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Author(s): Abhilasha Tamada1, Afaque Quraishi2, Smriti Adil*3



    1School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    2School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    3School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    *Corresponding Author Email-

Published In:   Volume - 4,      Issue - 1,     Year - 2022

Cite this article:
Abhilasha Tamada, Afaque Quraishi, Smriti Adil (2022) Preliminary examination of bioactive components and bioactivity of Hardwickia binata Roxb. extracts obtained through conventional extraction methods. NewBioWorld A Journal of Alumni Association of Biotechnology, 4(1):7-12

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 NewBioWorld A Journal of Alumni Association of Biotechnology (2022) 4(1):7-12               


Preliminary examination of bioactive components and bioactivity of Hardwickia binata Roxb. extracts obtained through conventional extraction methods


Abhilasha Tamada, Afaque Quraishi and Smriti Adil*

School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India.,,

*Corresponding Author Email-




Article history:


25 March 2022

Received in revised form

14 June 2022


18 June 2022




Hardwickia binata, phytochemical,

plant extract.


The phytochemical constituents, quantitative antioxidant, and total phenolic content of Hardwicikia binata Roxb. plant parts were investigated in the present study. Methanolic extracts of H. binata leaves and stems were obtained using the traditional extraction methods, soxhlet and maceration. Preliminary tests for phytochemicals, revealed the presence of flavonoid, steroids, tannin and terpenoids, tannins in the stem and, flavonoids and phenols in the methanolic extracts of the stem and leaves, respectively. Saponin was found in the leaves and stem parts, but no alkaloids were identified in either. The high antioxidant content of the H. binata stem and leaf methanolic extract was proven by the total antioxidant assay. Furthermore, total phenol quantification among the extracts revealed that the plant's stem extract was rich in phenols. DPPH scavenging activity showed the potential antioxidant capability of H. binata parts. Overall, the preliminary findings revealed the bioactive components and antioxidant properties of H. binata leaves and stem parts; further detailed examination and screening of the plant may prove it to be a potential source of medication in the pharmaceutical field.


Graphical Abstract:

OI: 10.52228/NBW-JAAB.2022-4-1-3



The use of medicinal herbs, shrubs, and trees, that are the best reservoirs of bioactive compounds in plants, dates back to the dawn of time to the humankind (Asfaw et al. 2022). They have been used as therapeutic or remedies to ailments in India since ancient times (Gunaselvi et al. 2010). With its mega biodiversity, India has various indigenous traditional medical systems which includes Ayurveda, Amchi, Siddha and Unani (Srivastava et al. 2021). The traditional scriptures of Ayurveda, such as the compendia, lexicons, pharmacopoeia, treaties, samgraha grantha and others provides the information about the medications. According to ancient texts such as the Charak Samhita, a specific herb has various actions to heal illness, and every plant may have a superior effect and other comparatively auxiliary effects (Saxena and Vikram 2004). Alkaloids, flavonoids, tannins, and phenolic compounds are by far the most potent bioactive constituents in plants (Edeoga et al. 2005). These phytoconstituents derived from medicinal plants holds a promise to be used as allopathic disease treatment alternatives, and they seem to have a great capacity for healing and curing a variety of ailments (Dewangan and Acharya 2017). Traditional medicine, on the other hand, were administered in the form of crude extracts or a mixture of chemical compounds for several years. These approaches may be unsafe because they do not isolate probable poisonous or present as an unusable substance along with the active chemical constituent (Patel and Patel 2016). As a result, knowledge of phytochemical constituents, pharmacological activities, extraction, isolation, refinement, and structural elucidation of bioactive compounds is critical for the development of potent drugs (Patel and Patel 2016).

Hardwickia binata Roxb., commonly known as Anjan (in Ayurved as Anjana; in Siddha/Tamil as Katudugu or Kodapalai) (Gunaselvi et al. 2010; Shingade and Kakde, 2021). It is an attractive medium or large deciduous ornamental tree with graceful drooping branchlets. It belongs to the Caesalpiniaceae family and is represented by a single species, Hardwickia binata Roxb. In folk medicine, it is used to treat diarrhoea, leprosy, worm infection, indigestion, leucorrhoea, chronic cystitis, gonorrhoea, cancer, and microbes (Shingade and Kakde, 2021). Its parts used include roots, leaves, bark, seed, wood, and husk. It is a medicinal plant that belongs to the endemic biodiversity category (Deshmukh and Ghanawat, 2020). The tannins from the bark are used in medicines to treat diarrhoea, worms, indigestion, leprosy, and as an appetiser. H. binata leaves extract have antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. And, the antimicrobial agents demonstrated by bioactive substances includes gonorrhoea, pneumonia, eye, and mycotic infections. Leaves of H. binata has been reported to effective in relieving headache and in constipation treatment (Gunaselvi et al. 2010). Also, the leaf and bark have been a medicinal source for the treatment of rheumatism (Kanaka et al. 2013). The natives of the Chhattisgarh Plains traditionally use its leaves to treat headaches (Oudhia 2003); the aqueous paste of the leaves is also applied dermally on painful parts as a remedy. Alongside, leaves are used as purgative and in the treatment of constipation.  Therefore, the present work aims at screening for the phytoconstituents and antioxidant potency in methanolic extract of H. binata stem and leaves parts, employing different extraction methods and hence has the present examination included the phytochemical analysis and antioxidant screening assay.

Materials and Methods

The leaves and stems of H. binata were collected for this study from a forest next to School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur. The collected fresh leaves and stems were sorted, cleaned with water to remove dirt and were then shade dried at a constant temperature of 40°C for absolute removal of moisture content. Dried plant parts were finely processed into powder and used in experimental purposes (Figure 1). Table 1 describes the plant part collection conditions and organoleptic features.

Two of the extraction methods were used to extract the bioactive compounds present in H. binata. For the maceration process, the powdered plant part and the solvent methanol were mixed in a 1: 10 w/v (sample: solvent) ratio and placed in a closed container for 72 hours (Pandey and Tripathi 2014). The extracts were collected and filtered after the aliquot was stirred at regular intervals to ensure adequate mixing of the sample with the solvent. The samples were mixed with the solvent in the same ratio as prepared for the maceration process and subjected to 10 cycles at the boiling point of the solvent in the Soxhlet extraction (or, hot extraction) method. The extracts were collected and filtered after the completion of the cycle.

% Yield of the extract

The yield percentage of H. binata extracts, macerated leaf (ML), macerated stem (MS), soxhlet extracted leaf (SL), soxhlet extracted stem (SM), were calculated on a dry weight basis using the following formula:

Secondary metabolites screening 

For preliminary phytochemical screening, various extracts of H. binata were qualitatively tested for alkaloids (Mayer’s test, Wagner’s test), terpenoids, saponin (foam test), tannins, flavonoids (alkaline reagent test), phenols (ferric chloride test), steroids (Salkowski’s test), carbohydrates (Fehling’s test), proteins (Ninhydrin test) (Harborne 1973; Trease and Evans 1989; Ayoola et al. 2008; Chaturvedi et al. 2011; Singh and Saxena 2011; Yadav and Agrawal 2011; Mushtaq et al. 2014).

Antioxidant assay of H. binata extract

2,2-diphenyl-2-picrylhydrazyl (DPPH) assay

Shimada et al. (1992) method was followed for the assessment of DPPH radical scavenging activity in H. binata extract. 1 ml of DPPH solution was added to 1 ml of different concentrations of test samples (20, 40, 60, 80 and 100 µg/ml of H. binata methanolic extract). Also, control (without the extract) was prepared. The reaction mixture was incubated in dark for 30 min at room temperature. The absorbance was measured at 517 nm and compared with the standard ascorbic acid. The percentage DPPH decolorization of the sample was determined according to the following equation:

Percent of DPPH inhibition

where AbsControl and AbsSample are the absorbance values of the control and test samples, respectively.

Determination of total antioxidant activity

For the evaluation of the total antioxidant activity, phosphomolybdenum method described by Prieto et al. (1999) was employed. To the 1.0 ml of the test extract, 1.0 ml of the phosphomolybdate reagent (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) was mixed. Then, the tubes were covered and incubated at 95°C for 90 min in a thermal block. After heating, the solution was cooled and brought to room temperature; the absorbance was read at 695 nm using a spectrophotometer along with the standard. Total antioxidant capacity was calculated from the standard graph and expressed as milligram of ascorbic acid equivalence (AAE) per gram of extract.

Determination of total phenolic content

Folin Ciocalteau's (FC) method was used to determine the total phenolic content of H. binata extracts (Singleton and Rossi 1965). FC reagent was added to 1 ml of the sample extract. After 3 min, 1 ml of sodium carbonate was added to the above mixture, and the final volume was made up to 10 ml with distilled water. After 90 min of incubation in dark, the absorbance of the tubes was measured at 725 nm against a blank. A standard calibration curve was plotted using gallic acid to quantify the total phenolic in the extract. The results were expressed in milligram of catechol equivalence (CE) per gram extract.

Statistical analysis

All the experiments were conducted in triplicates and one-way analysis of variance (ANOVA) was performed for the data analysis. Using, Duncan’s Multiple Range Test (DMRT) in SPSS version 16.0 tests of significance at 0.05 levels were checked. Data were realized as Mean ±Standard error (SE) of the determinants.


Table 1. Collection of plant material and organoleptic features of H. binata

S. No.


Plant part

Plant part collection

Physical appearance




Pre- processing







Small, bifoliate, sage green

Dark green, fine sticky powder






Thin, slight sap, brownish

Dark brown, coarse powder

Result and Discussion

The present work focussed on the preliminary screening of phytochemicals and assaying antioxidant activities in methanolic extracts of H. binata leaves and stem part, extracted through maceration and soxhlet extraction methods. The following sub-heads shows the obtained results:

Percentage Yield extract


Figure 1: Post-harvested and preliminary processed H. binata plant parts (a) Leaves (b) Stems

The percentage yield obtained from methanol-extracted MS, ML, SL and SM of H. binata using maceration or soxhlet method is tabulated in Table 2. From the obtained data, the highest percentage extractive potential was found in the case of maceration method. In addition, among the tested samples, percentage yield extract was found to be higher in methanolic extracts of leaves and stem parts of H. binata that showed percentage yield of 22.90% and 21.06%, respectively. Previously, yield of 31.9% was obtained in the leaves methanolic extract of H. binata and was much higher in comparison to petroleum ether extract (2.2 ug/ml), following maceration extraction method (Hamid et al. 2018). In another study, only 0.9% and 1.8% yield was obtained in methanolic and hexane extracts of H. binata plant material (Ali et al. 1999).

Phytochemical screening

Various qualitative phytochemical tests were performed on methanolic extracts of H. binata stem and leaves parts for the identification of active chemical constituents. In the present study, phytochemical screening revealed that the two extraction methods inspected have potentially isolated the various bioactive compounds present in the studied plant, H. binata. Saponin and carbohydrates were found to be present in all of the methanolic extracts of H. binata that were screened. Bioactive constituents screened in leaves part showed presence of phenol but not flavonoid in SL, however, in comparison ML was found to contain phenol and flavonoid as well. Flavonoid, steroid and tannin were obtained in MS, while flavonoid, steroid and terpenoid were present in SS extract.  Although, all the extracts showed negative results for alkaloids.

In earlier investigation, different parts of H. binata have been investigated for the presence of chemical constituents as well as secondary metabolites that account for their therapeutic properties. It was reported that the ethanolic extracts of the H. binata leaves and seeds contained flavonoids, glycosides, phenol, saponins and, tannins in H. binata leaves, though alkaloids were absent in both the parts (Sharanabasappa et al. 2007; Shingade and Kakde 2021). Sharanabasappa et al. (2007) identified bioconstituents flavonoids, phenols, saponins and tannins in leaves ethanolic extracts of H. binata. Besides, similar findings were reported among previous results that showed saponins, tannins, steroids, coumarin, flavonoids, however, alkaloids and anthraquinones were absent in the ethanolic extract obtained via soxhlet method (Gunaselvi et al 2010). In similar, root bark exudates methanolic extracts of H. binata (soxhlet method) contained amino acids, carbohydrates, fixed oils, fats, glycosides, proteins, alkaloids, flavonoids, phytosterols phenolic compounds, saponins, tannins (Prabakaran et al. 2014). Also, Deshmukh and Ghanawat (2020) reported in their paper the presence of steroids, glycosides in methanolic extracts of H. binata leaves and stem, however absence of carbohydrates, proteins and alkaloids revealed through phytochemical screening. Deshmukh and Ghanawat (2020) conducted phytochemical screening, and Fourier Transform Infrared (FTIR) of H. binata leaves extract. The investigation revealed the presence of numerous phytochemical components including proteins, flavonoids, glycosides, tannins, phenolic compounds, quinones, steroids, volatile oils, but not alkaloids. In addition, FTIR spectroscopic analysis of H. binata crude powder confirmed the presence of alcohol, aliphatic amines, alkanes, alkenes, alkyl halides alkynes, amides, amines, aromatics, carboxylic acids, esters, ethers, phenols in leaves.

Antioxidant activity

H. binata antioxidant activity was measured using DPPH and was compared with the activity of standard antioxidant ascorbic acid. The DPPH antioxidant assay percentage showed varying inclination among the plant extracts. ML and SL showed increasing antioxidant activities with increasing extract concentration from 20-100 μg/ml with highest values at 100 μg/ml of 92.58 and 87.79, respectively. On the other hand, the antioxidant activities of MS and SS were found to be similar at a various range of concentrations, although the activity was found to be greater than 90% for all the concentrations and the activities were also comparable to the standard. The antioxidant capacities of all the examined samples are presented in Figure 2.

Previous studies on H. binata leaves methanolic extracts, tested for antioxidant activity ranging from 3.12 μg/ml -50 μg/ml. DPPH free radical scavenging assay that was measured after 40, 80 and 120 min showed highest scavenging activity against DPPH at 50 μg/ml concentration of the extract (Hamid et al. 2018). The activity was noted to be greater than 65 μg/ml after 40 and 80 min and 70 μg/ml after 120 min. In addition, the activity of H. binata leaves extracts was higher compared to other plant species leaves extracts that included Acaia siberiana, Bahunia rufescens, Pongamia pinnata and Prosopis juliflora.

Quantitative assay of antioxidant and phenolic content

Methanolic extracts of different parts, extracted via different extraction process resulted in contrasting total antioxidant activity among the various extracts. ML and SS showed the highest values of 16.23 and 16.56 mg/ml for the assay, on the other hand, total antioxidant activity was found to be low in the case of SL and MS. Data represented in Figure 3. High total phenol values were detected in the stem part of H. binata i.e., MS and SS with values 0.52 and 0.51, while less phenolic content was found to be present in leaves extract SL and ML, being the values 0.29 and 0.19, respectively (Figure 4). Previous work reported the total phenolic content in 80% methanolic extracts of H. binata was 0.94 μg/ml (Hamid et al. 2018)

ANOVA (μg/ml concentration)
























Figure 2: DPPH scavenging activity of H. binata plant part extracts at different concentrations. Data expressed as Mean ±Standard error (SE) and different letters denotes significant differences from each other compared at p< 0.05.  ML, macerated leaf; MS, macerated stem; SL, soxhlet-extracted leaf; SS, soxhlet-extracted stem; AA, ascorbic acid (Standard).

Total antioxidant content (mg AAE/ gram extract)






Total Antioxidant




Figure 3: Total antioxidant content in methanolic extract of H. binata plant part. Data expressed as Mean ±Standard error (SE) and different letters denotes significant differences from each other compared at p< 0.05. ML, macerated leaf; MS, macerated stem; SL, soxhlet-extracted leaf; SS, soxhlet-extracted stem; AAE ascorbic acid equivalence






Total Antioxidant





Figure 4: Total phenolic content in methanolic extract of H. binata plant part. Data expressed as Mean ±Standard error (SE) and different letters denotes signifcant differences from each other compared at p< 0.05. ML, macerated leaf; MS, macerated stem; SL, soxhlet-extracted leaf; SS, soxhlet-extracted stem; CE, catechol equivalence.

Table 2. Yield percentage of methanolic extract of the plant parts of H. binata



Plant part

Initial weight (g)

Final weight (g)

Dried extract weight (g)

% Yield extract (g)

























Note: ML, macerated leaf; MS, macerated stem; SL, soxhlet-extracted leaf; SS, soxhlet-extracted stem



The current study included phytochemical analysis, antioxidant screening assays to determine the preliminary pharmacological potentialities of various parts of the studied plant H. binata. The qualitative phytochemical content of dried methanolic extracts of selected plant leaves and stem parts was determined. The percentage yield extract obtained was found to be greater in the case of leaves extract. The results showed that the soxhlet extraction method extracted more biologically important phytoconstituents than the maceration method. Furthermore, methanolic extract revealed the presence of bioactive compounds in the stem much more than in the leaves. The total antioxidant assay revealed that the methanolic extracts of the stem and leaves of H. binata have high antioxidant activity.

Conflict of Interest

Authors have no conflict of interest to declare.


Authors would like to express deep gratitude to the Head of the Department, School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh for providing laboratory and other facilities to conduct present research work.


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