QUALITATIVE PHYTOCHEMICAL SCREENING, EVALUATION OF ANTIOXIDANT AND ANTI-INFLAMMATORY POTENTIALS OF VITEX PINNATA L. LEAF EXTRACT

The purpose of the study was to screen phytochemicals and assess the antioxidant as well as anti-inflammatory properties of Vitex pinnata leaf extracts. Alkaloids, glycosides, tannins, terpenoids, saponins, phlobatannins, steroids, quinine, phenol, coumarins, and flavonoids were found during the phytochemical investigation. V. pinnata leaf extract exhibited an IC 50 value of 154.33 µg/ml in the DPPH free radical scavenging assay (standard: IC 50 = 25.48 µg/ml) IC 50 value of 218.8 µg/ml in the albumin denaturation inhibition assay (standard: IC 50 = 33.74 µg/ml). According to the findings , the leaves of V. pinnata contain a variety of pharmaceutically important secondary metabolites capable of scavenging DPPH and inhibiting albumin denaturation, demonstrating their antioxidant and anti-inflammatory properties.


Introduction
Phytochemicals are some bioactive compounds produced by plants, commonly known as "secondary metabolites."Secondary metabolites are found in almost every parts of the plant, such as barks, leaves, stems, roots, flowers, fruits, and seeds (Bansode and Salalkar 2015).It is well known that plants produce these chemicals to save themselves from adverse environmental situations as well as protect themselves against pests and diseases (Ajuru et al. 2017).
A substantial quantity of antioxidants is produced by plants such as polyphenols, flavonoids, anthocyanins, and phenolics in the form of secondary metabolites.These antioxidants can avert oxidative stress, a process that can create cell damage.Considering the growing interest in evaluating the antioxidant potential of natural products, it is necessary to study the phytochemistry of plants with antioxidant activity (Sati et al. 2010).
A chemical that can reduce inflammation has an anti-inflammation property.Most of the palliatives are synthetic anti-inflammatory drugs, which can reduce pain by shrinking the inflammation.These antiinflammatory drugs have been found comparatively effective and safer than that of sedatives.There are various plant species that have antioxidant as well as anti-inflammatory properties that can be used in medicinal fields.Medicines derived from plant sources are generally considered to be risk-free for health, but using untested plant products as medicines can often be harmful (Farzaei et al. 2020).
 Author for correspondence: animesh.biswas@cu.ac.bdVitex pinnata L. which belongs to the family Lamiaceae (formerly under the family Verbenaceae), is indigenous to south and south-east Asia, particularly forests in tropical regions.In Bangladesh, it has been reported from Chattogram and the Chattogram Hill Tracts.It is a slow-growing, small to moderate-sized evergreen tree with quadrangular branches and a crown that often spreads.In tropic conditions, flowering and fruiting are almost constant from January to December (Thenmozhi andSubasini 2016, Prasetyaningtyas et al. 2021).
In Indian traditional medicine, V. pinnata is used to oust intestinal worms, as an antipyretic, analgesic, antiinflammatory, antioxidant, wound healer, antibacterial, and to treat stomach aches (Thenmozhi and Subasini 2016).Sometimes the young leaf shoots are eaten raw to treat fever and hypertension.A bark decoction is used to treat stomach aches.A root tea is taken for body aches, backaches, and fatigue (Thenmozhi and Subasini 2016).A poultice of the leaves is used to cure fever and wounds, and Barks are occasionally used for treating wounds and to reduce spasms (Thenmozhi and Subasini 2016).No report was found on the phytochemical analysis of Vitex pinata obtained from Bangladesh.Moreover, it is believed that this plant is becoming rare in Bangladesh (Mia 2009).Considering all these factors, this research has been carried out.

Collection and preparation of plant materials
Plant materials were collected from the Chittagong University campus, examined carefully, and identified.A herbarium specimen (CUMED 20191) has been deposited for future reference.The leaf samples of V. pinnata were used for the qualitative estimation of secondary metabolites.The disease-free and fresh leaves were selected and thoroughly washed with tap water three times and finally with distilled water, air dried, and later dried in an oven at 60°C for 72 hours.It was then ground into coarse powder using a grinding machine and stored in an airtight bag at 4°C.Five hundred grams of leaf powder were macerated in methanol with constant shaking using a shaker.Then the macerates were filtered through filter paper and concentrated by a vacuum evaporator.For future utilization, the crude extract was kept at 4°C.Alkaloid tests were performed with fresh leaf samples.

Alkaloid test
Rapid spot testing (Webb 1949) was used with a slight modification (Aplin and Cannon 1971) to carry out the alkaloid detection test.In the qualitative test of alkaloids, alkaloid-detecting reagents were used.Five grams of fresh leaf were chopped and pasted with a mortar and pestle to moisten with 10 ml of 2% HCl.Then the mixture was heated in a water bath at 60ºC for one hour, and then the extract was filtered through filter paper.To detect alkaloids, we mixed 2 ml of each extract with 1 ml of 1% HCl in five test tubes, then added 6 drops of Meyer's, Wagner's, Dragendroff's, Hager's, and Tannic Acid reagents to each test tube, and observed yellowish, brownish red, orange, yellow, and reddish precipitation, respectively.The reagents were prepared by following standard methods (Cromwell 1955).If the plant extract reacts with different chemical reagents and produces the described colors, it is considered the presence of alkaloids.
Flavonoids: A portion of the powdered plant sample was heated with 10 ml of ethyl acetate over a steam bath for 3 min.The mixture was filtered, and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution.A yellow coloration was observed, indicating a positive test for flavonoids.
Saponins: About 2 g of crude powder was boiled with 20 ml of distilled water in a water bath and filtered.Ten ml of filtrate were mixed with 5 ml of distilled water and shaken vigorously for a stable, persistent froth.The persistent froth indicates the presence of saponins.
Tannins: If dark green or deep blue color develops in a mixture of 5 ml of extract and 1 ml of 5% ferric chloride, it indicates the presence of tannin in the extract.

Phenols:
The presence of phenols was revealed by the emergence of a blue or green color after a few drops of 10% aqueous ferric chloride were added to the plant extract.
Terpenoids: The 0.5 g of crude extract was diluted in 5 ml of methanol and combined with 2 mL of chloroform.Seven ml of concentrated H2SO4 added to the mixture result in the formation of a reddish-brown interface, which denotes the presence of terpenoids.
Steroids: Five ml of methanol were added to 0.5 g of crude extract.One ml of this solution was taken into a test tube.Ten ml of chloroform was added to it, and then an equal amount of concentrated H2SO4 was added from both sides of the test tube.The presence of steroids was determined by the yellow-green fluorescence of the H2SO4 layer and the red-colored upper layer.

Glycosides:
In 5 ml of methanol, the crude extract (0.5 g) was dissolved.Two ml of methanolic extract were combined in a test tube with ten ml of 50% HCl.It was then heated for 30 minutes in a water bath.Five ml of Fehling's reagent were added to the mixture, and it was then boiled for five minutes.The presence of glycosides was assumed based on the brick-red color of the precipitation.
Cardiac glycosides: Five ml of plant extract were added along with 2 ml of glacial acetic acid, one drop of ferric chloride solution, and 1 ml of concentrated H2SO4.A brown or green ring indicates the presence of cardiac glycosides.
Quinine: One ml of plant extract was mixed with 1 ml of H2SO4, and the mixture was left to stand for a while to acquire color.The appearance of a red color indicates the presence of quinine.
Coumarin: One ml of extract was added to one ml of 10% NaOH, and the mixture was let to stand for a while.The emergence of a yellow color indicates the presence of coumarin.

Phlobatannin:
The presence of phlobatannins was detected when two ml of the aqueous solution of the crude sample were added to dilute HCl.

Methods for antioxidant activity test
The antioxidant activities of a methanolic crude extract of V. pinnata leaf and the standard antioxidant ascorbic acid were assessed by comparing the free radical scavenging effect of the 2, 2-diphenyl-1picrylhydrazyl (DPPH, MWt.394.32) (Brand-Williams et al. 1995).Crude extracts of V. pinnata leaf were used to prepare a range of concentrations (50,100,150,200, and 250 µg/ml) in methanol.Ascorbic acid in different concentrations (50,100,150,200, and 250 µg/ml) was prepared in methanol and a 0.004% DPPH solution was prepared by dissolving the DPPH in methanol.
Three ml of the DPPH solution were mixed with 3 ml of the extract solution and the standard solution separately.These solution mixtures were kept in the dark for 30 minutes.The degree of DPPH purple decolorization to DPPH yellow indicated the scavenging efficiency of the extracts.The absorbance of the DPPH solution was measured at 517 nm using a UV-visible spectrophotometer.Ascorbic acid served as a positive control.The lower absorbance of the reaction mixture indicated higher free radical scavenging activity.The scavenging activity against the DPPH was calculated using the following equation:

Standard and test solution preparation
A stock solution comprising 10 mg/ml of ascorbic acid and plant extract was made up.The estimation of antioxidant activity was carried out in triplicate.For methanolic crude extract, 15 test tubes were taken.Besides, 15 test tubes were taken for standard and 3 for control.Each test tube was labeled as 50, 100, 150, 200, and 250 µg/ml.Three ml of methanol were added to each test tube.Then 50, 100, 150, 200, and 250 µg/ml of the concentrated stock solution were added using a micropipette, and finally three ml of the 0.004% DPPH solution were added to the test tubes.

Methods for anti-inflammatory activity test
The inhibition of albumin denaturation technique was used to investigate the anti-inflammatory properties of V. pinnata (Mizushima andKobayashi 1968, Sakat et al. 2010).The reaction mixture consisted of test extracts (50 µg/ml, 100 µg/ml, 150 µg/ml, 200 µg/ml and 250 µg/ml) and a 5% aqueous solution of egg albumin.The pH (5.6 ± 0.2) of all the reaction mixtures was adjusted by 1N HCl.The sample extracts underwent a 20 minute incubation period at 37°C and a 20 minute heating period at 60°C.A spectrophotometer was used to measure the turbidity at 660 nm after the samples had been cooled.Three replications of the experiment were run (Shinde et al. 1999).The following equation was used to calculate the anti-inflammatory activity:

% inhibition =
Where, A = Absorbance of 5% egg albumin solution and methanol (Control) B = Absorbance of 5% egg albumin solution and plant extract (test groups) or, B = Absorbance of 5% egg albumin solution and acetyl salicylic acid (standard solution).

Results
Preliminary qualitative phytochemical tests were done using the leaf extract of V. pinnata.The intensity of presence of alkaloid contents and eleven other phytochemicals in the extract of the test sample was expressed by plus (+) signs.One plus (+) indicated the lowest, two pluses (++) indicated moderate, three pluses (+++) indicated substantial, and four pluses (++++) indicated the highest quantity of phytochemicals in the samples.The absence of the secondary metabolites was indicated by the minus (-) sign.Tables 1 and 2 show the results of the qualitative phytochemical analysis.Results from Table 1 indicate the presence of a substantial to moderate amount of alkaloids in the leaf extract of V. pinnata.

Antioxidant activity
Antioxidant activity of V. pinnata leaves was carried out by the DPPH scavenging assay (Brand-Williams 1995).Five various concentrations (50,100,150,200 and 250 µg/ml), were utilized in this investigation.The results of the DPPH scavenging assay indicated that the leaves were potentially active as antioxidants.Later, the scavenging activity was compared with that of ascorbic acid, a common antioxidant.The study measured the percentage of scavenging action at five different concentrations (50,100,150,200 and 250 µg/ml) is presented in Table 3. Results showed that the highest mean scavenging activity of ascorbic acid was 94.23±0.23%at the concentration 250 µg/ml and the lowest mean scavenging activity was 51.92±0.72%at concentration 50 µg/ml.In the case of methanolic crude extracts of V. pinnata leaves, the highest mean scavenging activity was 80.77±0.21%at a concentration of 250 µg/ml and the lowest mean scavenging activity was 13.46±0.04%at a concentration of 50 µg/ml.
For comparison, different extract concentrations (50,100,150,200, and 250 µg/ml) were placed on the Xaxis, and the relative scavenging activity (RSA) of ascorbic acid and mehanolic crude extract of V. pinnata leaf was placed on the Y-axis (Fig. 1).A sequential change occurred as concentration increased, and the highest concentrations showed the highest RSA.The leaf sample's IC50 value was 154.33 µg/ml, while that of ascorbic acid was 25.48 µg/ml.

Anti-inflammatory activity test
Most biological proteins lose their biological function when denatured.Denaturation of proteins is a welldocumented cause of inflammation.The ability of methanolic plant extracts to denature proteins was studied as part of the investigation into the mechanism of anti-inflammation activity.It had a modest impact on preventing heat-induced albumin denaturation when present in high doses.Acetyl salicylic acid had the maximum inhibitory activity (90.89±0.009%)at a concentration of 250 µg/ml.The lowest inhibitory activity for methanolic crude extracts of leaves was 14.29±0.009%at a concentration of 50 µg/ml, and the most inhibition activity was 57.14±0.002%at a concentration of 250 µg/ml.For comparison, different extract concentrations (50,100,150,200, and 250 µg/ml) were plotted on the Xaxis, and the percentage inhibition of albumin protein denaturation by methanolic crude extractives of V. pinnata leaf and acetyl salicylic acid was plotted on the Y-axis (Fig. 2).The highest concentrations showed the highest inhibition, and the graph showed a sequential change with the increase of concentrations.The leaf sample and acetyl salicylic acid had IC50 values of 218.8 µg/ml and 33.74 µg/ml, respectively.

Discussion
In the present research, V. pinnata leaves were screened for different secondary metabolites.For the detection of alkaloids, five alkaloid-detecting reagents were used, namely Dragendroff's reagent, Tannic acid reagent, Wagner's reagent, Hager's reagent, and Mayer's reagent.Leaf extract gave different results with different reagents.Dragendroff's reagent showed the presence of alkaloids in the extract of V. pinnata leaf in a moderate quantity, which is supported by another study (Nuraskin et al. 2020).But another study found negative results for alkaloids in the extract of V. pinnata leaf while treating with this reagent (Thenmozhi and Subasini 2016).The tannic acid reagent showed the best result in alkaloid detection.V. pinnata leaf extract showed a moderate result with Wagner's reagent.A study found a negative result, whereas another study reported a positive result with Wagner's reagent (Thenmozhi and Subasini 2016).Treatment with Hager's reagent showed a substantial quantity of alkaloids in the extract of V. pinnata leaf.This is supported by another experiment conducted by Thenmozhi and Subasini (2016).Mayer's reagent also showed the presence of alkaloids in moderate quantity in V. pinnata leaf extract.Meyer's experiment yielded variable results (Thenmozhi andSubasini 2016, Nuraskin et al. 2020).
In addition to alkaloids, the qualitative assessment of eleven other secondary metabolites (glycosides, tannins, terpinoid, saponin, phlobatannin, steroids, cardiac glycosides, quinine, phenol, coumarin, and flavonoids) was done in V. pinnata leaf extract.The present study confirms the presence of all those secondary metabolites.Other studies (Thenmozhi andSubasini 2016, Nuraskin et al. 2020) supported the presence of phenol, tannin, steroids, flavonoids, terpenoids, and saponins.Though the presence of glycoside was recorded in the present study, it was absent in the previous report (Thenmozhi and Subasini 2016).
By analyzing previous studies and present research, it can be postulated that the leaf extract of V. pinnata contains different types of secondary metabolites, including alkaloids, glycosides, tannins, terpinoid, saponin, phlobatannin, steroids, cardiac glycosides, quinine, phenol, coumarin, and flavonoids.Differences have been observed in some other experiments compared to the current investigation (Thenmozhi and Subasini 2016).These variations may be due to differences in extraction methods and/or plant environmental conditions.
Plants have been known to be a potent source of medicinal compounds and antioxidants.The oxidative stress that is created by free radicals can have an impact on the pathophysiology of many diseases, including cardiovascular failure, atherosclerosis, inflammation, carcinogenesis, reperfusion injury, and neurological conditions (Aruoma 1998).Alternative sources of antioxidants from natural origins, particularly plants, are currently in demand due to the growing safety concerns with the consumption of synthetic antioxidants (Stankovic et al. 2016).Different endogenous or external activities in the human body can produce free radicals (Lobo et al. 2010).DPPH, hydrogen peroxide, hydroxyl, superoxide anions, and nitric oxide radicals are a few examples of these radicals.On the other hand, excessive free radicals can destroy the immune system and cause a variety of illnesses, such as cataracts, thrombus formation, cancer, diabetes mellitus, renal failure, neurodegenerative diseases, cardiovascular diseases, liver diseases, rheumatoid arthritis, atherosclerosis, angina pectoris, aging, metabolic disorders, DNA damage, and many more (Nigri et al. 2004, Schetter et al. 2009).When DPPH radicals interact with an antioxidant or other substances those gives protons, the absorbance of DPPH is decreased.In order to examine the radical scavenging capacities of various natural compounds, DPPH radicals are widely used (Satyanarayana and Eswaraiah 2010).This study was aimed at determining whether the leaf extract of V. pinnata could be a good source of antioxidants or not.Results showed that the leaf extract of V. pinnata contained antioxidants, which was supported by other studies (Shafie et al. 2020).
Protein denaturation is characterized as a process in which the secondary and tertiary structures of the protein become misaligned, and it happens as a result of heat, a strong acid or base, an organic solvent, or a concentrated inorganic salt (Leelaprakash and Dass 2011).The inflammation process is dependent on the activities of enzymes, specifically proteins.Any substance capable of protein denaturation is considered to have anti-inflammatory agents.The egg albumin method, which uses denaturation to test the antiinflammatory activity of herbal medicine, is a low-cost alternative (Dharmadeva et al. 2018).The present experiment showed the presence of anti-inflammatory competency in the leaf extract of V. pinnata.Previous studies also supported the presence of anti-inflammatory competency in the leaf extract of V. pinnata (Nam 2006, Thenmozhi andSubasini 2016).By analyzing the previous and present studies, it can be concluded that the leaf extract of V. pinnata contains bioactive compounds with antioxidant and anti-inflammatory properties.
of control (DPPH solution without the sample).B = Absorbance of the DPPH solution in the presence of the sample (extract / ascorbic acid).

Table 1 .
Qualitative tests for alkaloids of V. pinnata leaves.

Table 2 .
Qualitative test for eleven phytochemicals of V. pinnata leaves.

Table 3 .
DPPH scavenging activity of methanolic leaf extract of V. pinnata and standard solution.

Table 4 .
Albumin protein denaturation assay for anti-inflammatory activity against the methanolic crude extract of V. pinnata leaf.