INTRODUCTION:

Alstonia scholaris R. Br. (Apocynaceae), commonly known as “devil tree/ Chhatim” is glabrous, evergreen, perennial herbaceous ornamental plant which grows up to 40m in height¹. The plant is native to tropical area like Asia, Southern Bangladesh, India, Nepal, China, Sri Lanka, Australia and Pakistan but also grow dry tropical to sub-temperate climate². The plant mainly blossoms well in territories where yearly rainfall is around 100–150cm, as moist habitat is favourable for its growth. The rough, tessellated corky grey mature bark contains whorled branches which are copiously marked with lenticels. The leathery leaves are 7 in a whorl, 23cm long and 8cm in width, bluntly acuminate, narrowly obovate, apex usually rounded. Upper surface of leaves is dark green but the lower surface is pale whitish green. Greenish white flowers are small in size, umbellate and arranged in branched manner. Seeds are oblong, with ciliated margins, and ends with tufts of hairs 1.5–2cm. The ovaries are distinct and pubescent^3-4. According to the Ayurvedic system of medicine, the plant is used as potent blood purifier. The plant is also used as the main ingredient of 'Kashim' in Unani system of medicine. Conventionally, leaves of the plant is used the treatment of cough, chronic bronchitis, asthma, fever and other respiratory infections. Milky juice of the plant is mainly used to cure ulcers, wounds and rheumatic pain. Decoction of the bark is used for falciparum malaria, asthma, hypertension, lung cancer and pneumonia where the powdered bark cures malaria, diarrhea, and dysentery^5-6. The active constituents reported in this plant include alkaloids likes Scholarin, Alschomine, isoalschomine, scholaricine, 2-(demethylschoarine); akumillan type indole alkaloids;2, 3 secofernanetriterpenoids; alstonamine acids A and B; flavonoids like kaempferol, quercetin, isorhamnetin, kaempferol-3-O-β-d-galactopyranoside; Strictamine, Voacristine, erythrodiol, uvaol, oleanolic acid, β-amyrin acetate, α-amyrin acetate, stigmasterol, squalene, leucoanthocyanins, reducing sugars, simple phenolics, steroids, saponins and tannins⁷. A number of pharmacological activities are reported in this plant like anti-diabetic, anti-inflammatory, anti-oxidative, anti-bacterial, anti-microbial, anti-fungal, anti-spasmodic, antinociceptive, anti-viral, diuretic, anthelmentic and urinary tract disorder⁸.

According to Literature review, the plant has many potent phytochemicals which exhibit pharmacological activities. Among various disease conditions, diseases resulted from oxidative stress are on the rise. Plant possesses natural antioxidants that can reduce free radicals and thereby bring relief from diseases resulted from oxidative stress. The aim of the present study is therefore to determine in vitro antioxidant activity and also to ascertain the total alkaloid, phenolic and flavonoid content in the leaf extracts of Alstonia scholaris R. Br.

MATERIALS AND METHODS:

Plant material:

Fresh leaves of the plant Alstonia scholaris R. Br. was collected from the Medicinal Garden of Netaji Subhas Chandra Bose Institute of Pharmacy, Tatla, Chakdaha, Nadia, West Bengal, India, and authenticated by Dr. Suchandra Samanta Mandal, M.Sc. (Botany), KU, M Phil. (Education), KU, Assistant Professor, K. Bed College, Krishnanagar, West Bengal, India (Cert/01/ 23).

Chemicals and reagents:

The chemicals, solvents and reagents used in the study were of standard analytical grade obtained from S.D Fine Chem Ltd., Mumbai and Loba Cheme, Mumbai.

Pharmacognostical studies:

Macroscopic studies:

Macroscopic examination was carried out for the freshly leaves material. Organoleptic evaluation of plants material such as color, size, odor, taste, surface characteristic was observed. The results are depicted in Table1. Their identity was confirmed by comparing their macroscopic and characters reported in the literature^4,9.

Preparation of extract:¹⁰

The plant material was dried thoroughly in shade condition at room temperature. It was then subjected to size reduction. 250gm of the plant material was defatted with petroleum ether. The marc was thoroughly dried and subjected to cold maceration process with chloroform to remove chlorophyll (24hrs.) and finally extracted with methanol. The methanolic extract obtained was dried at temperature below 40°C to obtain concentrate of the crude extract. The extract was kept in a suitable container with proper labelling inside a desiccator for further use.

Phytochemical screening:¹¹

Different qualitative chemical tests were performed on the methanolic extract to identify the various active constituents that are present in the leaves of the plant Alstonia scholaris R. Br. Phytochemical screening was performed using standard procedures. The results are depicted in Table 2.

Quantitative estimation of total alkaloid content:¹²

The total alkaloid content was estimated by gravimetric method of Harborne. 20gm of powdered sample accurately weighed and took it into 250ml of stoppered conical flask.100ml of 10% acetic acid in ethanol was added and conical flasks were covered properly and allowed to stand for 4hrs. It was filtered through Whatman fine filter paper. Filtrate was concentrated on water bath at 60º C to one quarter of its original volume. Concentrated aqueous ammonium hydroxide solution was added dropwise to the samples in order to precipitate the alkaloids. The pellets (precipitates) were collected and washed with 15% ammonium hydroxide solution. The precipitates were dried in an oven at 60ºC for 30 minutes.

The percentage of alkaloid was calculated using following formula:

% = W/Y *100 (Where W= weight of the alkaloid extracted in grams Y= weight of the powdered drug material in grams).

Alkaloid content was calculated and expressed as percent of the weight of the sample analysed.

Quantitative estimation of total flavonoid content by colorimetric method:^13-14

The amount of flavonoid is assessed by the standard procedure mentioned by Abdel-Hameed, (2009) using Quercetin as standard flavonoid compound.

Preparation of calibration curve of Quercetin:

Accurately weighed Quercetin (10 mg) was dissolved in 10ml of methanol and the solution was appropriately diluted to obtain a final concentration of 1mg/ml. The standard plot was prepared by taking different volumes from final stock solution of Quercetin in 10ml volumetric flask to obtain the final concentration of 20, 40, 60, 80 and 100µg/ml of Quercetin, respectively.

Estimation of total flavonoid content:¹⁵

The total ﬂavonoid content present in the plant extracts was determined by using the method reported by Chang et.al. (2002). To the plant extract (1ml, 1000μg/ml) methanol (3ml, aluminium chloride (0.2ml, 10%), potassium acetate (0.2ml,1M) and distilled water (5.6 ml) were added. The reaction mixture was incubated at room temperature for 30 min and the absorbance was measured at 415nm using an UV – visible spectrophotometer. Quercetin (20–100μg/ml) was used as standard. Total ﬂavonoid content was calculated from the standard plot of quercetin and expressed as quercetin equivalents per gram of dry weight of extract (mg QE/g).

Quantitative estimation of total phenolic content by colorimetric method:^16-17

The total Phenolic content is determined by Folin-Ciocalteu’s colorimetric method based on the procedure of Tawaha, Alali, Gharaibeh, Mohammad & El-Elimat, (2007), with slight modifications.

Preparation of calibration curve of Gallic acid:

Accurately weighed Gallic acid (10mg) was dissolved in 10ml of methanol to obtain a concentration of 1mg/ml. The standard plot was prepared by taking different volumes from final stock solution of Gallic acid (1 mg/ml) in 10ml volumetric flask to obtain the final concentration of 20, 40, 60, 80 and 100µg/ml of Gallic acid, respectively.

Estimation of total phenolic contents:

Gallic acid is used as standard for phenolic compounds. The plant extract (1ml, 1000μg/ml), 2.25ml of distilled water and 1.25ml of 0.2 N Folin-Ciocalteu’s reagent is added and after five minutes 1 ml of saturated sodium carbonate is added to the above mixture. The resulting blue-colored solution is incubated at 30ºC for 1.5hr. with occasional shaking and finally the absorbance is measured at 765 nm in UV-Spectrophotometer.

The total phenolic content is expressed as gallic acid equivalents (GAE) in mg/g of fresh material.

Statistical analysis used:

All the qualitative and quantitative analyses were replicated in three independent assays, and the results were reported as a mean±standard deviation.

DPPH (1,1-Diphenly-2-picryl-hydrazyl) Free radical scavenging activity^18-25

Preparation of Sample Solutions:

The dried extract was dissolved in methanol to a final concentration of 100µg/ml. From stock solution 2, 4, 6, 8 and 10ml of the solution were taken in five volumetric flasks and serially diluted, final volume of each volumetric flasks was made up to 10ml whose concentration was then 20, 40, 60, 80, 100µg/ml respectively.

Preparation of Standard Solutions:

Ascorbic acid was used as a standard and dissolved in distilled water to make the stock solution with the same concentration (100µg/ml) of extract. The different concentrations of standard 20, 40, 60, 80, 100µg/ml were prepared.

Evaluation of Free Radical Scavenging Activity:

50µM of DPPH solution was prepared using methanol. 2 ml of the DPPH solution was mixed with different concentrations of sample and standard solution separately (2ml each). These solution mixtures were incubated for 30min in dark place and absorbance was measured in 517nm using spectrophotometer. Methanol (2ml) with 2ml DPPH solution was used as control. Control was prepared containing the same volume without sample and standard. Methanol was used as blank. The absorbance was recorded and % inhibition was calculated by using the following formula –

% Inhibition = (A₀−A₁) / A₀× 100, where A₀ is the absorbance of control solution (contains all the test reagents except the test compound) and A₁ is the absorbance of plant extracts or standard.

A plot was Constructed between concentration vs % reduction in absorbance of DPPH and calculated the value of IC₅₀. IC₅₀ is the concentration of the sample required to scavenge 50% of DPPH free radicals.

RESULTS AND DISCUSSION:

Macroscopic characters:

Macroscopic characters of Alstonia scholaris R. Br. leaves shown in the following Table.

Table 1. Macroscopic characters of Alstonia scholaris R. Br. Leaves

Characters	Observation
Colour	Dark green colour on the both surface
Odour	Bitter
Taste	Characteristic
Shape	Oblong-lanceolate or obviate bluntly acuminate
Margin	Smooth
Arrangements	Bunch of 5-7, spreading, very shortly stalked
characteristic	Four to seven in a whorl with a blunt tip.

Qualitative chemical tests of the extract:

Preliminary phytochemical analysis of different extracts indicated the presence of alkaloids, flavonoids, carbohydrates, tannins, and other compounds (Table 2.)

Table 2. Qualitative Chemical Tests of the Methanolic Extract of Alstonia scholaris R. Br. Leaves

Test		Methanolic Extract
Alkaloids	Dragendroff’s Test	+
Amino Acids	Millon’s Test	+
Carbohydrates	Molisch’s Test	+
	Barfoed’s Test	+
	Test for Pentoses	+
Flavonoids	Alkaline reagent Test	+
Flavonoids	Zinc hydrochloride Test	+
Glycosides	General Tests	+
	Borntrager’s Test	-
	Modified anthraquinones Test	+
	Froth formation Test	+
Tannins	Chlorogenic acid Test	+
	Ferric Chloride Test	+
	Gelatin Test	+
Proteins	Xanthoproteic Test	+
Steroids	Libermann-Burchard Test	+
Steroids	Salkowski Test	+

[“+” = Present and “-” = Absent]

Total alkaloid content:

The total alkaloid content was estimated by gravimetric method of Harborne. The total alkaloid content was done in duplicate. The total alkaloid content was found to 6.77±1.09mg/g of the sample.

Total flavonoid content:

The total flavonoid content in plant extract was examined with the help of spectroscopic method using aluminum chloride using Quercetin as standard. A five-point calibration curve was plotted for Quercetin in the concentration range of 20-100µg/ml. The regression equation (y = 0.0105x - 0.1552) for the five-point standard curve for Quercetin showed an excellent coefficient of correlation (R² = 0.9985) with low intercept value. The total flavonoid content is expressed as Quercetin equivalent. Total ﬂavonoid content was calculated from the standard plot of quercetin and expressed as quercetin equivalents per gram of dry weight of extract (mg QE/g).

The total flavonoid content was found to be 24.83±0.21 mg of QE/g of the sample.

Figure 1. Calibration curve of Quercetin at concentration range 20 -100 µg/ml

Total phenolic content:

The total Phenolic content in plant extract was examined by using Folin - Ciocalteu’s reagent. Five-point calibration curve was plotted for Gallic acid in the concentration range of 20-100µg/ml. The regression equation (y = 0.0086x + 0.0175) for the five-point standard curve for Gallic acid showed an excellent coefficient of correlation (R² = 0.998). Total phenolic content was calculated from the standard plot of gallic acid and expressed as mg gallic acid equivalents per gram of dry weight of extract (mg GAE/g). The total Phenolic content was found to be 42.19±0.43mg of GAE/g of the sample.

Figure 2. Calibration curve of Gallic acid at concentration range 20 -100 µg/ml

Table 3. DPPH Radical scavenging activity of Methanolic extract of Alstonia scholaris

Concentration (µg / ml)	DPPH Radical Scavenging activity (% inhibition)
Concentration (µg / ml)	Methanolic Extract of Alstonia scholaris	Ascorbic acid
20	29.40 ± 0.41	36.11 ± 0.33
40	40.27 ± 0.55	47.52 ± 0.57
60	52.35 ± 0.70	56.92 ± 0.63
80	62.29 ± 0.60	66.31 ± 0.78
100	73.43 ± 0.78	74.49 ± 0.89
IC₅₀	57.19 ± 0.64	46.88 ± 0.53

Values are expressed as mean ± SD, n = 3.

Antioxidants react with DPPH, a stable free radical (purple colour) which was reduced to DPPH‐H (this leads to the discoloration of DPPH solution from violet to yellow) and as consequence the absorbance was decreased from the DPPH radical to the DPPH‐H form. Hence, the more rapidly the absorbance decreases, the % of inhibition increases and the more potent the antioxidant activity of the extract. The percentage of DPPH scavenging activity of methanolic extract of Alstonia scholaris was presented in Table 3. The plant extract exhibited a maximum DPPH scavenging activity of 73.43±0.78% at 100µg/ml whereas for Ascorbic acid (standard) was found to be 74.49±0.89% at 100µg/ml. The IC₅₀values of the methanolic extract of Alstonia scholaris and Ascorbic acid were 57.19±0.64µg/ml and 46.88±0.53µg/ml respectively.

CONCLUSION:

The present work was carried out to estimate the total alkaloid, flavonoid and phenolic content present in the methanolic extract of leaves of Alstonia scholaris R.Br. Further antioxidant activity was also determined by using DPPH radical scavenging method. Preliminary phytochemical studies confirmed the presence of alkaloids, amino acids, carbohydrates, flavonoids, glycosides, tannins, proteins and steroids. The total alkaloid content was found to be 6.77±1.09mg/g of the sample, total flavonoid was found to be 24.83±0.21mg of QE/g of the sample while total Phenolic content was 42.19±0.43 mg of GAE/g of the sample. The IC₅₀values of the methanolic extract of Alstonia scholaris and Ascorbic acid were 57.19±0.64µg/ml and 46.88±0.53 µg/ml respectively.

From the above results, we can conclude that the leaves of Alstonia scholaris possess polyphenolic compounds, flavonoids that are a natural source of antioxidants, which might be able to cure the oxidative stress-related diseases. However, further, In-vivo study, isolation and characterization of the phytoconstituents are required to identify the active compounds responsible for antioxidant activity.

CONFLICT OF INTEREST:

The authors’ Dr. Bhawana Sati, Dr. Nilip Kanti Deb, Soumya Saha, Dr. Jhuma Deb, Shownok Dey and Doyel Mukherjee declare no conflict of interest, financially or otherwise.

ACKNOWLEDGEMENTS:

The authors are grateful to Dr. Suchandra Samanta Mandal, M.Sc. (Botany), KU, M.Sc. (Education), KU, M Phil. (Education), KU, Assistant Professor, K. Bed College, Krishnanagar, West Bengal, India and management of Netaji Subash Chandra Bose Institute of Pharmacy for providing the facilities to work.

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Received on 24.09.2024 Revised on 18.01.2025

Accepted on 28.04.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5669-5674.

DOI: 10.52711/0974-360X.2025.00819

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