Qualitative and Quantitative Phytochemical analysis, Fluorescence Study and TLC of Barleria prionitis L. and B. cristata L. (Acanthaceae)

Ashwini Sirsat¹ , Pratiksha Umale¹ , Rupali Shirsat² , Dipak Koche¹

¹Department of Botany, Shri Shivaji College of Arts, Commerce and Science, Akola (MS) 444003 India

²Department of Botany, Shri Dr. R. G. Rathod Arts and Science College, Murtizapur, Dist- Akola (MS) 444107 India

Corresponding Author Email: ashcoolsir09@gmail.com

DOI : https://doi.org/10.51470/ABP.2025.04.03.72

Abstract

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1. Introduction

Medicinal plants represent a rich reservoir of bioactive compounds that play a vital role in traditional healthcare systems and modern drug discovery. Members of the family Acanthaceae are widely recognized for their therapeutic applications, largely attributed to their diverse secondary metabolites, including alkaloids, flavonoids, phenolics, terpenoids, glycosides, and steroids [1,2]. Barleria prionitis L. and B. cristata L. hold significant ethnomedicinal value across India and Southeast Asia, where they are traditionally used for treating respiratory ailments, inflammation, wounds, fever, gastrointestinal disorders, and liver abnormalities [3].

Barleria prionitis L., commonly known as Vajradanti, is documented for its anti-inflammatory, antimicrobial, antioxidant, hepatoprotective, and wound-healing properties [3,4]. Phytochemical investigations of this species have previously revealed the presence of iridoid glycosides, phenolic acids, flavonoids, terpenoids, and steroids, which contribute to its pharmacological effects [5]. Barleria cristata L., popularly known as Philippine violet, also possesses notable medicinal qualities, including antioxidant, antidiabetic, antimicrobial, and analgesic potential, attributed to its diverse phytocompounds [6]. Despite their medicinal importance, comprehensive comparative studies on the phytochemical composition of their leaf and stem powders, especially using multiple solvent systems, remain limited.

Phytochemical screening is an essential preliminary step in identifying medicinally relevant plant constituents. The solubility of these compounds greatly depends on the polarity of the extraction solvents. Polar solvents like methanol and ethanol are known to extract a broader spectrum of phytochemicals, including phenolics, flavonoids, and glycosides, whereas non-polar solvents tend to extract terpenoids, steroids, and lipophilic components [7]. Evaluating extracts using different solvents therefore provides a clearer understanding of phytochemical diversity and distribution within plant tissues. Such analysis is fundamental for validating traditional uses and for guiding the isolation of pharmacologically active compounds.

Fluorescence analysis of crude plant drugs further serves as a rapid and reliable diagnostic tool in pharmacognosy. Many phytoconstituents either fluoresce naturally or exhibit fluorescence upon treatment with acidic, alkaline, or oxidizing reagents, producing characteristic colour changes under visible and ultraviolet light[8,9]. These fluorescence patterns create a diagnostic fingerprint that aids in crude drug identification, detection of adulteration, and quality control—an essential requirement in herbal standardization [10].

Thin-Layer Chromatography (TLC) is another indispensable technique used to separate complex mixtures of plant compounds. It provides insight into the number and nature of chemical constituents present in crude extracts and helps detect similarities across species by comparing retention factor (Rf) values [22,23]. TLC profiling is especially useful in chemotaxonomic studies and in preliminary selection of extracts for further chromatographic purification and phytochemical isolation.

Given the traditional significance and pharmacognostic potential of Barleria prionitis and B. cristata, the present study aimed to conduct a comparative analysis of their qualitative and quantitative phytochemical composition, fluorescence characteristics, and TLC profiles from leaf and stem powders using multiple solvent systems. Such an integrated evaluation not only strengthens authentication and quality control of these medicinal species but also provides a foundational basis for future phytopharmacological exploration.

• Materials and Methods:

2.1 Plant Material Collection and Authentication:

Fresh leaves and stems of Barleria prionitis L. and Barleria cristata L. were collected from (insert site; e.g., Akola district, Maharashtra) during the flowering season. Plant materials were authenticated by a qualified taxonomist, and voucher specimens were deposited in the departmental herbarium for future reference. The collected samples were washed, shade-dried for 10–15 days, and powdered using a mechanical grinder following standard pharmacognostic procedures [10].

2.2 Preparation of Plant Extracts:

Powdered leaf and stem samples (10 g each) were extracted separately using four solvent systems: aqueous, methanol, ethanol, and chloroform. Extracts were prepared by cold maceration for 48 hours with intermittent shaking, followed by filtration using Whatman No. 1 filter paper. Solvent systems were selected based on polarity gradient for effective recovery of a broad range of phytochemicals [2].

2.3 Qualitative Phytochemical Screening:

Phytochemical tests for alkaloids, flavonoids, phenolics, tannins, terpenoids, saponins, steroids, proteins, and carbohydrates were performed using standard protocols[2, 13].

2.4 Quantitative Estimation of Major Phytochemicals:

Total Phenolic Content (TPC)

TPC was estimated using the Folin–Ciocalteu method, with gallic acid as reference standard. Absorbance was recorded at 765 nm using a UV–Vis spectrophotometer [16,22].

Total Flavonoid Content (TFC)

TFC was determined using the aluminum chloride colorimetric assay, with quercetin as the standard. Absorbance was measured at 415 nm [14].

All values were calculated in mg/g dry weight and expressed as mean ± standard deviation.

2.5 Fluorescence Analysis:

Fluorescence characteristics of powdered leaf and stem samples were studied following the method of [15,16].

2.6 Thin Layer Chromatography (TLC) Profiling:

TLC analysis was performed for methanolic and ethanolic extracts of both species following the protocol of Wagner and Bladt [11]. Both extracts were spotted onto silica gel 60 F254 plates and developed in solvent systems optimized for various phytochemicals. The mobile phase used was Petroleum ether: Acetone: Formic acid (70:20:10). Developed plates were visualized under daylight, UV 254 nm, UV 365 nm, and after spraying with anisaldehyde–sulfuric acid reagent. Rf values were recorded for each visible band.

• Results

The present investigation evaluated the phytochemical richness, fluorescence characteristics, and chromatographic behaviour of leaf and stem samples of Barleria prionitis and Barleria cristata. Results obtained from qualitative and quantitative phytochemical screening demonstrated considerable variation in the presence and concentration of secondary metabolites across different solvent extracts. Fluorescence analysis further revealed distinct colour responses under visible and UV light, providing diagnostic features useful for crude drug identification. In addition, TLC profiling highlighted the diversity of phytoconstituents through varied numbers of spots and Rf values among extracts. These combined findings offer comprehensive insights into the chemical composition and pharmacognostic attributes of the two Barleria species.

3.1 Qualitative phytochemical analysis:

3.1.1Qualitative Phytochemical analysis of leaf powder extract:

The preliminary phytochemical screening of powdered leaf samples of the selected ethnomedicinal plants— Barleria prionitis L. andBarleria cristata L.  was carried out using four different solvent extracts: aqueous (Aq. E), methanol (ME), ethanol (ET), and chloroform (CL). The results of the phytochemical analysis are summarized in Table-1.

The findings revealed that the selected species are rich in secondary metabolites, particularly alkaloids, terpenoids, phenolics, flavonoids, cardiac glycosides, reducing sugars, and steroids. These phytoconstituents were detected more consistently in the methanolic and ethanolic extracts compared to aqueous and chloroform extracts. Notably, carbohydrates and proteins & amino acids were absent in all solvent extracts across all species, with the exception of a few isolated observations.

In Barleria prionitis L., the aqueous extract (Aq. E) of leaf showed positive results for reducing sugars, saponins, tannins, flavonoids, phenolics, and carbohydrates. Both methanolic and ethanolic extracts exhibited the presence of cardiac glycosides, reducing sugars, saponins, flavonoids, phenolics, andcarbohydrates. The chloroform extract of the leaf tested positive for terpenoids, reducing sugars, and steroids.

The powdered material of Barleria cristata L. showed the presence of cardiac glycosides, terpenoids, saponins, tannins, flavonoids, and steroids in the aqueous extract. The chloroform extract tested positive for terpenoids, reducing sugars, flavonoids, phenolics, and steroids, while the methanolic and ethanolic extracts exhibited the presence of all tested phytochemicals except carbohydrates and proteins & amino acids were present, except for reducing sugars, saponins, and flavonoids. The chloroform extract of the leaf revealed the presence of cardiac glycosides, terpenoids, reducing sugars, saponins, flavonoids, and steroids.

Among the species studied, Barleria prionitis L. showed a particularly high presence of terpenoids and phenolics in both leaf and stem, especially in methanolic and ethanolic extracts. Carbohydrates, proteins, and amino acidswere generally absent in all species and solvent extracts, with the exception of B. prionitis (Aq. E, ME, and ET extracts).

3.1.2. Preliminary phytochemical analysis of stem powder extract:

The preliminary phytochemical analysis of the stem powdered material of the selected ethnomedicinal plants—Barleria prionitis L., and Barleria cristata L., was conducted using four solvent systems: aqueous (Aq. E), methanol (ME), ethanol (ET), and chloroform (CL). The results are presented in Table 2.

The phytochemical screening revealed that all the selected plant species tested positive for terpenoids, reducing sugars (strong reaction), flavonoids, and steroids in all four extracts. Aqueous and ethanol extracts demonstrated a broader spectrum of phytochemical constituents, followed closely by methanol extracts, which were also rich in several bioactive compounds. Flavonoids were consistently detected in the aqueous, methanolic, and ethanolic extracts of all species. Steroids were present in all solvent extracts across all species. In contrast, tannins, carbohydrates, and proteins & amino acids were absent in all solvent extracts of both selected plants.

In Barleria prionitis L., the stem powder showed positive reactions for terpenoids, reducing sugars, saponins, flavonoids, phenolics, and steroids in aqueous extracts. The methanolic extract was positive for alkaloids, flavonoids, phenolics, steroids, and showed strong reactions for cardiac glycosides, terpenoids, and reducing sugars. Ethanol and chloroform extracts both tested positive for terpenoids, reducing sugars, and steroids.

In Barleria cristata L., the aqueous extract showed the presence of alkaloids, cardiac glycosides, reducing sugars, terpenoids, flavonoids, and steroids. The methanolic extract was positive for cardiac glycosides, terpenoids, saponins, flavonoids, and steroids. The ethanolic extract exhibited positivity for all tests except saponins, tannins, and phenolics. The chloroform extract demonstrated the presence of terpenoids, reducing sugars, saponins, and steroids.

Overall, terpenoids, reducing sugars, flavonoids, and steroids emerged as the most consistently detected phytochemicals across all stem extracts of the selected species. Methanolic and ethanolic solvents demonstrated higher extraction efficiency for multiple phytochemicals, whereas chloroform and aqueous extracts exhibited selective extraction patterns. The absence of tannins, carbohydrates, and proteins & amino acids in all extracts highlights a common chemical profile among the studied Acanthaceae species.

3.2 Quantitative analysis of major phytochemicals:

The crude content of major phytochemical compounds in Barleria prionitis L and, B. cristata L., It has been found that among all four tested phytochemicals, selected plant showed higher level of phenolics followed by flavonoids. The observation found in both plants leaf extract showed higher content than stem in phenolics though same trend followed in stem powder.The content level of phenolics was significantly higher in leaf powder of B. prionitis leaf (101.55 ± 0.34) and less in B. cristata (78.3 ± 1.4), while the stem extract showed the same trend.As we see flavonoids content in leaf extract of B. prionitis (1.13 ± 0.46) is slight more than B. cristata (1.01 ± 0.50). The comparative account of quantitative analysis of these five plants is presented in (Table 3).

3.3Fluorescence analysis of B. prionitis and B. cristata L. Leaf and Stem powder:

Both leaf and stem powders of B. Prionitis L. showed noticeable colour and fluorescence changes with different chemicals. Strong fluorescent green/yellow colours appeared under UV light, especially with NaOH, HCl 50%, and HNO₃, indicating phenolics and flavonoids. Acids like H₂SO₄ produced dark brown to black colours. FeCl₃ gave brown tones, confirming tannins. Leaf powder of B. cristata L. showed green to fluorescent yellow colours under sunlight. With water and acids, colours varied from yellow to moss green. Strong acids (H₂SO₄) produced blackish colours. NaOH and HNO₃ caused bright fluorescent reactions, indicating rich phenolic and flavonoid content. FeCl₃ produced brown, confirming tannins.

3.4 TLCanalysis of the crude powder extracts:

The TLC analysis was performed to identify the number of compounds extracted from the powder samples in their methanolic and ethanolic extract. The mobile phase used was Petroleum ether: Acetone: Formic acid (70:20:10).The methanolic extract of B. prionitis L. leaf and stem showed 5 and 5 spots respectively, whereas B. cristataL. showed 5 and 3 spots respectively. The leaf extracts of both plants share three compounds in common (Rf value 0.13, 0.11 and 0.07). The account of TLC analysis of methanolic extract of selected plants are given below in (Table 5).

The ethanolic extract of all plants showed more spots as compare to methanolic extract. B. prionitis leaf and stem showed 6, 5 spots whereas B. cristata L. leaf and stem showed 6, 1, 7 and 5 spots respectively. In both selected plants leaf extracts showed more spots than stem. The leaf extracts of both plants shares two common compounds having RF value 0.28, 0.10, where the stem share only one compound common having RF value 0.12. The comparative account of TLC analysis of ethanolic extract of five plants are given below in (Table- 6).

• Discussion:

The comparative analysis of extraction efficiency across solvents revealed methanol and ethanol to be superior in isolating the majority of chemical constituents from both leaf and stem tissues. This finding is consistent with earlier reports where polar solvents such as methanol and ethanol demonstrated higher efficiency in solubilizing alkaloids, glycosides, flavonoids, phenolics, and terpenoids compared to aqueous or chloroform solvents [7]. The stronger ability of alcohol-based solvents to penetrate plant cell walls and dissolve both moderately polar and polar constituents explains their consistent yield of positive phytochemical results in the present study.

The phytochemical screening confirmed the presence of major secondary metabolites, including alkaloids, terpenoids, phenolics, flavonoids, cardiac glycosides, reducing sugars, saponins, tannins and steroids. These classes of compounds are widely reported for their pharmacological activities such as antioxidant, antimicrobial, anti-inflammatory, hepatoprotective, and cardioprotective effects [2,15,16]. The absence of carbohydrates, proteins, and amino acids in B. cristata highlights the dominance of secondary over primary metabolites in the studied taxa.

Among the investigated plants, Barleria prionitis exhibited a particularly high abundance of phenolics and terpenoids in both leaf and stem extracts, especially when methanol and ethanol were used as solvents. This is significant, as phenolic compounds and terpenoids are key contributors to the plant’s traditional use to treat inflammatory disorders, wound healing, and hepatoprotection [18].

Fluorescence analysis of the powdered drug with different chemical reagents exhibited striking variations in coloration under visible and ultraviolet (UV) light. Such changes in fluorescence are attributed to the chemical nature of phytoconstituents, as many secondary metabolites fluoresce naturally or upon derivatization with reagents  [8, 9]. The distinctive colors observed under UV light serve as a simple, rapid, and reliable pharmacognostic parameter for the identification and quality control of crude drugs [10]. The unusual variations in fluorescence response of the studied plants confirm the presence of diverse groups of phytochemicals and validate their diagnostic value in standardization studies  [19]

The presence of diverse phytochemicals in all studied species underscores their therapeutic potential and validates their ethnomedicinal usage. Flavonoids and phenolic compounds contribute significantly to antioxidant and anti-inflammatory effects, while terpenoids and steroids are associated with antimicrobial and cytotoxic activities [20,21].

The fluorescence and phytochemical screening results collectively provide a pharmacognostic fingerprint for the studied plants. Such fingerprints are crucial for crude drug authentication, prevention of adulteration, and for guiding future isolation and characterization of bioactive compounds [15,20].

• Conclusion:

The study confirms that Barleria prionitis and Barleria cristata are rich in key secondary metabolites, especially phenolics and flavonoids, with methanolic and ethanolic extracts showing highest yields. Distinct fluorescence reactions and diverse TLC profiles further support their pharmacognostic value. Overall, both species exhibit strong chemical potential, validating their traditional medicinal use and suitability for future bioactive compound isolation.

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