Pharmacognostical Standardization of Balamulachurna-Root Powder of Sida rhombifolia subsp. retusa (L.) Borssum

Background: Ksheerabala tailam is well known Ayurvedic herbal formulation which is used in inflammations, pains and neurological disorders. The primary botanical source for Ksheerabala tailam is Balamulachurna. The literature refers to Balamulachurna as either of the root powders of Sida cordifolia, Sida acuta, Sida cordata or Sida rhombifolia subsp . retusa . To accomplish the clinical efficacy of Ksheerabala tailam , standardization of Balamulachurna is necessary. Objectives: The study is aimed to establish pharmacognostic standards of Balamulachurna with respect to Sida rhombifolia subsp. retusa root powder . Materials and Methods: Pharmacognostical standardization was carried out by focusing on organoleptic, macroscopic, microscopic, preliminary phytochemical evaluation, heavy metal assessments, fluorescence and HPTLC studies. Results: The roots macroscopic features were noticed as; an elongated taproot with few lateral roots, thick, woody, brown-coloured and bitter to taste. Organoleptic characteristics revealed the nature of the root powder as cream in colour with a pleasant odour and slightly bitter taste. The powder microscopic study specified the presence of fibres, fibre–sclereids, tracheids, vessel elements and calcium oxalate crystals. Physicochemical studies assessed total ash (7.5%), water-soluble ash (4.46%), alkalinity of water-soluble ash (0.4 ml) and acid-insoluble ash (3.3%). The preliminary phytochemical analyses revealed the presence of glycosides, alkaloids, phenols, flavonoids, tannins, terpenoids, and quinones. The HPTLC analyses exhibited various R f values with the diversity of multiple phytoconstituents. Conclusion: The scientific data generated by the study is beneficial for adequately identifying and establishing standards for using Sida rhombifolia subsp. retusa as a primary drug for maintaining the quality and purity of the Ksheerabala tailam , an Ayurvedic formulation.


INTRODUCTION
India has an ancient, dynamic, and diversified cultural past.The concept of health and healing plays a significant role in this culture and tradition.8][9][10][11][12][13] According to the National Medicinal Plants Board (NMPB), bala is the third-most-used medication in the Ayurvedic pharmaceutical sector.It is primarily collected from the wild. [14]The literature review referred to the roots of Sida rhombifolia subsp.retusa as Bala in Kerala. [15]Bala is a common herbal remedy with a variety of applications.[16][17] It is also the primary component of Ksheerabala tailam, therapeutic oil made from Sida rhombifolia subsp.retusa (Figure 1).The musculoskeletal system is treated with this oil in cases of sickness as a rejuvenator, which promotes tissue recovery and relieves pain, stiffness, inflammations, and other adverse effects.It also nourishes the nerves, spinal cord, and brain.16][17] The practice and demand for Ayurvedic medicine have rapidly expanded during the past few decades.Nowadays, in the same way, adulterations have increased with problems associated with their purity, quality, safety, and efficacy.[19][20][21][22] The present study aims to standardize the Balamulachurna with the use of pharmacognostic tools.

Collection of the material
Roots of Sida rhombifolia subsp.retusa was collected from hilly areas of Kottayam, Kerala and identified by comparing it with the herbarium specimen deposited at Sri Venkateswara University, Tirupati.The herbarium accession number is SVUBS1258. [14,16,17]

Powder preparation
Shade-dried roots were sliced into small pieces and powdered using a grinding mill.8][19]

Macroscopic and organoleptic evaluation
The crude roots were examined with the naked eye for macroscopic characteristics.0][21][22]

High-performance thin-layer chromatography
The methanolic extract of root powder was applied manually in triplets on a TLC plate and subjected to a solvent system having toluene: ethyl acetate (6:4).It was saturated for 45 min in CAMAG® Flat Bottom Chamber of 10 x 10 cm.These plates were kept in the HPTLC instrument and analyzed under UV light at 366 nm and 254 nm; daylight and bands were observed.

Macroscopic characters
Roots are elongated taproots with a length of 20-25cm, and the diameter is 1 cm 2 to 1.5 cm 2 .These were thick, hard to touch, brown-coloured and bitter to taste.Lateral roots were slender and few in number (Figure 1).

Organoleptic characteristics
The root powder is cream in colour, coarse fibrous powdery in nature, pleasant odour and slightly bitter taste (Figure 1 and Table 1).The root powder was subjected to powder microscopy, and noticed the presence of rosette crystals of calcium oxalate, starch grains, collenchyma cells and fibres (Figure 2: A, B, C, D).

Root Maceration
The dried root was subjected to maceration and following elements were noticed.(Figure 2: E and F).

Fibres
Fibres are primarily narrow with thick walls and narrow lumen, 545 mm long and 12 mm wide (Figure 2: D, E and F).

Fibres -Sclereids
These cells resemble the fibres but have a wider lumen shorter than the fibres, 250-450 m long, and 25 m thick (Figure 2: E and  F).

Tracheids
They were elongated, thick-walled cells with tapering ends; lateral wall pits well developed, bordered and abundant; 210 m long (Figure 2: E and F).

Vessel elements
They are narrow, cylindrical, and relatively long.Perforation plate is simple, mostly oblique, one-sided tailed or non-tailed, tail short, thick and 220-260 m long (Figure 2: E and F).

Physicochemical parameters
The pH, ash and extractive values are depicted in Table 2 and Table 3.
In heavy metal analysis, Arsenic (As) and Lead (Pb) content were found to be less than 2 to 20 ppm respectively.
Powder analysis [7][8][9][10] Root powder was treated with different reagents like distilled water, 5% aqueous NaOH and 60% aqueous sulphuric acid, and the powder was pressed between filter paper for 24 hr.The results are documented in Table 4.

Fluorescence analysis
Fluorescence study is an essential tool for the standardization of powder drugs.Results in the form of colour variations were obtained by treating the plant powder with different reagents like NaOH, HNO 3 , H 2 SO 4 , FeCl 3 , HCl, picric acid and acetic acid under ordinary light and ultraviolet (UV) light at 254 and 366 nm (Table 5).

Preliminary phytochemical screening
This study was accomplished for qualitative screening through methanolic and water extracts (Table 6).

HPTLC screening
Root extracts of the samples were spotted in triplets on the silica gel "G" plate (Figure 3).These plates were developed utilizing the mobile phase as toluene: ethyl acetate (6:4).Various spots were identified under UV light at 366 nm (Figure 3

DISCUSSION
Ayurveda is the most widely used system in the Traditional Indian system of medicine.In Ayurveda system of Medicine, Ksheerabala tailam is a compound formulation used to treat various diseases.The primary botanical source for this formulation is Balamulachurna, [14][15][16][17] root powder of Sida rhombifolia subsp.retusa.The root powder is subjected to different pharmacognostic standardization methods to maintain the quality of the formulation without any adulteration.
The observations from organoleptic, macroscopic, microscopic, physico-chemical, heavy metal assessments and fluorescence studies revealed some specific results for the drug plant, Sida      [14][15][16][17] The qualitative analysis of preliminary phytochemical screening from methanolic extracts revealed that the plant is a good source of glycosides and flavonoids, followed by alkaloids, phenols, tannins, terpenoids, and quinones.
HPTLC is an essential tool to standardize the product by developing fingerprint profiles, quantifying phytoconstituents and determining impurities.The R f values produced by HPTLC analysis are specific for Sida rhombifolia subsp.6][17]

CONCLUSION
The study on Balamulachurna with the scope of Sida rhombifolia subsp.retusa root powder successfully established the pharmacognostic standards.The morphological, microscopical, physicochemical, preliminary phytochemical and chromatographic parameters of this controversial drug would definitely support the identification of the species.It is also helpful in determining its purity and quality standards during the preparation of Ayurvedic formulations.Further, this type of data could be employed amicably in evaluating pharmacopoeial standards and adulteration in terms of quality assurance of drugs with techniques like HPTLC.
: A), 254 nm (Figure 3: B), under Iodine vapours (Figure 3: C) and derivatized with Anisaldehyde sulphuric acid (Figure 3: D).Different R f values are summarized in Table 7.The densitograms were developed for the root powder of Sida rhombifolia subsp.retusa plant (Figure 3: E).TLC studies of the root powder showed six major spots under U.V. light (366) nm with the R f values as; 0.36 (red), 0.54 (red), 0.57 (blue), 0.71 (red), 0.86 (blue) and 0.99 (red); under Iodine, vapours shows one spot at R f value 0.71 (brown) and under visible region after derivatizing with anisaldehyde sulphuric acid shows one spot at R f value 0.71 (purple).

Figure 1 :
Figure 1: A. Morphology of the plant; B. Fresh roots; C. Dried roots; D. Root powder.

Figure 3 :
Figure 3: A. TLC chromatogram under UV light at 366 nm; B. TLC chromatogram under UV light at 254 nm; C. TLC chromatogram under Iodine vapors with visible region; D. TLC chromatogram derivatized with anisaldehyde sulphuric acid; E. Densitogram showing the separation of peaks of root powder at UV 366nm.