Pharmacognostic, Physicochemical, Phytochemical and Chromatographic Characterization of Vaividanga Choornam, A Siddha Polyherbal Formulation

P. Parvathy¹*, M. Natarajan², B. Neethu Kannan³, G. S. Lekha⁴, A. Kanagarajan⁵

¹Research Associate (Siddha) - II, Siddha Regional Research Institute,

(Under C.C.R.S, Ministry of Ayush, Govt. of India) Thiruvananthapuram, Kerala - 695012.

²Research Assistant (Chemistry), Siddha Regional Research Institute,

(Under C.C.R.S, Ministry of Ayush, Govt. of India) Thiruvananthapuram, Kerala - 695012.

³Assistant Research Officer (Botany), Siddha Regional Research Institute,

(Under C.C.R.S, Ministry of Ayush, Govt. of India) Thiruvananthapuram, Kerala - 695012.

⁴Research Officer (Siddha), Siddha Regional Research Institute,

(Under C.C.R.S, Ministry of Ayush, Govt. of India) Thiruvananthapuram, Kerala - 695012.

⁵Assistant Director, Siddha Regional Research Institute,

(Under C.C.R.S, Ministry of Ayush, Govt. of India) Thiruvananthapuram, Kerala - 695012.

*Corresponding Author E-mail: parvathyamaldev@gmail.com

ABSTRACT:

Standardization of herbal formulations as determined by the concentration of their activecomponents is essential to appraise the quality of drugs. The current study intends to investigate the phytochemical and physicochemical analyses of Vaividanga Choornam, a traditional Siddha polyherbal composition. This Vaividanga choornam contains the ingredients like Vaividangam (Embelica ribesBurm.), Milagu (Piper nigrumL.), Chukku (Zingiber officinale Rosc.), Omam (Trachyspermum ammi (L) Sprague), and Nilavaagai leaves (Cassia angustifolia Vahl. - syn. Cassia senna). It treatspitha diseases, constipation, indigestion, flatulence, burning sensation, bronchial asthma, and Kapha diseases. It is also an effective medicine for treating intestinal helminths caused by heavy worm burdens in young children. This study aims to estimate the quality of Vaividanga Choornam by performing pharmacognostic studies, preliminary phytochemical analysis, physiochemical parameters like ash value, extractive value, and loss on drying as per pharmacopoeial laboratory for Indian medicine guidelines, and also with the help of tests done with High-Performance Thin Layer Chromatography (HPTLC), FTIR Spectroscopy, and UV -Vis spectrometric analyses. The results obtained fromthose analyses showed the presence of various phytochemicals. The results indicate that the drug is of adequate quality and may be used to establish pharmacopeial standards of the medicine.

KEYWORDS: FTIR Spectroscopy, HPTLC, Pharmacognostic analysis, Physicochemical analysis, Phytochemical analysis, Siddha formulation, Standardization, Vaividanga Choornam.

1. INTRODUCTION:

Globally, there has been a resurgence in herbal medicine. The widespread manufacturing of Siddha formulations indicates the current increase in interest in the Siddha system, an old and indigenous system of Indian medicine that is mostly practiced in South India.¹

Vaividanga Choornam is a polyherbal Siddha formulation used in treating pitha diseases, constipation, indigestion, flatulence, burning sensation, bronchial asthma, and Kapha diseases². It is also a very effective medicine to treat intestinal helminths caused by heavy worm burden in young children³. The medicines used in the siddha system are time-tested and used by generations in South India⁴. However, one of the barriers to the great acceptance of medical preparations in this system is the need for standard quality control profiles. Standardization of the drug will bear witness to its excellent quality and reliability. Therefore, it is of the utmost importance to provide confirmation of their identity and demonstrate that they are pure, high-quality, and quantity.⁵ By identifying the constituents under a microscope and employing chemical parameters such as physicochemical and phytochemical analysis, UV visible spectroscopy, FTIR spectrometry, and HPTLC fingerprinting profile, this study aims to verify and standardize Vaividanga Choornam.

2. MATERIAL AND METHODS:

2.1 Standard Operating Procedure for Preparation:

Vaividanga Choornam, a Siddha polyherbal formulation, is prepared based on the Kannusamy Paramparai Vaidhyam.² Textbook Cassia anguistifolia leaves were collected from a village, near Thirunelveli district of Tamil Nadu, and other raw drugs, such as vaividangam, omum, chukku, and milagu, were collected from the local market and identified and authenticated by the Pharmacognosy department of Siddha Regional Research Institute, Thiruvananthapuram. Cassia anguistifolia leaves were powdered, sieved, then purified by the steam boiling method in cow’s milk three times and dried in the shade.All other ingredients (Table 1) were dried, powdered separately, and passed through a 250µ sieve. Then, all the ingredients were mixed in specified proportions geometrically to get a uniform mixture and preserved in a tightly closed container. Table 1 lists the constituents of Vaividanga choornam, along with their names and quantities. It is given in the dose of thirikadi alavu (800-1000mg)⁶ twice or thrice daily with jaggery.

2.2 Organoleptic evaluation:

Organoleptic characteristics of Vaividanga Choornam, such as color, odor and taste, were noted.A tiny portion of a powdered sample was placed in glycerin and left at room temperature for two hours, then examined using a bright field microscope (Meswox, India) with 10X and 40X objectives.

Figure 1: Ingredients of Vaividanga Choornam – Vaividangam, Omum, Chukku, Milagu

Figure 2: Nilavaagai Leaves

Figure 3: Prepared drug, Vaividanga Choornam

2.3 Quality Evaluation of Vaividanga choornam:

2.3.1 Physicochemical parameters:

Vaividanga Choornam was evaluated for purity and caliber using WHO standards.¹² Physico-chemical parameters such as Total Ash, Acid Insoluble Ash, Water Insoluble Ash, Alcohol Soluble Extractives, and Water Soluble Extractives were measured.¹³ Soxhlet extraction was used to extract the extracts using ethanol under reduced pressure at room temperature.

2.3.2 Qualitative phytochemical parameters:

Standard protocols were followed for the initial phytochemical examination of proteins, alkaloids, carbohydrates, terpenoids, flavonoids, quinones, phenol, terpenoids, tannins, steroids, glycosides, and saponins.¹⁴

Table 1: Composition of Vaividanga Choornam²

Sl. No	English Name	Tamil name	Botanical name	Active compounds	Parts used	Quantity (gm)
1	False black pepper	Vaividangam	Embeliaribes Burm.	Embelin, quercitol, fatty ingredients, alkaloid, schristembine- a resinoid, tannins, and volatile oils.⁷	Fruits	35gms
2	Ajwain/ Ajowan	Omam	Trachyspermum ammi (L.) Sprague	Thymol, thymine, limonene, glycosides, saponins, and flavones.⁸	Fruit	35gms
3	Dry ginger	Chukku	Zingiber officinale Rosc.	Zingiberene, ß-bisabolene, ß-sesquiphellandrene, ß-curcumene, ß-farnesene, paradols, ß-shogaol, phytosterols.⁹	Rhizome	35 gms
4	Black pepper	Milagu	Piper nigrum L.	Piperine,chavicine, piperdine and piperettine¹⁰.	Fruits	35gms
5	Senna	Nilavaagai	Cassia angustifolia Vahl. (syn.C. senna)	Apigenin-6,8-di-C-glycoside, aloe emodin, tinnevellin glycoside, isorhamnetin-3-O- beta-gentiobioside, kaempferol, D-3-O-methyl inositol, and sucrose¹¹	Leaves and pods	70gms

2.3.3 High-performance thin-layer Chromatographic (HPTLC) profile:

HPTLC is a widespread micro-analytical separation and determination technique for herbal drugs. A solvent system of Formic acid, ethyl acetate, and toluene (5:2:0.1) was chosen for best constituent separations. The test samples were placed on silica gel 60 F254 sheets, inserted into CAMAG developing chamber, air dried, and scanned at 254 and 366nm using the TLC Scanner 4. Then the plate was heated at 1050⁰C, derivatized with a vanillin-sulphuric acid, and studied at 575nm to record Rf values and fingerprint data.

2.3.4 Ultra Violet-Visible (UV-Vis) Spectroscopy:

A spectroscopic analysis in the Ultra Violet-Visible region was conducted on the alcohol extract of the test material. Using an Analytical Technologies UV/VIS spectrophotometer (Model: UV3120), the extract was scanned at wavelengths between 200 and 900nm, and the matching characteristic peaks were found and recorded.

2.3.5 FTIR Spectroscopy:

The drug was subjected to FT-IR analysis using KBr pressed disk technique on the Analytical Technologies FT-IR spectrophotometer (Model: INFRA 3000-50), and the distinctive peaks were found and noted.

3. RESULTS AND DISCUSSION:

Drug standardization is essential to assess a medicine's effectiveness, involving organoleptic characteristics, physico-chemical and phytochemical parameters, UV visible spectroscopy, FTIR spectrometry, and HPTLC fingerprinting.

3.1 Organoleptic evaluation:

The Choornam was solid, had an aromatic smell and an astringent taste, was fine in nature, soft to the touch, and had a brownish color. Its fineness suggests that it will absorb quickly, improving availability. An image of the prepared medicine is given in Figure 3.

Table 2: Organoleptic characters of Vaividanga Choornam

Sl. No	Specification	Character
1	State	Solid
2	Nature	Fine
3	Odor	Aromatic
4	Touch	Soft
5	Flow Property	Non-Free flowing
6	Appearance	Brownish
7	Taste	Astringent

The following cellular characters were observed in the Vaividanga choornam. Secondary thickenings of xylem vessels, such as annular and reticulate thickening, were observed. Stone cells of various morphotypes and starch grains of two morphotypes also were seen in the sample. The other diagnostic characteristics were Rosetteprismatic Calcium oxalate crystals and smooth trichomes. Calcium oxalate crystals are a biomineral found in many plant sections and have various activities, including storing calcium in the tissues, removing excess oxalate, detoxifying metals, and maintaining calcium homeostasis.Trichomes are cellular components that give plants their physical and chemical defense systems. They are epidermal cells with specific functions found on plants' aerial portions and are involved in several biological activities.¹⁵^,¹⁶In the present study, only non-glandular types of trichomes were observed.Spherical protuberances of epicarp were observed, which might have come from fruit ingredients in the sample. Perisperm cells were found to be the diagnostic character of P. nigrum, one of the single drugs of the formulation, Vaividanga choornam. Oleoresin cells, cortical cells enclosing starch grains, and brownish content were also found to come from Z. officinale.

3.2 Physico-chemical parameters:

Physical and chemical characteristics greatly aid in standardization. Table 3 contains the findings of the physicochemical examination of Vaividanga Choornam.

Table 3: Physicochemical evaluation of Vaividanga Choornam

Sl. No.	Parameters	Result
1.	LOD at 105⁰C %	11.20
2.	Total Ash %	5.65
3.	Acid insoluble ash %	0.18
4.	Water soluble ash %	1.71
5.	Sulfated ash %	9.51
6.	The pH of water extract (4% aqueous solution)	6.85
7.	Volatile oil %	1
8.	Alcohol soluble extractives %	11.87
9.	Water soluble extractives %	18.31
10.	Swelling index	4ml
11	Foaming index	<100

Vaividanga Choornam lost 11.20% of its moisture while drying, suggesting that its stability and shelf life may be shortened due to high moisture content.Vaividanga Choornam's Total Ash value was 5.65%, indicating its purity, and less than 1% of acid-insoluble ash, indicating the presence of less siliceous materials, ensuring the medication is free of impurities.¹⁷^,¹⁸The amount of water-soluble ash, which was 1.71%, indicated that the osmosis and diffusion processes were facilitated. It includes 9.51% inorganic impurities, as shown by the sulfated ash value of 9.51%. Alcohol soluble extractive value (11.87%) revealed that the medication contained a few polar chemical components, including fats, certain steroids, and lipids. In addition, the constituents of choornam had sugars, carboxylic acids, and tannins, which contributed more to the water-soluble extractive value (18.31%). This guarantees the test drug's high purity, lack of adulteration, and excellent quality. The drug's pH (6.85) indicates a mildly acidic nature. When administered orally, the drug's acidic composition aids in rapid absorption¹⁹.

3.3 Qualitative phytochemical parameters:

The qualitative phytochemical analysis of the Vaividanga Choornam revealed carbohydrates, alkaloids, tannins, terpenoids, phenols, glycosides, quinones, and saponins. Alkaloids have anticancer, antioxidant, anti-inflammatory, antibacterial, antidiabetic, ameliorative, neuro and cardioprotective, and antiemetic effects.²⁰ Triterpenoids contain anti-inflammatory, antioxidant, and apoptosis-regulating properties.²¹ Phenolic substances have anti-inflammatory, antipyretic, antiviral, antioxidant, and analgesic properties.²² Saponins lower blood lipids, lower the risk of developing cancer and improve the body's reaction to glucose.²³ The Vaividanga Choornam contains sugars or carbohydrates, which provide the body with rapid energy. All of these substances work together synergistically to increase the effectiveness of the test medication.²⁴

Table 4: Preliminary Phytochemical Evaluation of Vaividanga Choornam

Sl. No.	Natural products	Test performed	Results
1	Saponins	Foaming in water	+ve
2	Tannins	Lead acetate test	- ve
3	Terpenoids	Liebermann-Burchard test	+ ve
4	Phenols	Neutral FeCl3 test	+ ve
5	Steroids	Liebermann’s test	- ve
6	Glycosides		+ ve
7	Carbohydrates	Molisch test	+ ve
8	Alkaloids	Mayer s reagent test	+ ve
9	Flavanoids	Shinoda test	- ve
10	Proteins	Millon’s test	- ve
11	Quinones		+ ve

Note: 1. Hexane ext.; 2. Chloroform ext.; 3. Ethyl acetate ext.; 4. Methanol ext.; 5. Water ext.; + ve; positive and – ve; negative

3.4 High-performance thin-layer Chromatographic (HPTLC) profile:

HPTLC fingerprinting is a valuable tool to identify the plant and its phytocompounds accurately.²⁵ Sample analysis yielded the most accurate results in the Formic acid, ethyl acetate, and toluene (5:2:0.1) solvent system. The HPTLC profile showed ten distinct peaks corresponding to ten different compounds, with Rf values between 0.02 and 0.98 and percentage areas between 0.87 and 46.17%. In addition, four green, two blue, and one neon yellow bands were found in the bands, indicating the presence of several phytochemicals. The findings are presented in Table 5 and Figures 6, 7, and 8.

The sample's HPTLC fingerprinting investigation revealed nine noticeable peaks, each corresponding to a different phyto-component. The percentage area between the peaks' Rf values ranged from 0.04 to 0.92, between 5.55% and 33.07%. Multiple bands in the chromatogram may suggest the presence of a broad spectrum of phytochemicals/their classes, contributing to its efficacy and supporting its ethnomedical usage in treating respiratory and gastrointestinal ailments.²⁶ Also, the synergistic effect of these phytoconstituents contributesto the efficacy of the choornam.²⁷

3.5 Ultra Violet-Visible (UV-Vis) spectroscopy:

The UV-Vis spectra of the powder sample of Vaividanga Choornam were obtained by scanning the alcohol extract of the test material in the wavelength range of 200-900 nm using a UV-Vis spectrometer (Model: UV 3120). The UV-Vis spectra of the Vaividanga Choornam sample are shown in Fig. 4. Absorption peaks with their absorbance are shown. The UV-Vis profile of the Vaividanga Choornam sample showed peaks at 290 nm, 425 nm, and 665 nm with absorption. The results obtained in UV-vis spectra revealed the existence of several medicinally essential phytoconstituents.

Figure 9: UV vis absorbance spectra of Vaividanga Choornam

3.6 FTIR Spectroscopy:

Vaividanga Choornam was studied under Fourier transform infrared (FTIR) spectroscopy for therelevant functional group of phytoconstituents. This study helps project a standard spectrum of Vaividanga Choornam to minimize batch-to-batch variation and ensure the identity and quality of the medicine.

Figure 10: FTIR spectra of Vaividanga Choornam

4. CONCLUSION:

The parameters analyzed in the present study,such as Powder microscopy, physicochemical analysis, preliminary phytochemical analysis, UV-Visible spectrum analysis, FTIR Spectroscopy, and HPTLC fingerprinting analysisresults,provide quality standards for the drug Vaividanga Choornam. Additionally, the pharmacognostic traits of the sample described in this work could contribute to creating an appropriate monograph for its correct identification—assessing its quality and purity and determining the kind of adulteration in the medicine.

5. CONFLICT OF INTEREST:

Nil.

6. ACKNOWLEDGEMENT:

The authors are highly thankful to Director General, Central Council for Research in Siddha, for the support.

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Received on 29.05.2023 Modified on 23.09.2023

Research J. Pharm. and Tech 2024; 17(2):727-733.

DOI: 10.52711/0974-360X.2024.00113


Perisperm cells	Prismatic Calcium oxalate crystal	Group of simple andcompound starch grains	Rosette Calcium oxalate crystal	Pitted tracheid

Brownish content	Parenchyma cells with oleoresin content	Parenchyma cells with starch grains	Stone cell	Xylem vessel with reticulate thickening

Thick-walled polygonal cells	Spherical body on epicarp	Smooth trichome	Stone cell	Xylem vessel with annular thickening


Under UV short (254 nm)	Under UV long (366 nm)	Under white light after derivatization (575 nm)