenoids	Flavonoids	Terpenoids
3.9	22.6 ± 0.34	18.5 ± 1.02	16.6 ± 0.54	12.2 ± 1.32	37.4 ± 1.36
7.8	36.8 ± 0.88	31.4 ± 0.86	32.2 ± 0.78	18.4 ± 1.34	51.8 ± 1.56
15.6	52.2 ± 1.34	49.4 ± 0.82	42.4 ± 0.98	36.4 ± 1.20	56.4 ± 0.94
31.25	67.8 ± 1.04	62.2 ± 1.32	55.2 ± 1.66	52.2 ± 1.38	62.2 ± 1.02
62.5	76.4 ± 1.38	76.4 ± 1.66	62.6 ± 2.12	66.6 ± 1.18	71.4 ± 1.12
125	85.6 ± 1.82	82.4 ± 1.54	66.2 ± 1.94	72.4 ± 1.52	84.8 ± 0.92
250	82.4 ± 1.54	88.0 ± 1.92	76.4 ± 1.44	81.8 ± 1.82	92.6 ± 0.78
500	92.6 ± 1.26	97.2 ± 1.88	87.6 ± 1.78	89.4 ± 2.04	100.2 ± 0.42
IC50 (µg/ml)	15.6	31.25	31.25	31.25	7.8

Values were expressed as Mean ± S.E.

Antioxidant activity:

Leaves and fruit extract of Gymnosporia montana was evaluated to scavenge free radicals (DPPH) and standard vitamin C was used for this study. The results of these studies as shown in Table 3 where percentage inhibition of scavenging activity of leaves and fruit extracts and Vitamin C against DPPH radical were expressed as IC50 values. Lower value of IC50 value which may reflect and showed higher DPPH radical scavenging activity. As per the studies it may showed that leaves and fruit extract of Gymnosporia montana showed significant DPPH activity.

Inhibition of Protein Denaturation:

The results of this experiment as shown in Table 4 where flavonoids and terpenoids content from leaves and fruit extract having effectively inhibit protein denaturation (albumin) at higher doses as compared to control. Diclofenac sodium used as standard and produced a significant inhibition as compared to control.

Table 4: Effect of flavonoid and terpenoid from leaves and fruit extract of Gymnosporia montana on protein denaturation using BSA

Concentration (µg/ml)	Percentage inhibition (leaves extract)- Protein denaturation		Percentage inhibition (Fruit extract)- )- Protein denaturation		Percentage inhibition by Diclofenac sodium
	Flavonoids	Terpenoids	Flavonoids	Terpenoids
3.9	7.4	8.2	5.6	3.8	-
7.8	14.2	16.4	10.4	9.4	-
15.6	22.8	25.4	26.4	16.6	-
31.25	35.6	37.6	32.8	27.2	62.2
62.5	41.4	44.6	46.8	31.8	68.4
125	52.4	52.4	49.2	44.2	75.6
250	66.4	61.0	56.2	56.5	87.6
500	76.4	63.4	66.6	61.6	92.4

The percentage values were obtained using variable concentration of flavonoids and terpenoids against BSA

DISCUSSION:

The current scenario about several medicinal plant products and its metabolites (primary or secondary) is of great interest because such type of information may be collected or gathered and required for revealing its structure and may be considered as one of the major source of compounds for synthesizing new chemical constituents, which may be helpful for the development of drugs. In addition, various metabolites were isolated from fungi (e.g. antibiotics, toxins etc.) and bacteria, considered as major source of molecules for drug development ^{[19, 20]}. In view of this, we worked on secondary metabolites especially flavonoids and terpenoids (crude extract) isolated from the leaves and fruit extract of Gymnosporia montana

Firstly, preliminary phytochemical analysis of leaves and fruit extract of Gymnosporia montana is an indication of the presence or absence of metabolites in a plant extract. This phytochemical tests are totally based on visual inspection of colour or precipitation reaction (qualitative test); whereas, HPTLC assays were performed and efficiently confirming the presence of these secondary metabolites i.e. flavonoids and terpenoids. In the current study, leaves and fruit extracts of Gymnosporia montana were evaluated for the detection and evaluation its antimicrobial and anti-inflammatory activity. This medicinal plant (Gymnosporia montana) used as traditional medicine and associated with microbial infections as mentioned in the literature. So, this medicinal plant (leaves and fruit extract) was screened out and determining its antimicrobial activity including its cytotoxicity which may be considered them as key immunobiological activities which are mediated through secondary metabolites especially flavonoids and terpenoids in these leaves and fruit extract. In this stud, our studies may have suggested that crude flavonoids and terpenoids of Gymnosporia montana gave antimicrobial activity at higher doses in our bioassays. In comparison with fruit extract, flavonoids and terpenoids having higher antimicrobial activity as compared to control. In addition, this medicinal plant also showed cytotoxic effect at higher doses. However, it showed substantial antimicrobial activity in the disc diffusion method and microdilution assay, which may have prompted to use and applied them for the treatment of various infectious diseases.

Antioxidant based studies were performed to evaluated ad recognize its immunopharmacological activity of leaves and fruit extracts to scavenge free radicals. One of the free radicals i.e. DPPH has scavenging ability for measuring its antioxidant activity of samples e.g. antioxidant Vitamin C having higher DPPH scavenging property ^{[21, 22]}. Our findings were indicated that flavonoids and terpenoids of leaves and fruit extract having phenolic compounds in large amount which showed encouraging antioxidant activity as compared to standard. Similarly, anti-inflammatory studies (in vitro) were conducted using this technique i.e. protein denaturation assay (e.g. Bovine serum albumin), considered them as one of the major cause of inflammation. From these studies, it may suggest that these extracts (flavonoids and terpenoids) from leaves and fruit showed significant anti-inflammatory properties. In short, flavonoids and terpenoid content from leaves showing better activity as compared to fruit extract.

CONCLUSION:

From this study, it may conclude that Gymnosporia montana containing several chemical compounds especially leaves and fruit extract in the form of secondary metabolites (e.g. flavonoids and terpenoids) with potent antimicrobial, antioxidant and anti-inflammatory potential. In this regard, it may confirm the plant (Gymnosporia montana) having active molecules in leaves and fruit extract for treatment of inflammatory diseases. Further studies were required to compare antimicrobial, antioxidant and anti-inflammatory potential of methanol extracts (having flavonoids and terpenoids) fractions and isolation of active molecules from leaves and fruit extract of Gymnosporia montana.

ACKNOWLEDGEMENT:

Authors are highly thankful to Infinity pathology laboratory and Department of Microbiology, VP Arts, science and Commerce College Baramati for conducting microbiology based studies.

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Received on 24.01.2022 Modified on 09.05.2022

Research J. Pharm. and Tech 2023; 16(4):1945-1950.

DOI: 10.52711/0974-360X.2023.00319

¹Department of Microbiology and Biotechnology, Graphic Era (Deemed to be) University, Dehradun.

²Infinity Pathology Laboratory, Katargam, Surat, Gujarat.

³Department of Botany, D.B.S. PG College, Dehradun.

1Department of Microbiology and Biotechnology, Graphic Era (Deemed to be) University, Dehradun.

2Infinity Pathology Laboratory, Katargam, Surat, Gujarat.

3Department of Botany, D.B.S. PG College, Dehradun.

¹Department of Microbiology and Biotechnology, Graphic Era (Deemed to be) University, Dehradun.

²Infinity Pathology Laboratory, Katargam, Surat, Gujarat.

³Department of Botany, D.B.S. PG College, Dehradun.