Phyto-Morphological and Phytochemical Insights: A Comprehensive Study of Hygrophila triflora (Roxb.) Fosb. and Sachet Leaves
1Research Officer (Ayurveda), Central Ayurveda Research Institute, Kolkata.
2Research Officer (Ayurveda), Central Ayurveda Research Institute, Kolkata.
3Research Officer (Botany), Central Ayurveda Research Institute, Kolkata.
4Research Officer (Chemistry), Central Ayurveda Research Institute, Kolkata.
5Research Officer (Chemistry), Central Ayurveda Research Institute, Kolkata.
6Director, Central Ayurveda Research Institute, Kolkata.
*Corresponding Author E-mail: shakti.imsbhu@gmail.com
ABSTRACT:
Hygrophila triflora (Roxb.) Fosb. and Sachet, a notable member of the Acanthaceae family, is a herbaceous plant indigenous to South Asia, commonly found along watercourses. This study delves into the taxonomic history, morphological diversity, ecological relevance, and physicochemical properties of Hygrophila triflora. Morphologically, the plant displays dimorphism in its leaves, with adaptations suited for both aquatic and terrestrial environments. Leaves were collected from Baruipur, West Bengal, and authenticated, followed by a series of processing and microscopic analyses to explore the plant's anatomical features. The physicochemical evaluations highlighted properties such as moisture content, ash values, and extractive values, establishing a foundation for the plant's quality assessment. High-Performance Thin Layer Chromatography (HPTLC) was employed to derive a comprehensive phytochemical profile, revealing distinct Rf values when observed at two wavelengths, 254 nm and 366 nm. Additionally, a qualitative analysis showcased the presence of various secondary metabolites, such as alkaloids and flavonoids, in different extracts of the plant. These findings contribute valuable insights into the anatomical, chemical, and potential therapeutic attributes of Hygrophila triflora, paving the way for deeper pharmacological investigations and potential applications in traditional and modern medicine. This research underscores the importance of continued exploration of indigenous plant species, elucidating their roles in ecosystems and potential contributions to scientific and therapeutic domains.
KEYWORDS: Hygrophila triflora, Physicochemical properties, Phytochemical profile, Medicinal plant, ethnobotany.
INTRODUCTION:
Hygrophila triflora, scientifically classified as Hygrophila triflora (Roxb.) Fosb. and Sachet, is a distinctive member of the Acanthaceae family known for its unique characteristics and ecological significance. This herbaceous plant is native to South Asia and is often found flourishing along the sides of watercourses.
Its intriguing features, including its aromatic nature and bilabiate pale blue flowers with a purple palate, make it a subject of interest for botanists, ecologists, and conservationists alike.
Originally described as Ruellia triflora by Roxburgh in 1832, the plant's taxonomic classification has evolved over time, and it is now officially recognized as Hygrophila triflora. This taxonomic journey underscores the complexity of plant classification and highlights the importance of continued research in botanical science.1
Hygrophila triflora exhibits morphological diversity, with dimorphic leaves that serve different functions. Submerged leaves are pinnately or bipinnately dissected, while aerial leaves are obovate to suborbicular and serrate-dentate. These adaptations allow the plant to thrive in both aquatic and terrestrial environments. Notably, the aerial leaves display glandular hairiness on the upper surface, providing an interesting avenue for studying adaptations to different ecological niches.
This herbaceous plant is predominantly found in the districts of Alappuzha, Thrissur, Kozhikkode, and Ernakulam in Kerala, India. Its habitat preference along the sides of watercourses underscores its dependence on water availability, making it a valuable indicator species for monitoring aquatic ecosystems. Understanding its ecological role, especially in the context of watercourse ecosystems, is critical for environmental conservation efforts. Hygrophila triflora exhibits distinct flowering and fruiting patterns, typically occurring from September to March. Its flowers, with their striking pale blue and purple coloration, contribute to its aesthetic appeal and may hold ecological significance in terms of pollination and interactions with local fauna.2
In this research paper, we delve deeper into the taxonomy, morphological diversity, and ecological significance of Hygrophila triflora. Through detailed observations and analysis, we aim to contribute to our understanding of this unique plant species.
In September 2021, matured leaves of Hygrophila triflora (Roxb.) Fosb. and Sachet were meticulously collected from their natural habitat in Baruipur, South 24 Parganas, West Bengal (geographical coordinates: 22.3508°N 88.4171°E). Underground root portions, present in tuft- like clusters, were carefully separated from the peripheral shoot region. To ensure the authenticity of the plant material, authentication was carried out by Dr. M. N. Das, a former scientist from the Department of Pharmacognosy at the Central Ayurveda Research Institute for Drug Development in Kolkata, West Bengal.
The collected leaves were subjected to a series of processing steps. Initially, they were washed with a 70% (v/v) aqueous ethanol solution and then dried at an ambient temperature ranging from 24°C to 27°C. A small section of the plant sample was reserved for macroscopic, organoleptic, and anatomical (transverse section) studies, while the remainder of the plant material was shade-dried and subsequently pulverized using a National SM 2000 grinder. The finely and coarsely powdered samples were stored in airtight, light-resistant containers at room temperature, following the guidelines outlined in the Ayurvedic Pharmacopoeia of India. The finely sieved (60 mesh) and coarsely powdered samples were utilized for various analyses, including powder analysis, physicochemical assessments, phytochemical investigations, and chromatographic examinations, all conducted in accordance with established standardmethods.
Organoleptic and morphological parameters such as texture, shape, size, color, and odor of the plant material were meticulously recorded through direct observation using a stereozoom microscope (Radical RS-Mr-3).
Fresh leaf samples were cross-sectioned with precision using a sharp diamond-edged blade. Subsequently, these selected sections underwent a graded ethanol treatment, progressing from 30% to absolute grade, with safranin stain. The treated sections were then mounted on slides in Canada balsam and examined under a digital trinocular compound microscope (Olympus CX 43) at 10X and 40X magnifications. Photomicrographs were captured to document the cytological characteristics.
Approximately 2 grams of fine dried powdered leaf sample were treated separately with various solutions, including aqueous saturated chloral hydrate (for maceration), 50% glycerin, phloroglucinol in concentrated HCl (for staining lignified tissues), and 0.02 N iodine reagent (for starch grains). These treated samples were then mounted on slides with 50% glycerin following established protocols.
Observations were made using a trinocular compound microscope (Olympus CX 43) at 10X and 40X magnifications, and photomicrographs were captured using a Magcam DC5 camera attached to the microscope.
This comprehensive methodology was employed to ensure the collection, authentication, and detailed examination of Hygrophila triflora plant material, laying the foundation for subsequent analyses and research investigations.3
Physicochemical Properties and HPTLC Profile of Hygrophila triflora (Whole Plant):
The physicochemical properties of the whole plant material were determined through a series of standardized tests These included assessments of parameters such as moisture content, total ash value, acid-insoluble ash, water-soluble ash, and extractive values in various solvents. All experiments were conducted following established procedures to ensure accuracy and reproducibility. For the HPTLC analysis, finely powdered plant material was extracted with suitable solvents, and the resulting extracts were analyzed using HPTLC. Silica gel plates were employed as the stationary phase, while a mobile phase composed of appropriate solvents was utilized for separation. The chromatographic plates were then developed, and the resulting chromatograms were visualized under ultraviolet (UV) light. Specific compounds and their Rf (Retention Factor) values were recorded, allowing for the creation of a characteristic HPTLC profile for Hygrophila triflora (Whole Plant). This profile serves as a valuable reference for the identification and quantification of phytoconstituents in the plantmaterial.
The physicochemical analysis and HPTLC profiling of Hygrophila triflora (Whole Plant) were carried out in accordance with established protocols and methodologies. These comprehensive analyses provide essential data for assessing the quality, purity, and chemical composition of this plant, which is of significant interest in various scientific and therapeutic applications.4
Hygrophila triflora, among the three plants studied, showed significant antioxidant activity, surpassing even Vitamin C, highlighting its potential for medicinal applications.5
In the investigation of Hygrophila triflora macroscopic characteristics, it was observed that the leaves are opposite, subsessile, and exhibit dimorphism. Submerged leaves measured 5-6 x 3-4 cm and displayed pinnate or bipinnate dissection. They were minutely hairy on the upper surface but gradually became glabrescent on the lower surface. Aerial leaves, on the other hand, were slightly smaller at 4-5 x 2-3 cm and had an obovate to suborbicular shape with obtuse tips. These leaves were serrate-dentate along the margins and possessed 6-7 pairs of lateral nerves. The upper surface of the leaves was densely covered in glandular hairs, while the lower surface exhibited sparse hairiness on the nerves. The coloration ranged from yellowish green to green, and the leaves displayed a soft fracture. Additionally, they emitted a slight aromatic odor and had a mildly bittertaste.
Figure 1a: Hygrophila triflora (Roxb.) Fosb. and Sachet flowering twig
Figure 1b: Hygrophila triflora (Roxb.) Fosb. and Sachet flowering twig
The transverse sections (T.S.) of Hygrophila triflora leaves revealed distinct anatomical features. The midrib exhibited upper and lower single-cell-layered epidermis with swollen bulbous epidermal cells. Moving towards the central vascular region, a cortical zone comprised of 4 to 6 parenchymatous cell layers surrounded the xylem vessels, phloem, and fibers. The lower cortex consisted of 2 to 4 cell layers followed by the lower epidermis. Notably, glandular trichomes were present in the epidermis, and characteristic cystoliths were observed in the cortical region. In the lamina, anisocytic stomata were evident on the upper epidermis, accompanied by numerous glandular trichomes with apical glands, mono to bicellular stalks, and prominent basal cells. Non-glandular tapering trichomes were also identified. The mesophyll consisted of spongy parenchyma compactly arranged with multiple glands, deposits, and small to elongated cystoliths enclosed within lithocysts. Groups of spiral xylem vessels were transversely distributed in the mesophyll tissue. [Figure 2a to 2e].
Fig : 2(a)
Fig :2(b)
Fig: 2(c)
Fig: 2(d)
Fig : 2(e)
Fig : 2(a-e) Transverse sections (T.S.) of leaf of Hygrophila triflora (Roxb.) Fosb. and Sachet
The fine powder derived from the leaves of Hygrophila triflora exhibited a greenish-brown coloration and possessed a slightly bitter taste along with a mildly aromatic odor. Examination of the powder microscopy revealed rectangular epidermal cells adorned with glandular and non-glandular covering trichomes. Glandular trichomes exhibited uni to tricellular stalks and brownish fan-shaped glands. Anisocytic stomata were also identified. Numerous spiral xylem vessels were frequently associated with long aseptate fibers. Furthermore, fragmented tissue from the lamina displayed profuse veins and mesophyll tissue. Prismatic crystals of calcium oxalate were abundant in thepowder.
These detailed findings shed light on the macroscopic, cytomorphological, and powder characteristics of Hygrophila triflora leaves, providing valuable insights into the plant's anatomical and phytochemical attributes. Such knowledge is essential for both taxonomic identification and the exploration of potential pharmacological and therapeutic applications of this plant. [Figure 3a to 3o].
a b
c d
e f
g h
i j
k l
m
n o
Figure 3 (a-o): Components of leaf powder of Hygrophila triflora (Roxb.) Fosb. and Sachet
a: Groups of epidermal cells with nonglandular covering trichomes (100X); b: Single fragmented covering trichome (100X); c, d: Fragmented single Glandular trichome with long stalk and apical gland(400X); e, f, g: Groups of fragmented spiral vessels with mesophyll tissue(100X); h, i, j: Groups of xylem vessels attached with fibres in mesophyll tissue (100X); k: Single thick walled fibre (100X); l: Fragmented tissue of lamina showing profuse veins and mesophyll tissue(100X); m,n,o: Prismatic crystals of calcium oxalate(400X)
The evaluation of various physicochemical parameters for the Hygrophila triflora sample yielded the following results:
Loss on Drying: The loss on drying was determined to be 9.95%, indicating the percentage of moisture content in the plant material. Notably, the API (Ayurvedic Pharmacopoeia of India) does not specify a prescribed range for this parameter.
Total Ash: The total ash content of the sample was found to be 13.60%. However, the API does not provide a specified range for total ash in the context of this plant material.
Acid Insoluble Ash: The acid insoluble ash content was determined to be 2.40%. Similar to the previous parameters, the API does not offer a prescribed range for acid insoluble ash in this case.
Alcohol Soluble Extractive: The alcohol soluble extractive content was measured at 5.10%. It's noteworthy that the API does not provide a specific range for this parameter concerning Hygrophila triflora.
Water Soluble Extractive: The water-soluble extractive content was determined to be 11.10%. As with other parameters, the API does not outline a defined range for water-soluble extractives in this context.
pH (10% Aqueous Suspension after 24hours): The pH of a 10% aqueous suspension, measured after 24 hours, was found to be 6.79. API does not offer a specified range for pH in the context of this plant material.
These results provide essential data on the physicochemical properties of Hygrophila triflora but also highlight the need for further standardization and the establishment of specific API ranges for these parameters to ensure consistent quality assessment in future studies and applications.
For the preparation of the sample, 2grams of Hygrophila triflora (Whole Plant) material was subjected to reflux with approximately 25mL of methanol for a duration of 1hour. Subsequently, the extract was carefully filtered using filter paper to remove any solid particulates. The resulting filtrate was then concentrated and set aside for the subsequent High-Performance Thin Layer Chromatography (HPTLC) analysis. The HPTLC analysis was conducted using a CAMAG HPTLC instrument from Switzerland. The software utilized for data analysis was CAMAG's winCATS version 1.4.6. A pre-coated silica gel plate was employed as the stationary phase. The specific plate used was TLC Silica Gel 60F254, manufactured by Merck on September 26, 2016, with Batch No. 1.05554.0007. The mobile phase consisted of a mixture of toluene, chloroform, and ethyl acetate in a ratio of 6:3:1 (v/v). The solvents utilized in this mobile phase were of GR (Guaranteed Reagent) grade and manufactured by MERCK, India. For the sample application, an aliquot of 5µL from the methanolic extract of Hygrophila triflora (Whole Plant) was applied as an 8mm band at a distance of 15 mm from the base of the TLC plate. The dimensions of the TLC plate used were 2.5 x 10cm.
The development of the chromatographic plate was achieved by placing it in a CAMAG Twin trough chamber. The development process extended up to a distance of 90mm on the plate. The plate preconditioning conditions were maintained at a temperature of 25°C, and the average relative humidity within the chamber was approximately 42%. For visualization and analysis of the chromatographic profile, the plates were observed at two distinct wavelengths: 254nm and 366nm. These well-defined parameters and conditions were employed for the HPTLC analysis, ensuring the accurate separation and identification of components within the methanolic extract of Hygrophila triflora (Whole Plant), and enabling a comprehensive assessment of its phytochemical profile.6,7,8,9
The High-Performance10,11 Thin Layer Chromatography (HPTLC) analysis of the methanolic extract of Hygrophila triflora (Whole Plant) revealed distinct chromatographic profiles when observed at two distinct wavelengths, namely 254nm and 366nm12,13.
Observed Rf Values at 254nm: The observed Rf (Retention Factor) values at 254nm were as follows: 0.02, 0.08, 0.14, 0.17, 0.25, 0.31, 0.35, 0.50, 0.60, 0.64, 0.72, 0.85, and 0.86. These values provide insight into the migration rates of different components present in the methanolic extract.
Observed Rf Values at 366nm: At a wavelength of 366 nm, the observed Rf values differed and were recorded as follows: 0.01, 0.03, 0.06, 0.16, 0.20, 0.29, 0.37, 0.49, 0.56, and 0.64. The variations in Rf values between the two wavelengths may indicate differential responses of the components to UV light absorption.
These HPTLC results serve as a valuable reference for identifying and characterizing the phytoconstituents present in the methanolic extract of Hygrophila triflora (Whole Plant). The distinct Rf values offer critical data for further analysis and elucidation of the plant's chemical composition, aiding in potential pharmacological and therapeutic investigations.
254nm 366nm
(a) 2D Densitogram at UV 254 nm
(B) 3D Densitogram at UV 254 nm
(C) 2D Densitogram at UV 366 nm
(D) 3D Densitogram at UV 366 nm
Fig- 5- HPTLC 2D and 3D densitograms of Hygrophila triflora (Whole plant) under UV 254 nm and UV 366 nm
A critical aspect of understanding the chemical composition and potential bioactive compounds within plant extracts involves qualitative analysis of secondary metabolites. Secondary metabolites are diverse, often specialized compounds that plants produce for various ecological roles, including defense against herbivores and pathogens, attraction of pollinators, and adaptation to environmental stressors.
Techniques such as chromatography, spectrophotometry, and spectroscopy are commonly employed to identify the presence of secondary metabolites, including phenolics, flavonoids, alkaloids, terpenoids, and saponins. Qualitative analysis allows researchers to ascertain the qualitative presence or absence of these compounds, providing initial insights into the phytochemical profile of plant extracts. Subsequent quantitative analysis techniques can then be applied to determine the concentration of specific secondary metabolites, helping to establish the potential pharmacological and therapeutic properties of plant materials. The interpreted results are mentioned in Table no 1:
Table No 1: Qualitative Analysis of Secondary Metabolites
|
Sl. No. |
Secondary Metabolites |
Test |
Hexane Extract |
Ethyl acetate Extract |
MeOH Extract |
|
1 |
Alkaloid |
a) Dragendroff Test |
Negative |
Negative |
Positive |
|
b) Mayers Test |
Negative |
Negative |
Positive |
||
|
2 |
Flavonoid |
a) NaOH Test |
Negative |
Positive |
Positive |
|
b) Pb(OAc)2 Test |
Negative |
Positive |
Positive |
||
|
c) Shinoda Test |
Negative |
Positive |
Positive |
||
|
3 |
Tannins |
a) FeCl3 test |
Negative |
Negative |
Positive |
This comprehensive study delved into Hygrophila triflora (Roxb.) Fosb. and Sachet, a South Asian herbaceous plant. We explored its taxonomy, morphological diversity, ecological importance, anatomy, and phytochemical composition[14]. The taxonomic journey of Hygrophila triflora highlights the intricacies of plant classification, emphasizing the need for ongoing botanical research. Its unique dimorphic leaves, suited for both aquatic and terrestrial habitats, underscore its adaptability, particularly in watercourse ecosystems. Anatomical examinations unveiled distinct features like glandular trichomes and cystoliths, shedding light on its structural adaptations. HPTLC analysis provided insights into its phytochemical profile, with potential implications for pharmacological investigations. Physicochemical assessments contributed to quality assessment and standardization. Qualitative analysis detected alkaloids, flavonoids, and tannins, suggesting medicinal potential. In essence, this research enhances our understanding of Hygrophila triflora, with implications for both botanical science and medicinal applications. Its role as an indicator species in aquatic ecosystems underscores its ecological importance, emphasizing the need for conservation. Continued exploration of indigenous plants like Hygrophila triflora is crucial for uncovering their multifaceted contributions
CONFLICTS OF INTEREST:
The authors have no conflict of interest regarding this investigation.
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Received on 25.09.2023 Modified on 19.12.2023
Accepted on 06.02.2024 © RJPT All right reserved
Research J. Pharm. and Tech. 2024; 17(3):1159-1165.
DOI: 10.52711/0974-360X.2024.00180