Physico-chemical standardization of Sitopaladi churna.

BACKGROUND: Standardization of a compound Ayurvedic formulation is a critical and essential issue to be considered in assuring the therapeutic efficacy and safety and to rationalize their use in the health care. Sitopaladi churna is a reputed polyherbal formulation of Ayurveda. It is prescribed for the treatment of pleurodynia, intercostal neuralgia, cold, cough associated with bronchitis, pneumonia, tuberculosis, viral respiratory infection, and in pharyngeal and chest congestion.
OBJECTIVE: The present study aimed at physico-chemical standardization of in-house and two marketed brands of Sitopaladi churna.
MATERIALS AND METHODS: In our investigation, in-house churna and two commercial brands of Sitopaladi churna were standardized based on powder microscopy, physico-chemical evaluations, thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) finger printing as per standard procedures.
RESULTS: The set parameters were sufficient to evaluate the churna based on various physico-chemical parameters.
CONCLUSION: The data evolved can be adopted for laying down the standards for the manufacturing units of Sitopaladi churna.

INTRODUCTION

Ayurveda, the traditional Indian medicine, is the “great tradition” with sound philosophical, experiential, and experimental basis. The Ayurvedic system touted as an “alternative system of medicine” has already gained worldwide attention due to increased side effects of drugs, lack of remedy for several chronic diseases, microbial resistance, high cost of synthetic drugs, and emerging diseases. These are some facts for renewed public interest in traditional medicines. With increasing demand for safer drugs, attention has been drawn to the quality, safety, efficacy, and standards of the Ayurvedic formulations.[1]

Ayurvedic pharmacy advocates the use of quality control tests to make sure that the formulated products adhere to the standards mentioned in Ayurveda. Most of the tests mentioned in ancient literature appear to be based on observation and seem subjective without valid scientific backing; therefore, formulation prepared may not have the desired quality and batch-to-batch consistency. Quality is the critical determinant of safety and efficacy of herbal medicines; however, herbal formulations rarely meet the standards of quality. Hence, there is a need for standardization, and development of reliable quality protocols for Ayurvedic formulations using modern techniques of analysis is extremely important.[23] The World Health Organization (WHO) has appreciated the importance of medicinal plants for public health care in developing nations and has evolved guidelines to support the member states in their efforts to formulate national policies on traditional medicine and to study their potential usefulness including evaluation, safety, and efficacy.[4-6] The present study deals with Sitopaladi churna (SPC), a polyherbal Ayurvedic formulation prescribed for pleurodynia, intercostal neuralgia, cold, cough associated with bronchitis, pneumonia, tuberculosis, burning sensation in extremities, supportive agent for allergy, viral respiratory infection, digestive impairment, and in pharyngeal and chest congestion.[78] The investigation was carried out to develop standardization parameters. The objectives include performing powder microscopic characterization, physico-chemical analysis, and thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) fingerprint profile for the quantification of piperine and cinnamaldehyde [Figure 1] in SPC samples.

Figure 1

Molecular structure of piperine and cinnamaldehyde

The two standards quantified by HPTLC have been reported to possess significant biological activities. Piperine is reported to have as an antidepressant, hepatoprotective, anti-metastatic, antithyroid, immunomodulatory, antitumor[9] antiplatelet[10] antioxidant[11] and antiamoebic[12] activities. Cinnamaldehyde is reported to show antidiabetic[1314] antifungal[15] antibacterial[16] anticancer[17] antimutagenic[18] and anti-inflammatory[19] activities.

MATERIALS AND METHODS

Collection and identification of plant materials

The raw drugs used in the in-house SPC-I formulation were procured from the local market of Udupi, Karnataka, India, and authenticated by botanist Dr. K. Gopal Krishna Bhat, Professor, Department of Botany, Poorna Prajna College, Udupi, Karnataka. A voucher specimen of the same was deposited in the museum of Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences. Commercially available brands of SPC [Baidynath Ayurved Bhawan Pvt. Ltd., Kolkata, India (SPC-II) and Dabur India Ltd., New Delhi, India (SPC-III)] were procured from local market. SPC-I was prepared according to Ayurvedic Formulary of India by mixing equal parts by weight of each of the five ingredients of formulation [Table 1].[7]

Table 1

Ingredients of Siwtopaladi churna

Chemicals

All the solvents and chemicals of analytical grade were purchased from E. Merck and S. D. Fine Chemicals, Mumbai. Piperine (purity 97%) and cinnamaldehyde (purity 98%) were purchased from Sigma-Aldrich, Bangalore, India.

Physico-chemical evaluations

Organoleptic parameters such as varna (color), gandha (odor), ruchi (taste), aakruti (shape), and parimana (size) were analyzed and recorded. Powder microscopy of shade-dried powder was carried out using Olympus BX 41 microscope.[20] Physico-chemical characteristics of SPC samples were analyzed by quantitative analysis for total ash, water -soluble ash, acid-insoluble ash, water-soluble extractives, alcohol-soluble extractives, foaming index, loss on drying, and pH (10% aqueous solution) as per standard techniques.[21] Micromeritic characteristics like bulk density, tap density, angle of repose, Haussner ratio and Carr's index were determined for SPC samples.[2223] Aqueous and methanol extracts of SPC samples prepared by hot extraction were used for screening of constituents like alkaloids, glycosides, flavonoids, tannins, sterols, terpenes, fixed oil, resin, protein, and gums.[20] Total percentage of reducing sugar, tannins, and flavonoids of SPC samples was determined.[24-26] Fluorescence analysis was carried out as per the method of Chase and Pratt.[27] SPC samples were analyzed for presence of heavy metals like lead (Pb), arsenic (As), cadmium (Cd), and mercury (Hg) by atomic absorption spectroscopy (AA 240, Varian, The Netherlands).[28]

Quantification of piperine and cinnamaldehyde by HPTLC densitometry

TLC conditions

Silica gel 60 F254 TLC plates with aluminum sheet support (0.2 mm thickness) (E. Merck) were used. The syringe was a 100 μL one (Hamilton) and spotting device used was Camag Linomat V spotter (Camag, Muttenz, Switzerland). The developing chamber was a Camag glass trough chamber (20 × 10 cm) previously saturated with mobile phase vapor for 30 min. Densitometry was performed with a Camag TLC Scanner 3 and Camag Reprostar 3 linked to Wincats software (V 3.15, Camag). The experimental conditions were temperature of 25 ± 2°C and relative humidity of 40%.

Preparation of standard solution

Piperine:

Stock solution of 1000 μg/mL of piperine was prepared in methanol. Aliquots (0.1–1 mL) of stock solution were transferred to 10 mL volumetric flasks and the volume of each was adjusted to 10 mL with methanol, so as to obtain standard solutions containing 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg/mL of piperine, respectively.

Cinnamaldehyde:

Stock solution of 100 mg/mL of cinnamaldehyde was prepared in methanol. Aliquots (0.1–1 mL) of stock solution were transferred to 10 mL volumetric flasks and the volume of each was adjusted to 10 mL with methanol, so as to obtain standard solutions containing 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mg/mL of cinnamaldehyde, respectively.

Preparation of sample solution

One hundred milligrams of methanolic extract of each of SPC-I, SPC-II, and SPC-III dissolved in 10 mL of methanol was used for quantification of piperine and cinnamaldehyde.

Preparation of calibration curves

Piperine:

10 μl of each of the standard solutions of piperine (100–1000 ng/spot) was spotted (band width: 6 mm) in triplicate on a TLC plate using an automated Linomat V applicator. The plates were developed in a twin trough chamber (20 × 10 cm) up to a distance of 9 cm using a mobile phase of toluene:ethyl acetate:formic acid (5:3.5:0.5 v/v/v) (12 mL) at a temperature of 25 ± 2°C and 40% relative humidity. The plate was air dried, and scanned at 342 nm in the absorbance-reflectance mode using a deuterium lamp. Calibration curve of piperine was obtained by plotting peak area versus applied concentration of piperine. Video densitometry of the chromatogram was carried out with the help of Camag Reprostar 3.

Cinnamaldehyde:

10 μl of each of the standard solutions of cinnamaldehyde (10–100 ng/spot) was spotted (band width: 6 mm, distance between the tracks: 12 mm) in triplicate on a TLC plate using an automated Linomat V applicator. The plates were developed in a twin trough chamber up to a distance of 9 cm using an optimized mobile phase of toluene:chloroform (8:2 v/v) (12 mL) at a temperature of 25 ± 2°C and 40% relative humidity. The plate was air dried, and scanned at 310 nm in the absorbance-reflectance mode using a deuterium lamp. Calibration curve of cinnamaldehyde was obtained by plotting peak area versus applied concentration of cinnamaldehyde. Video densitometry of the chromatogram was carried out with the help of Camag Reprostar 3.

Quantification of piperine and cinnamaldehyde in the SPC samples

Ten microliters of each of the methanolic extracts of SPC-I, SPC-II, and SPC-III was spotted in duplicate on a TLC plate using an automated Linomat V applicator. The plates were developed, scanned, and peak areas and absorption spectra were recorded. The amounts of piperine and cinnamaldehyde were calculated using their respective calibration curves. The identity and purity of the bands of piperine and cinnamaldehyde in the sample extract track were checked by overlaying their UV absorption spectra at start, middle, and end positions of the band.

Method validation

International Conference on Harmonisation guidelines were followed for the validation of the analytical methods developed for precision, repeatability, and accuracy.[2930] Instrumental precision was checked by repeated scanning (n = 7) of the same spot of piperine (500 ng/spot) and cinnamaldehyde (50 ng/spot) expressed as relative standard deviation (%RSD). The repeatability of the method was confirmed by analyzing 500 ng/spot of piperine and 50 ng/spot of cinnamaldehyde individually on a TLC plate (n = 5) and expressed as %RSD. The inter-day and intra-day variation of the method was studied by analyzing aliquots of standard solution containing 300, 500, and 700 ng/spot of piperine and 30, 50, and 70 ng/spot cinnamaldehyde on the same day and on different days and the results were expressed as %RSD. For the evaluation of limit of detection (LOD) and limit of quantification (LOQ), different concentrations of the standard solutions of piperine and cinnamaldehyde were applied along with methanol as blank and they were determined on the basis of signal-to-noise (S/N) ratio. LOD was measured at an S/N of 3:1 and LOQ at an S/N of 10:1. The specificity of the method was measured as per Bhandari et al.[31] The accuracy of the method was assessed by performing recovery study at three different levels (50, 100, and 150% addition of piperine and cinnamaldehyde). The percentage recovery and the average percentage recovery for each standard were calculated. Specificity was ascertained by analyzing reference compounds and samples. The bands for piperine and cinnamaldehyde from sample solutions were confirmed by comparing the Rf and spectra of the bands to those of the standards.

RESULTS AND DISCUSSION

Physico-chemical evaluation

The samples of SPC were found to be brown-colored, moderately fine powder, with a pleasant smell and spicy taste. All the samples passed through 60 mesh size and not less than 50% passed through 85 mesh size. Microscopic characterization [Figures 2a–i] revealed the presence of big siliceous crystals (Bambusa arundinacea); epidermis of testa was composed of yellow-brown prosenchymatous cells with pitted walls, and globules of volatile oil with underlying hypodermis and epidermis were present (Elettaria cardamomum); phloem parenchyma was associated with occasional phloem fiber and big isolated oil cell, and group of lignified cork cells was associated with pericyclic fiber, tannin contents (Cinnamonum zeylanicumm); stone cell with a broad lumen was present and group of spiral vessels formed a vascular strand and perisperm cells (Piper longum). Quantitative physico-chemical analysis for the SPC samples was performed for the parameters like total ash, water-soluble ash, acid-insoluble ash, water-soluble extractives, alcohol-soluble extractives, loss on drying, and pH [Table 2]. Micromeritic parameters of SPC samples were also analyzed and the data are depicted in Table 2. Phytochemical constituents like alkaloid, carbohydrates, flavonoid, tannins, saponins, and fats in each of the SPC samples [Table 3] were identified through qualitative analysis. Aqueous and methanolic extracts of SPC samples were studied spectrophotometrically for spectrum measurement, and colorimetric analyses like total carbohydrate sugar by phenol sulfuric acid reagent, total tannin by Folin–Denis reagent, and total flavonoid by aluminum trichloride reagent were carried out [Table 4]. Fluorescence analysis was carried out to check the chemical nature of drug with different reagents; data are depicted in Table 5. Heavy metal contents of SPC samples were found to be within permissible limits, except Pb in SPC-I [Table 6].

Figure 2

Powder microscopy of Sitopaladi churna

Table 2

Physico-chemical analysis of Sitopaladi churna samples

Table 3

Preliminary qualitative analysis of Sitopaladi churna samples

Table 4

Total carbohydrate, tannin, and flavonoid content of Sitopaladi churna samples

Table 5

Powder fluorescence test of Sitopaladi churna samples

Table 6

Heavy metal estimation in Sitopaladi churna samples

TLC fingerprint and co-chromatography

TLC fingerprint profile is a systematic representation of all the constituents of sample resolved in the given chromatographic system. It gives a semi-quantitative sketch of the chemical profile of the sample. In the present work, we developed simple, convenient, and time-saving TLC methods for co-chromatography with two marker compounds, viz. piperine and cinnamaldehyde. The proposed methods were further validated and used for the quantification of these compounds. The spot at Rf 0.69 was identified as piperine with the help of TLC chromatograms of its standard using toluene:ethyl acetate:formic acid (5:3.5:0.5 v/v/v) as the mobile phase [Figure 3]. The separation of piperine in SPC sample extracts is depicted in [Figures 4–6]. The identity of the band of piperine and in SPC-I, SPC-II, SPC-III extracts was confirmed by overlaying its UV absorption spectra with that of the standard compound [Figure 7].

Figure 3

TLC densitometric chromatogram of standard piperine at 342 nm (Rf 0.69)

Figure 4

TLC densitometric chromatogram of SPC-I at 342 nm; peak 4: piperine (Rf 0.69)

Figure 6

TLC densitometric chromatogram of SPC-III at 342 nm; peak 6: piperine (Rf 0.68)

Figure 7

Overlaying UV absorption spectra of piperine with corre-sponding band in the SPC sample extracts and standards

TLC densitometric chromatogram of SPC-II at 342 nm; peak 7: piperine (Rf 0.69)

The spot at Rf 0.35 was identified as cinnamaldehyde with the help of chromatograms of its standard using toluene:chloroform (8:2 v/v) as the mobile phase [Figure 8]. The separation of cinnamaldehyde in SPC sample extracts is depicted in [Figures 9–11]. The identity of the band of cinnamaldehyde in SPC-I, SPC-II, and SPC-III extracts was confirmed by overlaying its UV absorption spectra with that of standard compound [Figure 12]. The purity of the band in the SPC sample extracts was confirmed by comparing the absorption spectra recorded at start, middle, and end positions of the band. The video densitometric images of chromatoplate are depicted in [Figure 13]. Preliminary TLC and co-TLC indicated the possible presence of piperine and cinnamaldehyde due to the presence of P. longum and C. zeylanicumm in SPC. Hence, we quantified these two compounds from SPC samples.

Figure 8

TLC densitometric chromatogram of standard cinnamalde-hyde at 310 nm (Rf 0.35)

Figure 9

TLC densitometric chromatogram of SPC-I at 310 nm; peak 5: cinnamaldehyde (Rf 0.32)

Figure 11

TLC densitometric chromatogram of SPC-III at 310 nm; peak 3: cinnamaldehyde (Rf 0.33)

Figure 12

Overlaying UV absorption spectra of cinnamaldehyde with corresponding band in the SPC sample extracts and standards

Figure 13

Video densitometry of piperine and cinnamaldehyde. P1 and C1 are standard piperine and cinnamaldehyde, respectively; P2 and C2 are SPC-I samples; P3 and C3 are SPC-II samples; and P4 and C4 are SPC-III samples

TLC densitometric chromatogram of SPC-II at 310 nm; peak 4: cinnamaldehyde (Rf 0.32)

TLC densitometric quantification of piperine and cinnamaldehyde by HPTLC

The two standard compounds, viz. piperine and cinnamaldehyde, were quantified from SPC samples by TLC densitometric methods by HPTLC. The methods were validated for precision, repeatability, and accuracy. The linearity ranges for piperine and cinnamaldehyde were found to be 100–1000 and 10–100 ng/spot with correlation coefficients (r values) of 0.998 and 0.995, respectively [Table 7].

Table 7

Method validation parameters for the quantification of piperine and cinnamaldehyde by the TLC densitometric method

The TLC densitometric methods were found to be precise with %RSD for intra-day in the range of 0.98–1.18 and 1.50–1.76 and for inter-day precision in the range of 1.19–1.44 and 1.89–2.13 for different concentrations of piperine and cinnamaldehyde, respectively [Table 8]. This indicates that the methods were precise and reproducible. The LOD values for piperine and cinnamaldehyde were found to be 50 and 20 ng, respectively, and LOQ values were 150 and 40 ng, respectively [Table 7]. The average recoveries at three different levels of piperine and cinnamaldehyde were 99.36 and 97.99%, respectively [Table 9]. Piperine and cinnamaldehyde were quantified from SPC-I, SPC-II, and SPC-III at 342 and 310 nm, respectively [Table 10]. The analytical specifications for SPC based on the above results have been presented in Table 11.

Table 8

Intra-day and inter-day precision of piperine and cinnamaldehyde

Table 9

Recovery study of piperine and cinnamaldehyde by the TLC densitometric method

Table 10

Piperine and cinnamaldehyde content estimated in Sitopaladi churna samples

Table 11

Analytical specifications of Sitopaladi churna

CONCLUSION

The present work was carried out for the formulation and standardization of SPC. The in-house formulation was studied for various physico-chemical parameters, in comparison with the marketed samples. A TLC densitometric method has been developed for quantification of piperine and cinnamaldehyde from SPC using HPTLC. The developed and validated HPTLC methods are simple, precise, and accurate, and can be used for the quantification of piperine and cinnamaldehyde in herbal raw materials as well as in their formulations. Hence, these quality-control parameters and the developed HPTLC methods may be considered as a tool for assistance for scientific organizations and manufacturers in developing standards.