Powder microscopic, physicochemical and chromatographic approach for the quality control of anti-hypertensive drug Rattha Piththathirku

The present work aims to study powder microscopy, physicochemical and high-performance thin-layer chromatography photo documentation and fingerprint profiles of a Siddha drug, Rattha Piththathirku Kudinir Chooranam (RPK). The raw drugs were collected, authenticated and the RPK was prepared. Then the drug was investigated for powder microscopic characters, physicochemical parameters, Thin Layer Chromatographic photo documentation (TLC), High-Performance Thin-Layer Chromatographic (HPTLC) fingerprint profiles of successive n -hexane, successive chloroform, successive ethanol and hydro alcohol (1:1) extracts. The successive and hydro alcohol extracts of the drug displayed distinct TLC spots and HPTLC peaks which are distinct to this drug. pharmacodynamic


Introduction
Hypertension is a major health issue that leads to an increase in the risks of heart, brain, kidney and other organ related diseases. It is one of the major reasons for early death of public (1). According to the World Health Organization, it is estimated that 1.13 billion people are suffering from hypertension worldwide, and two-thirds of them belong to low and middle income countries (2). Traditional and complementary medicine plays an important role in maintaining good health due to its easy acceptance by the human body . Ratthapitham refers to Purpura i.e. hypertension. According to Siddha literature, 30 to 60 ml of medicine has to be taken twice daily for treating hypertension (5).
As hypertension is a common illness, and there are medications available in several brand names; thus quality control parameters are required for the analysis of commercial samples. Hence, it is decided to evaluate the powder microscopic, physicochemical values, document thin layer photography and record the HPTLC fingerprint profiles.

Plant material
Zaleya decandra root and Zingiber oficinale rhizome were collected from the Survey of Medicinal Plants Garden, Mettur, Tamil Nadu and were authenticated by referring floras and chemical test.

Formulation of RPK
The rhizome of Zingiber officinale and the roots of Zaleya decandra were washed well in running water and then shade dried. The dried samples were powdered and passed through a sieve no. 60. The powdered drugs were mixed thoroughly in the ratio of 1:1 (5).

Powder Microscopy
A pinch of the mixed powdered sample was mounted on a microscopic slide with a drop of 50% glycerol after treating it with 0.1 % chloral hydrate solution. Characters were observed using Nikon ECLIPSE E200 trinocular microscope attached with Zeiss ERc5s digital camera under bright field light. Photomicrographs of diagnostic characters were captured and documented (28).

Physico-chemical Analysis
All the physicochemical parameters were evaluated according to the standard methods (28).

Extract Preparation
RPK (4 gm) was subjected to successive extraction with 100 ml of each of n-hexane, chloroform and ethanol using Soxhlet apparatus. Extracts were concentrated to dryness. The total residues were resolvated with the corresponding solvents and made up to 10 ml, and transferred into sample vials for TLC application. For hydro alcohol extract, 1 gm of the drug was refluxed with 25 ml of aqueous ethanol (1:1 ratio) for 2 hrs, cooled, filtered and used for TLC in the sample vial.

HPTLC analysis
Successive n-hexane, chloroform and ethanol extract, 10 μl each, were applied on three different silica gel 60F254 pre-coated aluminium plate (6 × 10 cm) as 8 mm band 10 mm from the bottom. The plate containing n-hexane extract was developed using toluene: ethyl acetate: formic acid (8: 2: 0.5, v/v/v), chloroform extract plate using toluene: ethyl acetate: formic acid (6.0:4.0:1.0, v/v/v) and ethanol extract by using toluene: ethyl acetate : methanol: formic acid (3.0:4.0:3.0: 0.5, v/v/v/v) separately in pre-saturated twin trough chamber (10 × 10 cm). The developed plates were dried, and photographs were taken under UV, followed by scanning under λ 254 (absorbance mode, D2 lamp) and λ 366 (fluorescence mode, Hg lamp) respectively with a slit dimension 6 × 0.45 mm and scanning speed of 20 mm/s. The scanned plates were dipped in VSR and heated at 105°C till the appearance of coloured bands. Photographs were taken immediately at white light, followed by scanning at λ 520 (absorption mode, W lamp).

Powder Microscopy
The powder microscopic observation showed the following characters, Zingiber officinale: suberised cork; scalariform and pitted vessels; septate non lignified fibre; oleoresin and sac shaped starch grains; Zaleya decandra: thick-walled cork cells, fibrous layers, crystal fibres, pitted vessels, spiral vessels and heterogenous prismatic crystals (Fig. 1). The powder is pale yellow with a characteristic odour and taste.
Microscopic examination of the powdered compounded formulation revealed suberised cork, oleoresin and sac shaped starch grains which form the diagnostic characteristics of Zingiber officinale rhizome and thick-walled cork cells, crystal fibres together with heterogenous prismatic crystals could be attributed to Zaleya decandra root. Each species has its own diagnostic character, which can be used to confirm its identity. In the present study, the powder microscopic characterisation of the compound formulation has led to the effective diagnosis of the individual herbal drugs used in it.

Physicochemical results
The physicochemical values of the tested drug are presented in Table 1. Drug quality is markedly affected by moisture content denoted by loss on drying (LOD), which alters the shelf life of herbal drug. LOD value was found to be 10.38 ± 0.17 %. Total ash value was determined as 6.87 ± 0.09 %. Total ash contains physiological and non-physiological inorganic salts of phosphates, silicates and carbonates. Acid insoluble ash (AIA) value represents the amount of earthing matters present in roots and rhizomes. AIA was found to be 1.43 ± 0.09%. The alcoholic soluble extractive value was estimated as 17 ± 0.15%, whereas the water-soluble extractive value was 12.26 ± 0.16 %. The pH of the drug was determined as 4.62, which revealed the acidic nature of the drug. The physicochemical values of any herbal drug would vary and the same could be considered for the quality control of drugs (29)(30)(31).

TLC Photo documentation
In the TLC photo documentation of n-hexane extract of RPK ( Supplementary Fig. 1 The photo documentation of successive chloroform extract (Supplementary Fig. 2

Conclusion
The powder microscopic characters, TLC/HPTLC photos and fingerprint profiles are distinct to Rattha Piththathirku Kudinir Chooranam and the same could be applied for the quality control of the drug as pharmacopoeial reference standards.