|Year : 2018 | Volume
| Issue : 4 | Page : 212-219
Chemoprofiling of homoeopathic drug Holarrhena antidysenterica L.
Rakhi Mishra1, Manoj Kumar1, Binit Dwivedi1, BS Arya2, Renu Arya2, Anil Khurana2, Raj K Manchanda2
1 Dr. DP Rastogi Central Research Institute (H), Noida, Uttar Pradesh, India
2 Central Council for Research in Homoeopathy, New Delhi, Ministry of Ayush, Government of, India
|Date of Submission||19-May-2018|
|Date of Acceptance||26-Dec-2018|
|Date of Web Publication||08-Feb-2019|
Ms. Rakhi Mishra
Dr. DP Rastogi, Central Research Institute for Homoeopathy, A-1/1, Sector-24, Noida - 201 301, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Chemoprofiling of homoeopathic drug/tincture (HT) represents a comprehensive approach for evaluation of quality, purity, safety and efficacy of HT. This paper reflects the chemoprofiling of Homoeopathic drug Holarrhena antidysentrica L. Objective: The objective of this study is to standardise Holarrhena antidysenterica mother tincture by taking the samples from four different sources: Dr D. P. Rastogi, CRI (H) Noida (A) and three from market (labelled as B, C and D). Materials and Methods: The authentic sample of bark of Holarrhena antidysenterica supplied by the Centre of Medicinal Plants Research in Homoeopathy, Emerald, Tamil Nadu, India was used to prepare the mother tincture (as per the Homoeopathic Pharmacopoeia of India). The solvents used throughout the study, namely, ethanol, high-pressure liquid chromatography water, cyclohexane, chloromethane and diethylamine, were of analytical grade purity (MERCK Ltd.). Physicochemical properties, ultraviolet (UV) spectroscopy and high-performance thin-layer chromatography (HPTLC) chemoprofile of raw drug and mother tinctures were standardised and compared with market samples. Results: The present study reveals the moisture content (14.40%), total ash (4.65%), alcohol (18.0%), water extractive values (16.0%), total solids (1.47%), weight/ml (0.92 g) and alcohol content (60.6%). In UV spectroscopy, λmaxvalues were observed at 228 and 278 nm in HT. HPTLC analysis of in-house HT (A) and three market samples (B, C, D) was performed by using cyclohexane: chloromethane: diethylamine (7:3:1, v/v/v) as mobile phase. Under UV light (254, 366 nm) and in the presence of visualising agent Dragendroff, bands of active constituent were observed in all the four samples. However, excess amount of active constituents were found in in-house HT (a) rather than the market samples (B, C and D). Conclusion: The present physicochemical and phytochemical data may be considered as pharmacopoeia standards for the homoeopathic drug Holarrhema antidysentrica L.
Keywords: Drug standardisation, High-performance thin-layer chromatography, High-performance thin-layer chromatography fingerprint, Homoeopathic drug, Physicochemical, Ultraviolet
|How to cite this article:|
Mishra R, Kumar M, Dwivedi B, Arya B S, Arya R, Khurana A, Manchanda RK. Chemoprofiling of homoeopathic drug Holarrhena antidysenterica L.. Indian J Res Homoeopathy 2018;12:212-9
|How to cite this URL:|
Mishra R, Kumar M, Dwivedi B, Arya B S, Arya R, Khurana A, Manchanda RK. Chemoprofiling of homoeopathic drug Holarrhena antidysenterica L.. Indian J Res Homoeopathy [serial online] 2018 [cited 2019 Sep 17];12:212-9. Available from: http://www.ijrh.org/text.asp?2018/12/4/212/251914
| Introduction|| |
Holarrhena antidysenterica L. of the family Apocynaceae is also known as conessi bark in English, kutaja in Sanskrit, kura or kurchi in Hindi and found throughout many forests of India, in Travancore, Assam and Uttar Pradesh. Holarrhena antidysentrica Linn. f is a small deciduous tree, with brown bark. The bark contains a large number of alkaloids such as conessine, holonamine, kuchine, kurchicine, holarrhimine and conimine., It acts as a good astringent, anthelmintic, amoebicidal and has diuretic-like property. The bark and seeds have been used in the treatment of many diseases such as colic, dyspepsia, piles, dysentery, diseases of spleen, diseases of the skin, diarrhoea, anaemia, epilepsy, stomach pain and cholera. Kurchicin, an active principle of Holarrhena antidysenterica, is highly effective against causative microorganisms of diarrhoea and dysentery, especially amoebic types. It has huge medicinal values due to the presence of a large number of alkaloids. In Homoeopathy, Holarrhena antidysentrica is used for the complaints of both chronic and acute dysentery with large quantity of mucus, excessive blood, colic pain, fever, piles, leprosy and skin diseases, tenesmus, emaciation, loss of appetite and aversion of food colicky around the navel, aggravated by lying on the right side but better by lying on the left side. According to the Homoeopathic Pharmacopoeia of India, its bark is used for the preparation of mother tincture.
| Materials and Methods|| |
The plant material Holarrhena antidysenterica was supplied by the Centre for Medicinal Plant Research in Homoeopathy, Nilgiris, Tamil Nadu. In-house mother tinctures were prepared from authentic materials and the other three mother tinctures were purchased from market.
Chemical and reagents
All solvents used in this study were of analytical grade, and purified water was of high-pressure liquid chromatography grade. Post-chromatographic derivatisation of developed Thin-layer Chromatography (TLC) plates was done using Dragendroff's reagent. Dragendroff's reagent was prepared by dissolving 0.85 g-basis bismuth nitrate in 10-ml glacial acetic acid and 40-ml water under heating if necessary (Sol. A). Sol. B was prepared by dissolving 8-g potassium iodide in 30-ml water, and then stock solutions A + B solutions were mixed 1:1 (v/v) and 4-ml glacial acetic acid and 20-ml purified water were added. Freshly prepared solution of 10% sodium nitrite was used for better visibility of high-performance TLC (HPTLC) plate.
Preparation of standard mother tincture
100 g of coarsely powdered bark was taken and 635-ml alcohol and 400-ml water were added to make 1000 ml of mother tincture using the percolation method (as per the Homoeopathic Pharmacopeia of India).
CAMAG Spotting device – Linomat V automatic sample spotter; syringe: 10 μL (Switzerland); TLC chamber – Glass twin trough chamber (20 × 10); densitometer – TLC scanner 3 with visionCATS software; CAMAG; HPTLC plate – 20 cm × 10 cm, pre-coated silica gel 60F254 plate.
Phytochemical tests were conducted from the bark of Holarrhena antidysenterica to identify the various phytochemicals present in the plant material. The various tests, are described below and observations are recorded in [Table 1].
Test for flavonoids
Sulphuric acid (H2 SO4 test): The test solution was treated with concentrated H2 SO4. Formation of orange colour indicates the presence of flavonoids.
Tests for alkaloids
- Dragendroff's test: To 2–3 ml of the filtrate, add a few drops of Dragendroff's reagent. Formation of orange brown precipitate indicates the presence of alkaloids
- Mayer's test: To 2–3 ml of the filtrate, add a few drops of Mayer's reagent. Formation of cream precipitate indicates the presence of alkaloids.
Test for triterpenoids
Salkowski's test: To the test solution, add a few drops of concentrated sulphuric acid, shake well and allow to stand for some time. Red colour appears in the lower layer indicating the presence of sterols, and formation of yellow-coloured lower layer indicates the presence of triterpenoids.
Test for steroids
Salkowski's test: Chloroform solution of the extract when shaken with concentrated acid and on standing yields red colour.
Test for glycosides
Sodium hydroxide reagent: Dissolve a small amount of alcoholic extract in 1 ml water and add sodium hydroxide solution. A yellow colour indicates the presence of glycosides.
Test for saponins
Froth test- A pinch of the dried powdered plant material was added to 2–3 ml of distilled water. The mixture was shaken vigorously. Formation of foam indicates the presence of saponin.
Chemoprofiling by high-performance thin-layer chromatography analysis
Evaporate 25 ml of mother tincture on water bath to remove alcohol, basified with ammonia and extract with (3 × 20) ml of chloroform. Combine and concentrate chloroform layer to 2 ml. Carry out HPTLC of chloroform extract of mother tincture on silica gel 60 F254 pre-coated plate using cyclohexane: chloromethane: diethylamine (7:3:1 v/v) as mobile phase.,
The concentrated chloroform extracts (A, B, C and D) were used for the HPTLC study. The extracts were spotted in the form of band of width 8.0 mm with a microlitre syringe on pre-coated silica gel aluminium plate 60F254, using a Linomat V sample applicator. A constant application rate of 3 and 5 μL was employed. The slit dimension was kept at 6.00 mm × 0.30 mm, and 20 mm/s scanning speed was employed. The mobile phase (10 ml) consisting of cyclohexane: chloromethane: diethylamine (7:3:1 v/v/v) was taken for HPTLC analysis. Linear ascending development was carried out in a 20 cm × 10 cm twin trough glass chamber (Camag, Switzerland) saturated with the mobile phase at room temperature for 25 min. The length of the chromatogram run was 8 cm and, subsequent to the development, the TLC plates were dried in a current of air with the help of hot air dryer in a wooden chamber with adequate ventilation. Densitometric scanning was performed at 254 nm and 366 nm by reflectance scanning and operated by visionCATS software resident in the system.
Ultraviolet spectrophotometric studies
With spectrophotometer set at range 190–1100 nm, samples and standard were put in cuvettes. Before analysis, cuvettes were washed with ethanol, analysis was performed on Specord 200 plus spectrophotometer Analytical Jena AG, Konrad-Zuse-Str.1, 07745 Jena, Germany and Analytical Jena WinAspect software was used for the ultraviolet (UV) analysis.
Samples (in-house mother tincture used for UV analysis) were prepared by mixing 1 part of mother tincture and 99 parts of absolute alcohol (1:99) and filtered through membrane filter prior to UV analysis.
| Results|| |
The determined data under the physicochemical study for the raw drug are summarised in [Table 2]. Mother tincture preparation and its standardisation are summarised in [Table 3] and [Table 4], respectively. Qualitative phytochemical test loss on drying revealed the presence of water in the plant and also some volatile organic matter. Results of physicochemical studies are summarised in [Table 1], [Table 2], [Table 3], [Table 4].
High-performance thin-layer chromatography (fingerprinting)
Holarrhena antidysenterica in-house mother tincture was prepared in the laboratory and labelled as A and other three mother tinctures purchased from the market were labelled as B, C and D. The profile of chromatographic separation was scanned at 254 nm and 366 nm wavelength. At 254 nm, five spots appeared in in-house mother tincture (A) at Rf0.20, 0.35, 0.44, 0.73 and 0.87; five spots appeared in market sample (B) at Rf0.21, 0.33, 0.46, 0.72 and 0.86; four spots appeared in sample (C) at Rf0.34, 0.43, 0.74 and 0.88 and four spots appeared in sample (D) at Rf0.36, 0.42, 0.71 and 0.86 (all brown) [Figure 1]. While chromatogram scanned at 366 nm showed six spots in in-house mother tincture (A) at Rf0.21, 0.31, 0.53, 0.59 0.73 and 0.89, five spots appeared in market sample (B) at Rf0.32, 0.51, 0.58, 0.72 and 0.88; five spots appeared in sample (C) at Rf0.31, 0.54, 0.60, 0.72 and 0.90 and six spots appeared in sample (D) at Rf0.20, 0.31, 0.55, 0.59, 0.74 and 0.89 (all blue) [Figure 2], chromatogram after spray Dragendroff's reagent and then 10% sodium nitrite showed five spots in in-house mother tincture (A) at Rf0.28, 0.38, 0.43, 0.47 and 0.63; five spots appeared in market sample (B) at Rf0.27, 0.37, 0.43, 0.48 and 0.61; three spots appeared in sample (C) at Rf0.39, 0.42, 0.62 and five spots appeared in sample (D) at Rf0.28, 0.37, 0.42, 0.46 and 0.61 (all orange) [Figure 3].
|Figure 1: High-performance thin-layer chromatography fingerprints of chloroform extract of Holarrhena antidysentrica at 254 nm. Track A in-house sample; Track B, C, D market samples|
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|Figure 2: High-performance thin-layer chromatography fingerprints of chloroform extract of Holarrhena antidysentrica at 366 nm. Track A in-house sample; Track B, C, D market samples|
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|Figure 3: High-performance thin-layer chromatography fingerprints of chloroform extract of Holarrhena antidysenterica after derivatisation with Dragendroff's reagent. Track A in-house sample; Track B, C, D market samples|
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It is evident from the data that these are characteristics for the studied drug, which will help in the identification and authentication of the mother tincture. The HPTLC chemoprofiling of in-house mother tincture (A) and market sample (B, C and D) was almost similar. However, excess amount of active constituents were found in in-house homoeopathic drug/tincture (A) rather than the market samples (B, C and D). These may be considered as valuable standards in pharmacopoeia and act as vital fingerprint parameters to ensure the reliability and reproducibility of the drug.
Ultraviolet spectrophotometeric studies
UV absorption spectra (λmax) of in-house mother tincture of Holarrhena antidysenterica were found at 228 nm and 278 nm [Figure 4].
|Figure 4: Ultraviolet absorption spectra (λmax) of extract from homoeopathic drug/tincture of Holarrhena antidysentrica|
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| Discussion|| |
Physicochemical and Phytochemical analysis using various reagents showed the presence of secondary metabolites such as tannins, phenolic compounds, alkaloids and flavonoid. Physicochemical constants, namely, ash, extracted values and other parameters can be used as a reliable aid to check the identity, purity and strength. HPTLC chemoprofiling is done as an important tool for the authentication of herbal drugs and formulations. The results obtained from the study could be utilised for scientific validation and formulating standards for the quality assurance of the drug. In HPTLC chemoprofiling, the developed chromatogram and Rf values of bands will be specific for the drug with the selected solvent system. UV spectroscopic study exhibits prominent peaks, which serve as characteristic standards.
| Conclusion|| |
The present physicochemical and phytochemical study of Holarrhena antidysenterica L. reveals that its bark is rich in phytoconstituents. In UV spectroscopy, λmax values of active constituents of Holarrhena antidysenterica L. were observed at 228 and 278 nm in HT. HPTLC Chemo profiling study shows bands of active constituent were observed in all the four samples, in-house HT (A) and three market samples (B, C and D). However, excess amount of active constituents were found in in-house HT (A) rather than the market samples (B, C and D) [Figure 1], [Figure 2], [Figure 3]. Hence, the present physicochemical, phytochemical and Chemoprofiling fingerprinting data of Holarrhena antidysenterica L. may be considered as pharmacopoeia standards for the aforesaid drug.
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Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]