|Year : 2017 | Volume
| Issue : 2 | Page : 109-117
Comparative standardization study for determination of reserpine in Rauwolfia serpentina homoeopathic mother tinctures manufactured by different pharmaceutical industries using HPTLC as a check for quality control
Binit Kumar Dwivedi1, Manoj Kumar1, Anil Khurana2, Bhopal Singh Arya1, Echur Natrajan Sundaram2, Raj K Manchanda2
1 Dr. DP Rastogi Central Research Institute (H), Noida, India
2 Central Council for Research in Homoeopathy, New Delhi, India
|Date of Web Publication||8-Jun-2017|
Binit Kumar Dwivedi
DDPR Central Research Institute for Homoeopathy, A-1/1 Sector-24 Noida-UP-201301, New Delhi
Source of Support: None, Conflict of Interest: None
Background: Rauwolfia serpentina (L.) Benth. ex Kurz (Apocynaceae) (Indian snakeroot), popularly known as Sarpagandha (Sanskrit), is used for the treatment of insanity, fever, snake bites, anxiety and in neuropsychiatric conditions. The antihypertensive actions of Reserpine are a result of its ability to deplete catecholamines (amongst other monoamine neurotransmitters) from peripheral sympathetic nerve endings which are normally involved in controlling heart rate, force of cardiac contraction and peripheral vascular resistance. Objective: Comparative study of Reserpine content in R. serpentina homoeopathic mother tinctures manufactured by different pharmaceutical industries and in-house mother tinctures applying high-performance thin-layer chromatography investigative techniques to facilitate the use of correct species. Materials and Methods: The authentic samples of roots of R. serpentina were supplied by Centre of Medicinal Plants Research in Homoeopathy, Emerald, Tamil Nadu, India. Authentic plant material was used to prepare the mother tincture (as per Homoeopathic Pharmacopoeia of India). Reserpine (C33H40N2O9,M.P. 360°C, purity >99% w/w by high-performance liquid chromatography [HPLC]) was purchased from Sigma-Aldrich as a standard reference. The solvents for the study, namely, ethanol, HPLC water, toluene, ethyl acetate, diethylamine and chloroform were of analytical grade purity (MERCK Ltd.,), used throughout. Results: Five samples of mother tinctures were used for the study, in-house mother tinctures (labelled: D and E) of R. serpentina shows a higher amount of Reserpine content than the marketed samples (labelled: A, B and C). Conclusion: It may be concluded that mother tinctures prepared by authentic plants showed the excess amount of Reserpine rather than that of mother tinctures procured from the market.
Keywords: High-performance thin-layer chromatography, Homoeopathic mother tincture, Reserpine
|How to cite this article:|
Dwivedi BK, Kumar M, Khurana A, Arya BS, Sundaram EN, Manchanda RK. Comparative standardization study for determination of reserpine in Rauwolfia serpentina homoeopathic mother tinctures manufactured by different pharmaceutical industries using HPTLC as a check for quality control. Indian J Res Homoeopathy 2017;11:109-17
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Dwivedi BK, Kumar M, Khurana A, Arya BS, Sundaram EN, Manchanda RK. Comparative standardization study for determination of reserpine in Rauwolfia serpentina homoeopathic mother tinctures manufactured by different pharmaceutical industries using HPTLC as a check for quality control. Indian J Res Homoeopathy [serial online] 2017 [cited 2017 Sep 25];11:109-17. Available from: http://www.ijrh.org/text.asp?2017/11/2/109/207665
| Introduction|| |
Rauwolfia serpentina (L.) Benth. ex Kurz (Apocynaceae) is a plant whose roots are therapeutically used as a sedative, a hypnotic drug and in hypertension. Reserpine (an indole alkaloid) was isolated in 1952 from the dried root of R. serpentina (Indian snakeroot), had been known as Sarpagandha (Hindi and Sanskrit), which is used for the treatment of insanity, fever, snake bites, and in anxiety. The antihypertensive actions of Reserpine are a result of its ability to deplete catecholamines (amongst other monoamine neurotransmitters) from peripheral sympathetic nerve endings which are normally involved in controlling heart rate, force of cardiac contraction and peripheral vascular resistance (Pharmacology, An Introduction: Pharmaceutical Sciences, Pharmacology, Edition 6, 2011, Henry Hitner and Barbara Nagle).
Its molecular structure was elucidated in 1953 and natural configuration published in 1955; the first total synthesis was accomplished by R. B. Woodward in 1958. Santapau (1956) has studied botanical aspects of this plant. Kattel (1987) has reported phyllotaxical morphotypes. Variation of chemo-botanical characters in the indigenous collections of this plant was reported by Sethi et al. (1991).
R. serpentina mother tincture is used in Homoeopathy for treatment of blood pressure without any side effect, but in allopathic system of medicine, some side effects are reported such as nausea and vomiting, diarrhoea, shortness of breath, drowsiness, dizziness and headache.
The plant R. serpentina (L.) Benth ex Kurz (Apocynaceae) is a medicinally famous herb used in Ayurveda, Siddha, Unani, Homoeopathy and in Western systems of medicine., There are different types of alkaloids present in Rauwolfia namely, ajmaline, ajmalinine, ajmalicine, serpentine and serpentinite.Reserpine, Yohimbine, rescinnamine, reserpine, rauwolfinine, renoxidine, rescinnamine, reserpiline, sarpagine, serpentinine, tetraphyllicine and 3-epi-a-yohimbine have also been reported. It also contains small amounts of phytosterol and fatty substances. The root of R. serpentina was found to possess 0.1% of the active principle, Reserpine (indole alkaloid).
Number of factors relating to climate, altitude, rainfall and other conditions responsible for growth of plants affect the quality of active constituents present in a particular species even when it is grown in the same country. These conditions may produce major variations in the active constituents present in plants  and thus cause variation on the therapeutic efficacy. The understanding of how environmental factors affect the production of secondary metabolites will be of great importance for the conservation of medicinal plants and optimising field growth conditions for maximal recovery of active constituents. Resource availability theory suggests that the way a plant defends itself ultimately depends on resource availability and its intrinsic growth rate. This theory predicts that the rapidly growing plants in resource-rich habitats contain low levels of highly mobile secondary metabolites. Nitrogen is taken up early in the growing season in excess of the plant's need for growth. Excess nitrogen is available to be synthesised into N-based secondary metabolites.
In Homoeopathy, mother tinctures(Ф) are defined as the original substance prepared with the aid of alcohol, directly from crude drug. They are the precursors of the corresponding potencies of the respective drug and the starting point for the production of homoeopathic medicines.R. serpentina is one of the most important homoeopathic drugs being prescribed for various disorders including hypertension. Therefore, the present study is proposed to determine the quantity of the active constituent, Reserpine present in R. serpentina mother tinctures manufactured by different pharmaceutical industries were procured to ascertain whether there is uniformity or whether variation exists by applying high-performance thin-layer chromatography (HPTLC) technique studies.,,
| Materials and Methods|| |
The roots of R. serpentina were collected by Center of Medicinal Plants Research in Homoeopathy (CMPRH), Emerald, Tamil Nadu, and was authenticated by the staff of the Center of Medicinal Plants Research in Homoeopathy (CMPRH), Emerald, Ooty. The voucher specimen has been deposited in the herbarium and in the laboratory of DDPR Central Research Insitu te for Homoeopathy, Noida, Uttar Pradesh, India, for future reference. Authentic plant material was used to prepare the Mother Tincture. Reserpine (C33H40N2O9, M. P. 360°C, purity >99% w/w by HPLC) was purchased from Sigma Aldrich. The solvents ethanol, HPLC water, toluene, ethyl acetate, diethyl amine, chloroform were of analytical grade purity (Merck Ltd.).
Moisture content was determined by loss on drying method. Total ash, water-soluble ash, foreign matter and acid-insoluble ash parameters were performed as per methods recommended in Homoeopathic Pharmacopeia of India.
Determination of physical constants (raw drug standardisation)
Loss on drying
Loss on drying is the loss of mass expressed as percentage w/w. The test for loss on drying determines both water and volatile matter in the crude drug by IR balance. Moisture is an inevitable component of crude drug, which must be eliminated as far as possible.
An accurately weighed quantity of 2 g of powdered drug was taken in a porcelain dish. The porcelain dish was kept open in a vacuum oven, and the sample maintained at a constant temperature of 100°C. Then, it was cooled in a desiccator at room temperature. The procedure was repeated till constant weight on repeated weighing is observed. Percentage loss on drying was calculated using the following formula.
Determination of foreign matter
Weigh 100–500 g of the plant material under study and spread it out in a thin-layer. Inspect the sample with the unaided eye or with the use of a 6x lens and separate the foreign organic matter manually as completely as possible. Weigh the sorted foreign matter and determine the percentage of foreign matter from the weight of the drug taken.
Ash value is helpful in determining the quality and purity of a crude drug, especially in the powdered form. On incineration, crude drugs normally leave a quantity of ash as residue usually consisting of carbonates, phosphates and silicates of sodium, potassium, calcium and magnesium. The total ash of a crude drug reflects the care taken in its preparation of non-dissipation of non-volatile elements. A higher limit of acid-insoluble ash is imposed, especially in cases where silica may be present or when the calcium oxalate content of the drug is very high.
Determination of total ash value
Accurately 2 g of the powdered drug in a silica crucible, previously ignited and weighed. Incinerate by gradually increasing the heat to temperatures not exceeding 450°C for 4 h, until free from carbon, crucible is cooled and weighed. Calculate the percentage of ash with reference to air-dried drug using the following formula.
Determination of water-soluble ash value
The ash is boiled with 25 ml of water for 10 min. Filter and collect the insoluble matter on an ashless filter paper, wash with hot water and ignite in a crucible at a temperature not exceeding 450°C for 4 h. Cool in a dessicator and weigh. The difference in weight represents the weight of water-soluble ash. Calculate the percentage of water-soluble ash with reference to the air-dried drug using the following formula:
Determination of acid-insoluble ash value
Boil the ash for 10 min with 25 ml of 2M HCl. Filter and collect the insoluble matter on ashless filter paper, wash with hot water and ignite in a crucible at a temperature not exceeding 450°C for 4 h. Cool in a dessicator and weigh. Calculate the percentage of acid-insoluble ash with reference to the air-dried drug using following formula:
The results obtained with reference to air-dried drug are tabulated and observations are recorded in [Table 1].
Phytochemical tests were conducted on the roots of R. serpentina to identify various phytochemicals present in the plant material (A.K. Gupta et al., 2008).,, The various tests conducted are given below and the observations are recorded in [Table 2].
- Test for tannins (lead acetate test): To the test solution, a few drops of 10% lead acetate were added. Precipitate was formed, indicates the presence of tannins
- 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
- 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 flavonoids:
- Sulphuric acid (H2 SO4 test): The test solution was treated with concentrated H2 SO4. formation of orange colour indicates the presence of flavonoids
Test for phenolic compounds (ferric chloride test): To the test solution, few drops of ferric chloride test reagent were added. An intense green, purple, blue or black colour developed is taken as an evidence for the presence of tanninsTests for alkaloids:
- Dragendorff's test - To 2–3 ml of the filtrate, add a few drops of Dragendorff's reagent. Observe for formation of orange-brown precipitate
- Mayer's test - To 2–3 ml of the filtrate, add a few drops of Mayer's reagent. Observe for formation of precipitate
- Hager's test - To 2–3 ml of the filtrate, add a few drops of Hager's reagent. Observe for formation of yellow precipitate
- Wagner's test - To 2–3 ml of the filtrate, add a few drops of Wagner's reagent. Observe formation of reddish brown precipitate.
The results of phytochemical tests carried out are recorded in [Table 2].
Preparation of in-house mother tinctures
100 g of coarsely powdered root were taken, added 824 ml alcohol and 200 ml water to make 1000 ml of mother tincture using the percolation method (as per Homoeopathic Pharmacopoeia of India). This tincture was transferred to a suitable glass container and stored for further study.
Standardisation of mother tincture
Standardisation of mother tincture was conducted to identify the organoleptic and physicochemical properties of mother tincture (Banerjee, D.D. 2006, Augmented Textbook of Homoeopathic Pharmacy: B. Jain Publishers).
- Organoleptic properties
- Appearance: Clear liquid
- Colour: Yellowish brown
- Odour: Characteristic
- Sediments: Nil
- Weight per ml: 0.867–0.877 g
- Total solid: Not <1.0 percent w/v
- Alcohol content: 75.0–79.0 per cent v/v
- pH value: 5.9.
Quantification of Reserpine by high-performance thin-layer chromatography study
Quantification of Reserpine was done by HPTLC as mentioned below.
Preparation of standard Reserpine
Five milligrams of Reserpine was weighed in a 10 ml volumetric flask. To this, 5 ml chloroform and 5 ml ethanol were added to make final volume 0.5 μg/μl.
Preparation of sample
5 ml of mother tincture (about 4 g) was taken in 100 ml beaker, added 10 ml of distilled water and 0.2 ml concentrated hydrochloric acid; evaporated to dryness on water bath. Dissolved the residue in 2 ml of chloroform, methanol mixture (1:1) then carried out HPTLC analysis.
HPTLC system equipped with a sample applicator device CAMAG Linomat 5. CAMAG Twin Trough Chamber, Camag TLC Scanner and integration software (winCATS).
HPTLC Plate: Silica gel GF254 (Merck) 20 × 10 cm.
Mobile Phase: Toluene-Ethyl Acetate-Diethylamine (7:2:1, v/v/v).
Wavelength: 254 nm.
Standardisation of in-house mother tincture
CAMAG HPTLC system comprising Linomat 5 as sample applicator and TLC Scanner controlled by winCATS software was used for quantitative evaluation. Stationary phase used was silica gel 60 F254 and the mobile phase used was toluene-ethyl acetate-diethylamine (7:2:1, v/v/v). Samples and standard were applied as 8 mm bands with 6 mm distance between the tracks. Tank saturation and plate equilibrium were given with filter paper for 10 min. Ascending development for a distance of 80 mm in a Twin Trough Chamber was completed in approximately 15 min. Volume of standard mother tincture(Ф) was first optimised at 4 μl for quantification. The λ max of Reserpine was found to be 254 nm after taking the spectra of the standard of Reserpine. Quantitative measurement in the absorbance mode was done at 254 nm using a slit dimension of 6.00 mm × 0.45 mm.
The volume of the in-house mother tincture was optimised to 2 μl for quantification. It was then simultaneously applied with different concentrations of standard Reserpine, i.e., 4, 6, 8 and 10 μl. The method was found to be linear with a regression of 0.99983, and a standard deviation of 1.67% and the amount of Reserpine was calculated in the mother tincture.
Standardisation of the in-house mother tinctures
Standardisations of the mother tinctures were done using HPTLC method. In-house mother tinctures were chromatographed simultaneously along with three other mother tinctures available from the market at 2 and 5 μl, respectively, on the same plate for comparison [Figure 1], [Figure 2], [Figure 3]. Multiwavelength (MWL) scan was done for finding the optimum wavelength for scanning. The optimum wavelength was found to be 254 nm. The entire plate was further scanned at this wavelength for quantification and spectral match [Figure 4] and [Figure 5]. Many fractions of in-house mother tinctures were matched with the help of its characteristic spectra with that of other marketed samples. Individual λ max of each fraction was also found with the help of spectral scanning, and then the plate was scanned with these selected wavelengths in MWL mode. The pattern of the peaks was compared for the in-house mother tinctures and marketed samples. It was observed that the response for various concentrations of standard Reserpine was linear in the range of 100–500 ng with a coefficient of variation of 0.99983 and a standard deviation of 1.67%. Reserpine was quantified and the amount was calculated in individual mother tinctures. With this method, all available mother tinctures were compared and the active principle was quantified [Figure 6] and [Figure 7].
|Figure 1: High-performance thin-layer chromatography fingerprints of A, B, C, D and E samples (Rauwolfia serpentina) under UV 254 nm|
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|Figure 2: High-performance thin-layer chromatography fingerprints of A, B, C, D and E samples (Rauwolfia serpentina) under UV 366. nm|
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|Figure 3: High-performance thin-layer chromatography fingerprints of A, B, C, D and E samples (Rauwolfia serpentina) after derivatisation|
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|Figure 4: Overlay of absorption spectra of standard, in-house and market mother tinctures|
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|Figure 5: Chromatogram of standard, in-house and market mother tinctures|
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|Figure 7: Calibration curve of Reserpine in market samples and in-house mother tincture|
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Quantification of Reserpine in market samples and in-house mother tincture
5 ml each of mother tincture taken for analysis, i.e. 5 ml of mother tincture weight is 4 g or 4000 mg, i.e., 5 ml = 4000 mg. Therefore, 1 ml = 800 mg or 1 μl =0.0800 μg
Hence, final concentration (2 ml) by dissolving chloroform and methanol mixture is (1:1, v/v) ratio. Sample applied on plate for sample A, B and C is 5 μl each and for sample for D and E is 2 μl.
So final concentration on plate of
- Sample A = 0.800 × 5 = 4000 μg
- Sample B = 0.800 × 5 = 4000 μg
- Sample C = 0.800 × 5 = 4000 μg
- Sample D = 0.800 × 2 = 1600 μg
- Sample E = 0.800 × 2 = 1600 μg.
Calculation of Reserpine content in sample A
- 4000 μg sample (A) on plate
- Reserpine content from calibration graph = 337.56 ng or 0.33756 μg.
Percentage of Reserpine in sample A
4000 μg = 0.33756 μg
Calculation of Reserpine content in sample B
- 4000 μg sample on plate
- Reserpine content from calibration graph = 288.57 ng or 0.28857 μg.
Percentage of Reserpine content in sample B
4000 μg = 0.28857 μg.
Calculation of Reserpine content in sample C
- 10,000 μg sample (B) on plate
- Reserpine content from calibration graph = 366.26 ng or 0.36626 μg.
Percentage of Reserpine content in sample C
4000 μg = 0.36626 μg
Calculation of Reserpine content in sample D
- 1600 μg sample on plate
- Reserpine content from calibration graph = 538.18 ng or 0.53818 μg.
Percentage of Reserpine content in sample D
1600 μg = 0.53818 μg
Calculation of Reserpine content in sample E
- 1600 μg sample on plate
- Reserpine content from calibration graph = 465.11 ng or 0.46511 μg.
Calculation of Reserpine content in sample E
1600 μg = 0.46511 μg
The amount of Reserpine in R. serpentina in the in-house sample (D and E) and market sample mother tinctures (A, B and C) were calculated and presented in [Table 3].
| Discussion|| |
Repeatability of the method was checked by scanning 15 tracks of 2 μl volume in-house mother tincture. The coefficient of variation was found to be 0.0844. The percentage recovery of Reserpine was calculated using the above method. The average recovery values obtained were 96.6%–104.37%, which confirms that the method is validated. The HPTLC scanning of 'R. serpentina' mother tincture(Ф) obtained from manufacturer (A, B and C) and the in-house mother tinctures (D and E) had been done at 254 nm wavelength. The scanning report obtained after integration. From the results obtained after densitometry scanning, it was observed that the in-house mother tinctures (D and E) of R. serpentina shows a higher amount of Reserpine content than the marketed samples (A, B and C). It may be concluded that samples procured from the market are showing a lesser amount of Reserpine hence may not be up to the standard level. This quantification may lead to better quality checking of market samples which in turn will be responsible for better therapeutic efficacy.
| Conclusion|| |
It is concluded that in-house prepared mother tincture showed excess greater amount of Reserpine, i.e., 0.5381% and 0.0290% (D and E) in comparison to marketed samples (A, B and C).
Authors wish to express their gratitude to Dr. Raj K. Manchanda, Diector General, Central Council for Research in Homoeopathy, New Delhi, for his support and Mr. Dilip Charegaonkar, Mr. T. B. Thite and other Anchrom lab staff for help in HPTLC analysis.
Financial support and sponsorship
Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]