|Year : 2023 | Volume
| Issue : 3 | Page : 271-279
Establishment of botanical and phytochemical standards for quality assessment of Sida veronicifolia Lam. roots
Jyoti Dahiya, Navdeep Chourasiya, Rajendra Kumar Panda, Karan Vasisht, Maninder Karan
Department of Pharmacognosy, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, Punjab, India
|Date of Submission||24-Jan-2023|
|Date of Acceptance||20-Apr-2023|
|Date of Web Publication||16-Aug-2023|
Prof. Maninder Karan
Department of Pharmacognosy, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, Punjab
Source of Support: None, Conflict of Interest: None
BACKGROUND: Sida veronicifolia Lam. (Malvaceae), popularly known as Nagabala, is a well-known medicinal plant of the Indian system of medicine, traditionally used in a variety of health problems. In spite of the significant pharmacological activities of S. veronicifolia roots, no detailed botanical and chemical work is reported. In the present study, the authors aimed to develop the pharmacognostical and chemical standards using instrumental analytical techniques. METHODS: Sophisticated analytical techniques viz. TLC-densitometry, high-performance liquid chromatography, and Fourier-transform infrared spectroscopy were utilized in the present study. RESULT: The pharmacognostic parameters, physicochemical parameters, and chemical profiling have provided valuable information as a tool of identification of the plant. CONCLUSION: The quality standards developed in this study can serve not only for authentication of S. veronicifolia roots but also discrimination from other roots of Sida genus.
Keywords: Fourier-transform infrared spectroscopy, high-performance liquid chromatography, Sida veronicifolia Lam., TLC-densitometry
|How to cite this article:|
Dahiya J, Chourasiya N, Panda RK, Vasisht K, Karan M. Establishment of botanical and phytochemical standards for quality assessment of Sida veronicifolia Lam. roots. J Drug Res Ayurvedic Sci 2023;8:271-9
|How to cite this URL:|
Dahiya J, Chourasiya N, Panda RK, Vasisht K, Karan M. Establishment of botanical and phytochemical standards for quality assessment of Sida veronicifolia Lam. roots. J Drug Res Ayurvedic Sci [serial online] 2023 [cited 2023 Sep 23];8:271-9. Available from: http://www.jdrasccras.com/text.asp?2023/8/3/271/383698
| Introduction|| |
Sida veronicifolia Lam. (syn: Sida cordata (Burm. f.) Borss. Waalk. is a renowned medicinal plant of the Ayurveda and the Siddha system of medicine. In Ayurveda, the plant is also known as Nagabala, Bhumibala, Shaktibala, etc., and it is believed that the plant provides elephant-like strength, as in ancient literature, Naga refers to elephant probably because of its snake-like trunk., It is mentioned as an official substitute for Nagabala in AFI. It is a highly branched hairy herb of the Malvaceae family with a small main stem and sprawling long branches. The herb mainly grows as a weed all over the hotter parts of India and also in tropical and subtropical parts of the world.,Nagabala has the power to eliminate the imbalance of three doshas and provide strength to body. The powder of root with Arjuna bark is conventionally consumed with milk to treat heart-related and breathing problems, including cough. According to Charak Samhita, Nagabala roots are consumed as Rasayana with milk for one month, with an initial dose of 5 g, gradually increasing up to 40 g. Also, the leaves and roots of this plant have high wound-healing properties. Traditionally, Nagabala is used as a pain healer in childbirth, and it also reduces the period of postpartum bleeding. The roots are administered as a soup in many tribal areas in the last days of pregnancy. Pharmacological reports also revealed abortifacient outcomes of the whole plant in pregnant rats on oral administration during 15–17 days of pregnancy. The ethanol and aqueous extract of leaves of S. veronicifolia have shown antitumor activity against Ehrlich ascites carcinoma. The root of this plant is useful in leucorrhoea and infections of the genitourinary tract when taken as root juice (10–20 mL) and root decoction (50–100 mL). The exhaustive literature review revealed that no detailed work on the phytochemistry of this plant is reported except for a water-soluble fraction of an alcohol extract with a muscarine-like active component. In spite of the countless significant pharmacological roles of S. veronicifolia, no reports on pharmacognostical and phytochemical studies as quality standards have been communicated until now. Hence, in the present study, the quality control parameters for roots of S. veronicifolia have been developed. Several techniques like microscopy, chromatography, and spectroscopy have been utilized in the development of quality parameters. Moreover, chemical fingerprint profiling has been developed by three sophisticated techniques, namely: TLC-densitometry, high-performance liquid chromatography (HPLC), and Fourier-transform infrared spectroscopy (FT-IR) for a better understanding of the chemical nature of constituents present in the plant.
| Materials and Methods|| |
Collection, identification, and authentication of plant material
The roots of S. veronicifolia Lam. were collected in the month of July 2019 from the Medicinal Plants Garden of Panjab University, Chandigarh (latitude: 30°45′ 38.2248″N and longitude: 76°45′ 55.3968″E). The identity of the root was authenticated by NISCAIR, New Delhi, vide certificate no. NISCAIR/RHMD/Consult/2020/3627-28-4. The voucher sample as a whole plant (WP/1509) was also deposited at the museum-cum-herbarium of the Department of Pharmacognosy, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, for all future references.
Material, methods, and reagents
Reagents used in microscopic studies are phloroglucinol (Merck, Mumbai) and chloral hydrate (TCI Co. Ltd., Tokyo, Japan). For phytochemical studies, Dragendorff’s, Million’s, Biuret, and many other reagents of Loba Chemie Pvt. Ltd, Mumbai, were used. All samples for HPLC and mobile phase were prepared in HPLC-grade solvents. Precoated silica gel 60F254 plates (Merck India Ltd. Catalogue number: 1.05554.0007) of thickness 200 µm were used in TLC fingerprint studies. The FT-IR studies were done using a KBr (particle size of less than 2 µm) disc.
Plant sample preparation
The roots were thoroughly washed with distilled water following collection and dried at room temperature. Both fresh and dried root samples were used for morphological, organoleptic, and transverse section studies. The shade-dried roots were pulverized to obtain both the coarse powder (sieved with 25 #) as well as a fine powder (sieved with 60 #) and used in further studies. The samples were preserved in an airtight container, and extracts were stored in a refrigerator following standard guidelines throughout the study period.
Morphological and organoleptic studies
The morphology of the root samples was examined by the naked eye and also by using a simple microscope (Olympus make; model no: OIC DM) to note the characteristic features. The organoleptic characters were determined as per the API guidelines.,
For powder microscopy, a finely powdered root sample was treated with aqueous saturated chloral hydrate followed by treatment with phloroglucinol:HCl (1:1) to stain lignified tissues. The treated powder was also mounted in glycerine and observed under the trinocular microscope at 10×. Photographs of different cellular structures were taken with a camera (Magcam make; model no: DC14) attached with a microscope.,
Transverse section studies
The transverse section was performed by the free-hand section technique. The sections were mounted in glycerine and observed under a microscope. The thinnest section with the highest clarity was stained with phloroglucinol reagent, and histological analysis was carried out to observe different cellular structures and inclusions under a trinocular microscope (Olympus make; model no: CX21i) at 10× and 40× magnification. Photomicrographs were taken with a camera (Magcam make; model no: DC14) attached with a microscope.,
The physicochemical parameters like ash values, extractive values, loss on drying, and pH value (10% aqueous solution) of roots were determined by using coarse powder as per the standard guidelines of The Ayurvedic Pharmacopoeia of India (API). Additionally, extraction with different solvents of varying polarities, viz. petroleum ether, chloroform, ethyl acetate, and methanol, were also done by conventional maceration and reflux method, using 2 g of powdered drug sample and 100 mL of solvent. The maceration was done at room temperature and extraction by reflux at the respective boiling point of the solvent.
Preparation of extract
A coarsely powdered root sample (5 g) of S. veronicifolia was refluxed with 100 mL of methanol for 30 min in a water bath. The extract was filtered, and the solvent was recovered in a rotary vacuum evaporator to obtain a residue. The residue was completely dried in a vacuum oven. The dried extracts were redissolved in methanol and used for phytochemical screening, chromatographic and spectroscopic studies.
A part of the redissolved extract was treated with different reagents to detect the presence of phytochemical groups like alkaloids, flavonoids, phenols, etc.,
A volume of 10 µL of methanol extract (1 mg/mL) was applied as an 8-mm band on a precoated TLC plate using an ATS-4 applicator, CAMAG HPTLC system. The plate was developed in solvent system toluene: chloroform: ethyl acetate: formic acid (6: 2.5: 1: 0.5 v/v) to a distance of 8 cm in a twin-trough chamber saturated for 20 min. The developed plate was dried in a current of hot air, visualized under ultraviolet light (254 and 366 nm), and the profiles were captured as digital images. The same plate was further derivatized by dipping in an anisaldehyde-sulphuric acid reagent followed by heating at 110°C, and observation was made under white light. The profile was recorded as a digital image. The images of the TLC plate were taken using CAMAG Reprostar-3 fitted with Dx A 252 16 mm camera. CAMAG TLC Scanner and WINCATS software (version 1.4.5) were used to scan the chromatograms at wavelengths 254 and 366 nm.
High-performance liquid chromatography: fingerprint analysis
Alliance HPLC system comprising of 2695 separation module (Waters Corporation, Milford, Massachusetts) with an auto-sampler and 2996 PDA detector was used in the present study for HPLC fingerprinting. The best chromatographic separation was achieved on a reversed-phase C18 column, 150 × 4.6 mm, 5 µm (Waters Atlantis) at 25°C. A mobile phase with methanol: water (40:60) was optimized with a flow rate of 0.7 mL/min for 30 minutes. 10 µL of methanol extract (1 mg/mL) was injected, and peaks were detected at 210 nm (preoptimized wavelength with maximum peaks). The Empower 2 chromatography software was used for data acquisition and processing.
Fourier-transform infrared spectroscopy: fingerprint analysis
For FT-IR analysis, 2 mg of dry residue (methanol extract) was grounded with a weighed quantity of KBr in an agate mortar and pestle (particle size of less than 2 µm); the grinding was done under an IR lamp to avoid condensation of atmospheric moisture. After complete drying, the disc was prepared by pressing the grounded mixture at elevated temperature and pressure. A blank disc was also prepared with pure KBr and used as a reference pellet. The discs were analyzed with a spectrophotometer (Perkin Elmer make; RX1 FT-IR Spectrophotometer), and the spectra were taken in both transmittance and absorbance mode.
| Results|| |
Morphological and organoleptic studies
The roots are cylindrical, 10–15 cm in long and 0.7–1.2 cm in width, with rough texture with minute scars of lateral rootlets, branched with plenty of lateral roots, tortuous, fracture short in root bark and fibrous in wood, the root bark with a thickness of about 0.8 mm can be easily peeled off, externally brown in color, internally light brown, tasteless and odorless. [Figure 1] presents the morphology of the root of S. veronicifolia.
The fine powder of S. veronicifolia is light brown in color. Powder microscopy at 10× revealed the presence of fragmented thick-walled cork cells and parenchymatic cells with deposition of tannin and minute starch grains. Rosette crystals were found isolated as well as embedded in cortical parenchyma. Fibers, groups of fibers, and fibers with pitted vessels were also seen. Prismatic calcium oxalate crystals and spherical to oval-shaped starch grains were also observed, as presented in [Figure 2].
|Figure 2: Powder microscopy of roots of Sida veronicifolia. (A) Cork cells with brownish content; (B) parenchymatic cells treated with phloroglucinol; (C and D) rosette crystals deposited in cortical parenchyma and fibers; (E) pitted vessels; (F and G) fiber and scattered minute starch grains; (H) calcium oxalate crystal and minute starch grains; (I) group of cells with tannin deposition|
Click here to view
The transverse section of the root [Figure 3] showed a circular outline comprising the following tissues from periphery to center. Cork consisting four to five layered tangentially elongated polygonal cuticularized cells; phelloderm consisting thin-walled irregular cells; secondary cortex consisting thin-walled parenchymatous cells embedded with abundant rosette crystals and numerous phloem fibers; phloem narrow comprising of thin-walled cells; cambium narrow present in between phloem and xylem region; wood consisting of xylem, medullary rays, and pith; secondary xylem occupies the major part of the wood and is composed of xylem vessels, xylem parenchyma, and thin-walled cells. Xylem vessels are lignified, metaxylem wide, and round in shape; medullary rays run radially from the center part to the cortex, uni-biseriate, lignified, elongated radially and thin-walled; narrow pith present in the central part.
[Table 1] represents the compiled results of different quality parameters, namely ash values, loss on drying, pH value, and extractive values. The results were expressed in percentage as mean ± standard deviation (SD).
The phytochemical screening of methanolic extract revealed the presence of alkaloids, tri-terpenoids, flavonoids, steroids, and phenolics. The result of phytochemical screening is given in [Table 2].
Thin layer chromatography
A variety of combinations of solvents were tried as mobile phase to produce a resolved chromatogram of the methanolic extract prepared, as detailed in Section TLC-densitometry. The final composition of the mobile phase was selected as toluene: chloroform: ethyl acetate: formic acid in the ratio 6:2.5:1:0.5. Six bands were observed under short UV (254 nm), whereas nine bands were found under long UV (366 nm) at different Rf values before derivatization. After derivatization with an anisaldehyde-sulphuric acid reagent, nine bands were seen. The TLC fingerprint results are tabulated in [Table 3], and chromatograms are presented in [Figure 4] and [Figure 5].
High-performance liquid chromatography
[Figure 6] shows the HPLC fingerprint profile at 210 nm and represents 12 peaks with different Rt. The peaks are marked as Peak A to Peak L at Rt, ranging from 5.52 to 25.76 min. The area under the curve, obtained at different Rt, is summarized in [Table 4].
Fourier-transform infrared spectroscopy
The IR profiles of methanolic extracts of roots of S. veronicifolia are presented in [Figure 7], in transmittance (7-a) and absorption (7-b) mode in the range 400–4000 cm-1. The absorbance noticed at different wave numbers is summarized in [Table 5]. The transmittance mode spectra revealed distinct peaks at 3424.47, 2927.85, 2854.19, 1633.74, 1494.10, 1453, 1398.09, 1384.83, 1338.78, 1261.14, 1052.54, 938.81, 897.81, 721.02 cm-1.
| Discussion|| |
Nagabala (S. veronicifolia) is an important plant in the Indian system of medicine. The exhaustive literature survey revealed the pharmacological importance of the roots of Nagabala, but no detailed pharmacognostical, botanical, and chemical work was reported for this drug. Quality control by morphological and microscopical techniques plays an important role in the establishment of the appropriate identity of medicinal plants., Hence, the present study was planned to develop the botanical and phytochemical quality standards of the roots of S. veronicifolia. The morphological study revealed a tap root system with a thin and tapered end. Roots were terminally branched with a length of 10–15 cm and a thickness of 0.7–1.2 cm. The color of the roots was light brown, with no odor and taste.
To study the anatomical features, powder microscopy, and transverse section were done. The microscopic evaluation of powdered roots showed the presence of abundant rosette crystal deposition in the secondary cortex as the major diagnostic character. Other cellular structures like cork cells, starch grain, prismatic crystals, fibers, pitted vessels, and parenchyma cells with tannin content were also found. The transverse section was carried out to have a complete picture of the anatomical structure of the root to find the cell arrangement, cell positions, cell content, etc. The transverse section showed cork cells followed by the cortex, phloem region, cambium, xylem region, medullary rays, and pith from the periphery to the center. Abundant rosette crystals were found embedded in the secondary cortical cells. Wide metaxylem vessels occupy a major wood portion. Unibiseriate medullary rays with very narrow pith were also seen.
As per “The Ayurvedic Pharmacopoeia of India,” physicochemical studies are a vital tool for the standardization of plant drugs. The results of different parameters, including loss on drying, ash values, extractive values, and pH value, were determined and expressed as mean ± SD. The loss on drying was found to be 11.27%± 0.32%. Total ash value, water-soluble, and acid-insoluble ash values were 9.67% ± 0.24%, 4.65% ± 0.23%, and 2.76% ± 0.15%, respectively. The extractive values in methanol (25.76% ± 0.24% and 28.56% ± 0.18% in cold maceration and reflux extraction, respectively) were found to be highest, showing the presence of more polar components in the root. The same solvent was used for preparing the extract for further analysis. The preliminary phytochemical analysis revealed the presence of alkaloids, triterpenoids, flavonoids, steroids, and phenolics, and these phytochemical groups are also reported to occur in other Sida species.,, Alkaloids, steroids, and triterpenoids are the major phytochemicals, whereas flavonoids and phenolics are the minor phytochemicals.
The chemical fingerprint analysis has more potential to reveal the chemical nature of plant constituents in totality than the marker analysis; therefore, in the present study, three analytical techniques, namely TLC, HPLC, and FT-IR, were used to develop the chemical profile of S. veronicifolia roots. TLC fingerprint profile under 254 nm showed six black-colored bands at Rf values of 0.03, 0.1, 0.18, 0.25, 0.28, and 0.68 against the green background. Under 366 nm, seven light fluorescent blue bands at Rf 0.03, 0.1, 0.15, 0.18, 0.35, 0.50, and 0.56, one green band at Rf 0.68 and one red band at Rf 0.72 were observed as a result of fluorescence quenching. Derivatization with anisaldehyde-sulphuric acid reagent resulted in the appearance of seven purple bands at Rf 0.15, 0.18, 0.35, 0.43, 0.50, 0.87, and 0.91 and two orange bands at Rf 0.68 and 0.72; the purple bands support the presence of triterpenoid components. Further, HPLC was used to develop the fingerprint profile to have more details. In HPLC analysis, twelve peaks were noticed at λmax 210 nm, and the earlier reports have suggested mostly alkaloidal components at this wavelength. Further, to find the functional groups and their absorbance, preliminary FT-IR was done in both transmittance and absorbance modes. The FT-IR profile showed many peaks, with the highest intensity peak appearing in the region 1619–1612, giving an indication of –C=C– bonds in the chemical components of roots. The band at around 3400 cm-1 confirms the presence of N–H stretching of alkaloids. The peaks at 1380–1399 and 1000–1300 cm-1 indicated the presence of compounds with C–H or C–O bonds. The peak in the region of 2800–3000 cm-1 indicated the presence of –C=CH2– bonds suggesting the presence of long-chain alkanes/alkenes and their derivatives.,,
All technical information generated from different analytical techniques supported the presence of alkaloids and triterpenoid components as a major class of chemical constituents of S. veronicifolia roots. These results can be further used to chemically explore the S. veronicifolia roots for the isolation and characterization of chemical components. Further, important isolated components can be evaluated for their ethnopharmacological properties using in vitro and in vivo models. Moreover, the developed quality parameters can be used as a discriminative tool to differentiate the morphologically similar roots of the Sida genus, viz. Bala, Atibala, Mahabala, Nagabala, Bhumibala, Rajabala, and many more.,,,
| Conclusion|| |
The present study has generated a set of phyto-pharmacognostical quality parameters of S. veronicifolia roots following macroscopic, anatomical, microscopical, physicochemical, and chemical fingerprint (TLC, HPLC, and FT-IR) analysis. The generated data can serve as an investigative tool for the quality assessment, authentication, and distinguishing medicinally significant plant species of the Sida genus.
The authors are thankful to the Chairperson, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India, for providing all the necessary facilities.
Financial support and sponsorship
The financial support was provided by the UGC—India under Research Fellowship in Science for Meritorious Student Scheme to Jyoti Dahiya.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]