INTRODUCTION

Rauwolfia consists of the dried roots and rhizomes of

snake wood, Apocynaceae family, a small shrub found

in India, Pakistan, Burma, Thailand and Java. The

geographical source tends to affect the alkaloid content

and producers tend to prefer drugs from India or

Pakistan[1]. Reserpine the most important constituent is

contained in many other species of Rauwolfia[1]. The R.

serpentina (L.) Benth Ex Kurz[2] is utilized in an Indian

medicine for the treatment of assorted sickness[3] and are

principally utilized in the treatment of high blood

pressure[4-7], mental illness and snake bite[8], control of

anxiety[9], breast cancer[10] and psychiatry[11]. The

mother tincture contains not less than 0.10 and not more

than 0.30 percent of alkaloids[12]. The mother tincture

prepared from the powdered herbal drug and purified

water according to the method specified in Homeopathic

Pharmacopoeia of India[13] using alcohol. Reserpine was

first extracted from the roots of R. serpentina[14] in 1952

and has since been isolated from the roots of many

Rauwolfia species by other investigators[15]. Reserpine

blocks the absorption of norepinephrine from central and

peripheral axon terminals into storage vesicles resulting

in degradation of catecholamine and serotonin[16].

Reserpine is chemically a methyl

(1R,15S,17R,18R,19S,20S)-6,18-dimethoxy-17-(3,4,5-

trimethoxybenzoyl)oxy-1,3,11,12,14,15,16,17,18,19,

20,21-dodecahydroyohimban-19-carboxylate[16]. The

chemical structure of reserpine[17] and R. serpentina[18] is

shown in fig. 1.

Several studies on literature reveals that few methods

have been reported for Rauwolfia using different

Journal of Pharmaceutical Research and Therapeutics

2020; Volume 1 (Issue 01); 35-40

Research Paper

A Validated UV Spectrophotometry Method for the Quantification of Reserpine in Rauwolfia serpentina Mother Tincture

SUMAN SHRIVASTAVA1*, SAURABH SHRIVASTAVA2 AND S. J. DAHARWAL1

1University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur-492010, 2Shri Rawatpura Sarkar Institute of Pharmacy, Kumhari, Durg-490042, India

Shrivastava, et al.: A Validated UV Spectrophotometry Method for the Quantification of Reserpine

Abstract:

In the present work, the qualitative and quantitative analysis of reserpine in the roots and rhizomes of

Rauwolfia serpentine (Apocynaceae) is presented using different analytical approaches. Extracts of

Rauwolfia serpentina was first examined by UV spectrophotometry and the content of reserpine was

identified. It obeys the Beer-Lambert’s law in the concentration range of 2-10 µg/ml with λmax at 217 nm.

Using this method, the alkaloidal content in reserpine and marketed mother tincture containing reserpine

were determined. It could be used for routine analysis of marketed samples dealing with homeopathic drugs

for standardization of plant materials containing alkaloids and for alkaloid containing herbal drugs. The

method was validated for linearity, accuracy, precision, limit of detection, limit of quantification, robustness,

ruggedness according to the International Council for Harmonisation guidelines. The method was accurate

with recovery of 98.54-99.72 %. The intraday precision was 0.09-0.29 % and interday precision was 0.14-

0.5 %. The limit of detection and limit of quantification for reserpine was 1.05 and 3.19 µg/ml. The reserpine

content quantified from homeopathic mother tincture was found well within limits. The proposed method is

simple, selective and reproduce method for the estimation of reserpine in homeopathy mother tincture.

Keywords: Rauwolfia serpentina, Apocynaceae, Reserpine, UV spectrophotometry

*Address for Correspondence:

Suman Shrivastava

University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur-492010, India. E-mail: sumanshrivastava1991@gmail.com

Article History:

Received 03 April 2020 Revised 22 April 2020 Accepted 26 April 2020

J Pharm Res Ther 2020;01(01):35-40

35 Journal of Pharmaceutical Research and Therapeutics April-June 2020

techniques such as colorimetric method[19];

spectrophotometric method[20,21], thin layer

chromatography[22], high performance thin layer

chromatography[23-28], high performance liquid

chromatography[29-33], nuclear magnetic resonance

spectroscopy[34], atomic absorption spectroscopy[35]. A

tandem mass spectroscopy was also used such as liquid

chromatography-mass (LC-MS) spectrometry[36-41], gas

chromatography-mass spectrometry [42], HPTLC-MS[43],

HPTLC-HPLC method[44,45].

MATERIALS AND METHODS

Collection of plant material:

A fresh plant of R. serpentina root was collected from

the botanical garden of department. Root was collected

and washed under the tap water to avoid adhering dirt

then shade dried. The dried materials were powdered

into coarse powder by grinding in mechanical grinder.

Chemicals and reagents:

Reference standard reserpine was obtained as gift

sample from Chemical resources, Haryana. AR/ACS

reagent grade of methanol was used in the experiment

and procured from Central Drug House (P) Ltd., CDH

Fine Chemicals, New Delhi. Marketed mother tincture

of Rauwolfia was procured from local market of Raipur,

Chhattisgarh.

Instrumentation:

UV-Visible double beam spectrophotometer-1800

(Shimadzu, North America) with matched quartz cells (1

cm) was used in present study.

Standard preparation of reserpine:

Standard stock solutions of reserpine were prepared in

methanol at a final concentration of 1 mg/ml. About 10

mg of the standard was accurately weighed to prepare a

stock standard solution, dissolved with methanol in a 10

ml volumetric flask, and completed to volume. For stock

solution, 1 ml of the standard stock solution was

transferred and diluted into a 10 ml volumetric flask

using methanol to obtain the concentration of 100 µg/ml,

and processed beyond that to obtain a series of

calibration solutions at different concentrations.

Preparation of extract solution:

The root part of R. serpentina was taken and washed and

dried in an open environment. Then it was accurately

weighed (10 g) and it was immersed in 50 ml methanol

and then shaken twice daily for 10 days. The extract was

filtered through 0.45 µ filter paper and filtrate was

collected in a volumetric flask. One milliliter of extract

solution was dissolved in 10 ml of volumetric flask

adjust with 10 ml of methanol to get the concentration of

100 µg/ml. About 0.2 ml of above solution was further

diluted with methanol to make up the volume up to 10

ml to get the final concentration of 2 µg/ml.

Preparation of test solution of marketed mother

tincture:

One milliliter of marketed mother tincture was mixed

with methanol and volume was adjusted up to 10 ml to

get the concentration of 100 µg/ml. Pipette out 0.2 ml

from stock solution and diluted with methanol up to 10

ml to make the final volume of 2 µg/ml.

RESULTS AND DISCUSSION

Development and optimization of the method: Proper

wavelength selection of the method depends on the

nature of the sample and its solubility. To develop a

suitable method for quantitative determination of

reserpine, number of trials were done.

Selection of wavelength: The standard solution of

reserpine in methanol between 200-400 nm in UV

spectrophotometry and it exhibits maximum absorbance

at 217 nm, which was reported as λmax in the literature.

The spectrum of reserpine complies with the reference

spectra (fig. 2).

The analytical method validation of the

spectrophotometric method was carried out based on the

Fig. 1: Structure of (a) reserpine and (b) Rauwolfia serpentina plant

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parameters involves linearity, accuracy, precision,

ruggedness, sensitivity (as per ICH guidelines Q2

R1)[46].

The linearity was evaluated by analyzing the standard

solutions of reserpine at five different concentrations

levels (2, 4, 6, 8, 10 µg/ml). The wavelength of the above

solution scanned at 217 nm against the blank solution

prepared in the same manner without adding the drug. A

graph of absorbance versus concentration was plotted

and r2 was found to be 0.9972. The linear regression

equation was found to be 0.0511x+0.1064. The linear

calibration curve of the reserpine is listed in fig. 3 and

Tables 1 and 2. The linearity of the analytical response

of the studied range was excellent, with correlation

coefficient was higher than 0.99.

The recovery was performed by adding a known amount

of individual standards into a certain amount. Three

replicates of concentration 2, 4, 6 µg/ml were performed

for the test. The percent recoveries of reserpine ranged

from 98.54–99.72 %. The quantitative method for the

determination of indole alkaloids is feasible within

acceptable limits. The recovery study is summarized in

the Table 3.

The precision of the chromatographic system was

evaluated by 3 successive injections 2, 4, 6 µg/ml of

standard solutions of reserpine. The results were

expressed in % RSD as 0.09-0.29 % for intra-day and

0.14-0.5 % for inter-day (n=3). The precision was

verified by intra-day and inter-day variability. The intra-

day variability was performed by the successive

injections of three samples in the same day. The inter-

day variability study was conducted for three

consecutive days using the same solution. The RSD

values obtained from intra-day and inter-day precision

studies are summarized in Table 4. The RSD values were

less than 2 % in run-to-run and day-to-day analysis are

within the limits.

The sensitivity of the method was evaluated by

determining the limits of detection (LOD) and limit of

quantification (LOQ). The LOD was defined as the

lowest concentration of analyte which was determined

by the signal to noise ratio of atleast 3. The LOQ was

defined as the lowest concentration of analyte, which can

be quantified with signal to noise ratio of atleast 10.

These can be determined at n=3 times of standard

solution. The sensitivity of the method was determined

by UV is shown in Table 5.

Fig. 2: UV spectrum of reserpine

217 nm, 0.310 Abs.

Fig. 3: Linearity curve of reserpine

♦ Absorbance; ⸺ linear (absorbance)

TABLE 1: LINEARITY DATA OF RESERPINE S. No. Concentration (µg/ml) Absorbance (nm)

1 2 0.216

2 4 0.31 3 6 0.399 4 8 0.516 5 10 0.624 Mean 0.413 SD 0.16

TABLE 2: REGRESSION DATA S. No. Summary Values

1 Regression equation 0.0511x+0.1064 2 r2 0.9972

y = 0.0511x + 0.1064R² = 0.9972

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15

Ab

sorb

ance

Concentration (µg/ml)

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The effect of slight changes on absorption and recovery

at wavelengths 215 and 219 nm has been studied for the

reserpine. To test robustness of the method, small

changes in the chromatographic parameters were

deliberately made, which may affect the performance of

the method such as absorbance, but negligible changes

in the recovery were found. In ruggedness study, the

impact of adjusting personnel has been studied and this

was done by preparing solution and reading absorption

by another analyst in order to see the effect of changes

on the analytical method. Quantitation was not

significantly affected by changing scanning wavelength

±2 nm. Robustness study of reserpine is depicted in

Table 6. From stock solution, sample solution was

prepared containing reserpine 2 µg/ml and analyzed by

two different analysts using similar operational and

environmental conditions (n=3). The ruggedness study

was shown in Table 7.

The method specificity was assessed by comparing the

absorbance and absorption spectra of the reference

compounds in sample and standard tracks. On

comparing of spectra at peak start, peak apex and peak

end positions of the band, an acceptable correlation (r2=

0.94-0.99) was obtained between standards and sample

overlay spectra, which confirms the purity of reserpine

in the sample tracks (fig. 4). The method was quite

selective for reserpine since there was no other

interfering peak. The baseline did not show any

significant peak.

Quantification of reserpine in marketed mother tincture:

2 µg/ml of each sample were analyzed by the proposed

method, then by extrapolating from the absorbance data

the unknown concentration was determined. The

methods were developed and scanned at 217 nm for

quantitative evaluation. Contents of reserpine in sample

and marketed mother tincture were estimated using

linearity equation. Amount of reserpine present in R.

serpentina was shown in the Table 8.

In the present study, simple and accurate method was

established for the determination of reserpine in R.

TABLE 3: RECOVERY STUDY

Drug Initial Conc.

(µg/ml) Conc. added % Mean±SD (n=3) Recovery % % RSD

Reserpine

2 80 % 1.57±0.005 98.54 0.36

100 % 1.98±0.005 99.17 0.29 120 % 2.38±0.005 99.44 0.24

4 80 % 3.18±0.005 99.47 0.18 100 % 3.97±0.005 99.41 0.14 120 % 4.78±0.01 99.65 0.24

6 80 % 4.78±0.005 99.65 0.12 100 % 5.98±0.01 99.67 0.16 120 % 7.18±0.01 99.72 0.13

TABLE 4: INTRA-DAY AND INTER-DAY PRECISION

Analyte Conc.

(µg/ml)

Intra-day precision Inter-day precision

Mean±SD (n=3) % RSD % amt found±SD Mean±SD (n=3) %RSD % amt

found±SD

Reserpine

2 1.97±0.005 0.29 98.83±0.005 1.98±0.01 0.50 99 ± 0.01

4 3.97±0.005 0.14 99.17±0.005 3.98±0.005 0.14 99.58±0.005

6 5.97±0.005 0.09 99.61±0.005 5.98±0.01 0.16 99.67±0.01

TABLE 5: DETECTION LIMIT AND QUANTIFICATION LIMIT Parameters Reserpine (µg/ml)

LOD 1.05 LOQ 3.19

TABLE 6: ROBUSTNESS STUDY

Conc. (µg/ml)

At 215 nm At 219 nm

Absorbance Mean±SD % RSD Recovery % Absorbance Mean±SD % RSD Recovery %

2 0.216

0.217±0.0005 0.26 99.84

0.216

0.217±0.001 0.46 100 2 0.217 0.217

2 0.217 0.218

TABLE 7: RUGGEDNESS STUDY

Conc. (µg/ml)

Analyst 1 Analyst 2

Absorbance Mean±SD %RSD Recovery % Absorbance Mean±SD % RSD Recovery %

2 0.217

0.217±0.0005 0.26 100.15

0.218

0.218± 0.0005 0.26 100.61 2 0.217 0.218

2 0.218 0.219

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serpentina. The optimized UV spectrophotometry

method was applied to the qualitative and quantitative

analysis of reserpine in R. serpentina. Reserpine was

reported to be the most abundant component in

Rauwolfia. The method is validated in compliance with

ICH guidelines is suitable for estimation of reserpine

with excellent recovery, precision and linearity.

Therefore, taking a variety of complimentary

approaches to analysis can specifically and

comprehensively assessed the quality of homeopathic

medicine.

Acknowledgement:

We are thankful to the Director of University Institute of

Pharmacy, Pt. Ravishankar Shukla University, Raipur

for providing support and analysis. We are also thankful

to the Chemical resources, Haryana for providing

reserpine as standard drug.

Conflicts of interest:

The authors declare no conflicts of interest.

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