Research J. Pharm. and Tech.

13(1): January 2020

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Carbohydrates determination in the Snowdrops (Galanthus L.) herbal

pharmaceutical substances by TLC and UV-Spectrophotometry
Bokov D.O.1,2, Kulaeva I.R.3, Potanina O.G.4, Sergunova E.V.1, Bondar A.A.1, Evgrafov A.A.1,
Antsyshkina A.M.1, Krasnyuk I.I.1
1
Institute of Pharmacy, Sechenov First Moscow State Medical University, 8 Trubetskaya St., bldg. 2, Moscow,
119991, Russian Federation.
2
Laboratory of Food Chemistry, Federal Research Center of Nutrition, Biotechnology and Food Safety, 2/14
Ustyinsky pr., Moscow, 109240, Russian Federation.
3
Medical Institute, Chechen state University, 32 Sheripova St., Grozny, Chechen Republic, 364061,
Russian Federation.
4
Pharmaceutical chemistry and pharmacognosy chair, Рeoples’ Friendship University of Russia
(RUDN University), 6, Miklukho-Maklaya Street, Moscow, 117198, Russian Federation.
*Corresponding Author E-mail: fmmsu@mail.ru

ABSTRACT:
Plants of Galanthus L. genus (snowdrops) used in Russian medicine for obtaining neurological and
cardiovascular remedies, contain different types of carbohydrates (polysaccharides, monosaccharides) as
additional biological active substances group. In present research the carbohydrate composition and content were
investigated in the snowdrops herbal pharmaceutical substances (Galanthus woronowii Losinsk, Galanthus
nivalis L.). Optimal conditions for TLC analysis, the extraction of crude herbal drugs, were determined;
spectrophotometric procedures for quantifying the total carbohydrate content in terms of monosaccharides
(fructose, glucose) were successfully developed. The results of the investigation can be used in quality control
and standardization of crude herbal drugs containing carbohydrates, and for inclusion in pharmacopoeial
monographs.
KEYWORDS: carbohydrates, Galanthus woronowii Losinsk, Galanthus nivalis L., herbal pharmaceutical
substances, glucose, fructose.

INTRODUCTION: At the same time added sugars are responsible for

Presently, the determination of various types of
carbohydrates (CARB) in crude herbal drugs (CHD) or
herbal pharmaceutical substances (HPS), used for the
needs of pharmaceutical practice, attracts attention of
researchers in different fields of pharmaceutical science1-
4 and HD CARB composition. Notoriously CARB are
. In general, CARB are involved in the unique complex
of biologically active compounds (BAC), occurred in
every living plant, play great role in the plants and
animals life5.
Received on 08.05.2018 Modified on 21.08.2018
Accepted on 18.10.2018 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(1):243-249.
DOI: 10.5958/0974-360X.2020.00049.9
243
chronic diseases such as diabetes mellitus 6. It’s known,
homeopathic mother tinctures (HMT) – are HPSs that
used for production of homeopathic drugs (HD).
Pharmacotherapeutic effects of HD depends on the BAC
composition. Therefore, it is important to know HMT
divided into 2 groups: mono- and polysaccharides, which
are used widely in pharmacy and medicine7.
BAC complex of the Galanthus L. (Fam.
Amaryllidaceae J.St.-Hil.) genus contains CARB, but
CARB composition and content is not evaluated 8.
Therefore, it is very significant to investigate CHD and
HMT CARB composition of Galanthus woronowii
Losinsk. and Galanthus nivalis L., because CARB
possess a essential value on the pharmacological activity
of HD. Pharmacognostical analysis involves the study of
the morphological features and chemical composition of
CHD9-15.
 Research J. Pharm. and Tech. 13(1): January 2020
Today, one of the most universal and proper method for
estimation of CARB sum is spectrophotometric
determination. It is carried out after hydrolysis of
polysaccharides that decomposed to monosaccharides
(glucose, fructose) and obtaining colored complex with
specific reagent (anthrone, 2,4,6-trinitrophenol).
Anthrone method was developed for the CARB
quantification of in the different natural foodstuffs (raw
rice, black gram, green gram, guava, ground nut, banana,
grape, bean, carrot, milk)16. A UV-visible
spectrophotometry rapid method was developed for the
quantitative CARB estimation in the different seeds
(almond, peanuts, cashews, walnuts, pistachios, corn,
wheat, chickpeas, soya beans, barley)17-19. In another
study quantitative CARB estimation was performed in
the different natural foodstuffs (spinach, amaranthus,
Chinese spinach, curry leaves, sorrel leaves, drumstick
leaves, fenugreek leaves, mint, lettuce, coriander)20. This
method can be used for quantitative CARB estimation in
the different fruits (apple, custard apple, banana, papaya,
guava, pineapple, grapes, pomegranate, orange, kiwi
fruit, strawberries, mango)21. One of the newest method
for the CARBS determination (glucose, fructose,
sucrose) is LC/MS/MS22. Along with modern methods of
analysis (HTLC, OPLC, GC-MS, GC-IR, LC-MS, LC-
MS/MS)23, spectrophotometry remains in demand in
pharmaceutical analysis24.
MATERIALS AND METHODS:
The objects of research were samples collected during
flowering time – whole fresh plants of Voronov’s
snowdrop (Galanthus woronowii Losinsk) and common
snowdrop (Galanthus nivalis L.), harvested in March-
April 2016-2017 at the Botanical Garden of Sechenov
First Moscow State Medical University in Moscow
(Russia). HMT was produced from the whole flowering
plant according to the 3a method in general
pharmacopoeial monograph25. Qualitative CARB
determination was performed in the CHD and HMT
consistently: CARBs were identified by qualitative
reactions, then analyzed by thin layer chromatography
(TLC). The spectrophotometric investigation of
snowdrops HMT and CHD was carried out by
instrument – «Cary 50 Scan» Agilent Technologies
company (previously – Varian, USA), followed by
results computer processing with «Cary WinUV
Analysis Pack ver. 3.1» program for «Windows XP».
The working standard samples (WSS) of fructose (Fru)
and glucose (Glu) were used in the analysis as standards.
Extracts preparation for CARB determination by
TLC:.
A sample weight of GW (1) or GN (2) crushed in mortar
was placed in a 100 ml conical flask with ground joint
and extracted twice by hot purified water (boiling water
bath under reflux). Water extract was cooled, filtered
244
through a cotton filter into a 50 ml volumetric flask. The
solution volume was brought to initial volume with
purified water and thoroughly mixed. To remove
polyphenol compounds the extract was passed through a
10 mm column in diameter that was filled with
aluminum oxide (Al2O3) for chromatography,
Brockmann Activity II, basic, CAS Number 1344-28-1
(extract 1.2 A).
Aliquot of HMT GW (1) or GN (2) was placed in an
evaporation bowl, then was evaporated at a boiling water
bath up to dryness. Resulting solid residue was dissolved
in purified water. For removing the polyphenol
compounds the solution was subjected to a similar
procedure as extract 1.2 A (1.2 extract B).
Qualitative reaction for free CARB:
To confirm free CARB in the snowdrop samples
qualitative reaction for reducing sugars was performed
according Bertrand method with Fehling reagent26.
1.2 A and 1.2 B extracts, Fehling's solution (reagent 1
and reagent 2 mixture) were added in test-tube and was
heated at boiling water bath for 3 minutes.
Qualitative reaction for bounded CARB:
Determination for bounded sugars was performed as in
“Qualitative reaction for free CARB” after acid
hydrolysis of water extracts.
CARB hydrolysis (obtaining reducing CARB):
To 1.2 A and 1.2 B extracts the same volume of sulfuric
acid was added and heated at a boiling water bath for 5-7
minutes, after this procedure was cooled. Thus, the
obtained extract 1A', 2A' – from CHD, 1B ', 2B' - from
HMT GW or GN respectively.
The same volume of Fehling's solution was added to the
resulting 1.2 A' and B 1.2' extracts.
TLC was performed using “Sorbfil” ready-TLC plates
HPTLC-A-UV 10×15 (Sorbfil Imid Ltd, Russia).
Several mobile phases were tried for chromatography
procedure: ethanol (70%, 96%), purified water,
isopropanol - water (4: 1), isopropanol - water (3: 1)
were among them27. The camera saturation time with
solvent vapors was 45 minutes.
7µl of a 1 A extract was applied to the 1st point, a
similar amount of 2A extract – to the 2nd point, extract
1B - to the third point, extract 2B – to the 4th point, 3
µl a 5% Fru solution – to the 5-th point at the start line
of the 1st chromatographic plate (CP-1).
7µl of a 1 A' was applied to the 1st point, a similar
amount of 2 A' extract – to the 2nd point, 1 B' extract to
the third point, B 2 ' extract - to the 4-th point, 3µl of
 Research J. Pharm. and Tech. 13(1): January 2020

5% Glu solution – to the 5-th point at the starting line of solvent was added until the solution reaches the mark
the second chromatographic plate (CP-2). and stirred. The solution was allowed to settle for 24
hours and the clear resulting liquid was decanted from
After passing 14 cm through the front plate the CPs were the precipitate (reagent for analysis).
removed, CPs were dried at air and treated with CARB
detecting agents: anthrone reagent (AR), resorcinol Preparation of 2,4,6-trinitrophenol (TNP) solution 1%. 1
reagent (RR) and diphenylamine reagent (DR)27. CP-1 g of TNP was dissolved in 90ml of purified water in a
was submerged in AR No 1, then air dried and sprayed 100ml volumetric flask under heating, then same solvent
with AR No 2, heated at 108°C for 6 minutes. was added until the solution reaches the mark and
stirred.
Reagents for CARB TLC.
A solution of the Fru standard sample (SS). 0.1g of Fru Preparation of the sodium carbonate solution 20%. 20g
was placed in a 100ml volumetric flask, dissolved in 85 of anhydrous sodium carbonate was dissolved in purified
ml of purified water, then same solvent was added until water in a 100m volumetric flask, then same solvent was
the solution reaches the mark and mixed. added until the solution reaches the mark and stirred.

A solution of the Glu SS. 0.1grams of Glu was placed in
a 100 ml volumetric flask, dissolved in 85 ml of purified
water, then same solvent was added until the solution
reaches the mark and mixed.
Anthrone reagent (AR) was consisted of reagent No 1
and reagent No 2, which was applied to CP sequentially,
reagent No 1 with dipping, then reagent №2 with
spraying by an atomizer.
Reagent No 1: 0.9g anthrone (CAS Number 90-44-8)
was dissolved in 30ml of hot acetic acid. The reagent
used immediately after preparation. Reagent No 2: 60ml
of 96% ethanol, 9ml of phosphoric acid (ρ = 1.7) and 3
ml of purified water were added to 100 ml flask and
stirred.
Resorcinol reagent (RR). To 54ml of 2 M hydrochloric
acid solution was added 6ml of 96% ethanol and stirred.
0.6g of resorcinol (CAS №108-46-3) was dissolved in
the resulting solution.
Total carbohydrates (TCARB) determination in
terms of Fru in CHD:
Quantification of TCARB in the snowdrops CHD in
terms of Fru was performed according to the
pharmacopoeial monograph “Burdock roots”28. The
extraction was performed with hot purified water in a
boiling water bath under reflux, the resulting water
extract was purified from ballast and related undesirable
substances. 30% hydrochloric acid, 0.1% resorcinol
solution were added to this solution and heated by a
water bath. Absorbance of the interaction products
(CARB with resorcinol) was determined at a wavelength
of 482 nm. Absorbance of of Fru SS was determined
under the same conditions.
TCARB content (percentage) in terms of Fru and
absolutely dry CHD (X) calculated by the formula:

Diphenylamine reagent (DR). 12ml of n-butanol was where D- absorbance of the sample solution,
mixed with 12ml of methanol, 1.2g of trichloroacetic - specific absorption (Abs) index of the interaction
acid; then 0.48 g of diphenylamine (CAS № 122-39-4) products (Fru with resorcinol) in an acidic solution was
was dissolved in resulting solution. equal to 298,
m - mass of the CHD, grams
Reagents for TCARB determination.: W - loss on drying of CHD, %.
Preparation of the Glu reference solution (RS). 50µg
(accurately weighed) of Glu was dissolved in purified TCARB determination in terms of Fru in HMT:.
water in a 25ml volumetric flask, then same solvent was Quantification of TCARB in the snowdrops HMT in
added until the solution reaches the mark and stirred. terms of Fru was performed according to the
Array with Glu, based on anhydrous Glu in grams (m1) pharmacopoeial monograph “Burdock roots” . HMT
28

is calculated by the formula: evaporated, purified water was added, ballast and related
undesirable substances were removed from water extract
water. 30% hydrochloric acid, 0.1% resorcinol solution
were added to this solution and heated by a water bath.
where: m - Glu mass, g; W - moisture%. Absorbance of the interaction products (CARB with
Preparation of sodium hydroxide solution 40%. 40g of resorcinol) was determined at a wavelength of 482 nm.
sodium hydroxide was dissolved in purified water in a Absorbance of of Fru SS was determined under the same
100ml volumetric flask, after was cooled, then same conditions.
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 Research J. Pharm. and Tech. 13(1): January 2020

TCARB content (percentage) in terms of Fru (X)

calculated by the formula:

where D1; D0; m1; m0; W – similar to other one’s

described above.
where D - absorbance of the sample solution,
- specific Abs index of the interaction products (Fru RESULTS AND DISCUSSION:
with resorcinol) in an acidic solution was equal to 298, Free reducing CARB presence was proved in a test
a – HMT volume, ml. extract. The qualitative reaction was carried out with
Fehling reagent: the orange-red precipitate – cuprous
TCARB determination in terms of Glu in CHD: oxide (copper (I) oxide) sedimentated. The Bertrand test
Quantification of TCARB in snowdrops CHD in terms was positive both for the CHD extract (1.2 A) and HMT
of Glu was performed according to pharmacopoeial (1.2 B).
monograph “Flax seeds” 29. The CHD was extracted with
hot purified water at a boiling water bath under reflux, There was loss of orange-red precipitate in all 4 extracts
then hydrolysis with concentrated hydrochloric acid was after the hydrolysis when heated. The volume of
conducted. After neutralization procedure with 40% precipitate in extracts A 1.2 'and 1.2 D' exceeded the
sodium hydroxide, ballast and related undesirable other ones obtained with 1.2 A and 1.2 B extracts.
substances were removed from extract. Absorbance of
the interaction products of CARB with TNP (1% Thus, at the study preliminary stage the presence of free
solution) in an alkaline solution (sodium hydrogen and bound CARB in CHD and HMT of G. nivalis and G.
carbonate) was determined at a wavelength of 470 nm woronowii was approved.
after heating in a water bath in the resulting hydrolysate.
Absorbance of Glu SS was determined under similar The obtained data was confirmed by TLC method. AR is
conditions. specific reagent for keto- and pentoses. Ketopentoses
possess purple color, ketohexoses – yellow color,
TCARB content (percentage) in terms of Glu and ketoheptoses - yellow-orange color. The best separation
absolute dry CHD (X) is calculated by the formula: was performed with isopropanol-water (4: 1). The 1,2 A
and 1,2 B extracts possessed a yellow spot, (Rf value
coincides Fru – 0.68). CP was sprayed by RR (specific
for ketose), then heated at 90 ° C for 10 minutes, the
pink color spots were observed with similar location and
where D - absorbance of the sample solution; D - shape. The results are shown at Figure 1.
1 0
absorbance of Glu SS; m1 - mass in grams of CHD; m0 -
mass of the Glu sample of in terms anhydrous Glu in
grams; W - loss on drying of CHD,%.

TCARB determination in terms of Glu in HMT:

Quantification of TCARB in snowdrops HMT in terms
of Glu was performed according to pharmacopoeial
monograph “Flax seeds” 29. HMT was evaporated,
purified water was added, then hydrolysis with
concentrated hydrochloric acid was conducted. After
neutralization procedure with 40% sodium hydroxide,
ballast and related undesirable substances were removed
from extract. Absorbance of the interaction products of
CARB with TNP (1% solution) in an alkaline solution
(sodium hydrogen carbonate) was determined at a
wavelength of 470 nm after heating in a water bath in the
resulting hydrolysate. Absorbance of Glu SS was
Figure 1: TLC free CARB in CHD and HMT of GW and GN. 1 -
determined under similar conditions.
5% Fru solution; 2 - 1 A, an extract from CHD of GW; 3 - 1 B, an
extract from CHD of GN; 4 - 2 A, HMT of GW; 5. - 2 B, HMT of
TCARB content (percentage) in terms of Glu (X) was GN. Mobile phase: isopropanol: water (4: 1). Developer: anthrone
calculated by the formula: reagent (AR).

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 Research J. Pharm. and Tech. 13(1): January 2020

Figure 2: TLC bounded CARB in CHD and HMT of GW and GN.

Figure 3: The Abs spectra of the reaction products with resorcinol
1 - 5% Glu solution; 2 - 1 A ' an extract from CHD GW after
in the acidic environment of the standard working sample of Fru
hydrolysis; 3 - 1 B ', an extract from CHD of GN after hydrolysis;
(1), extracts from CHD of GN (2) and GW (3) HMT of GN (4) and
4 - 2 A' HMT of GW after hydrolysis; 5 - B 2', HMT of GN after
GW (5).
hydrolysis. Mobile phase: isopropanol: water (4: 1) Developer:
diphenylamine reagent (DR).
The maximum optical density of the interaction products
Free CARB are presented in CHD and HMT of GW and of Fru with resorcinol in an acidic media was achieved
GN. CP-2 was sprayed by DR (specific for aldose, after 20 minutes when heated on a water bath at 80 ° C
aldohexoses gave a brown color, aldopentoses – purple and did not change in 3.5 hours after followed heating.
color), 1,2A' and 1,2 B' extracts possessed a gray-brown Specific Abs index of the reaction products of Fru with
color spot (with Rf value of Glu – 0.61). Figure 2 shows resorcinol in an acidic solution at a wavelength of 483
that the free CARB contained in the CHD and HMT of nm is equal to 298.
two snowdrops species.
In order to establish the completeness of CARB
TCARB determination in terms of Fru: extraction from snowdrops CHD studied the effect of the
One method of quantitative CARB determination is factors: the type of extragent, ratio of CHD and
standardized methodology. It is based on the extragent, temperature, extraction time, extraction
measurement of the optical density of colored interaction multiplicity.
products of Fru with resorcinol after heating in an acid
Table 1: Relation between completeness of CARB extraction and
media 30, 31, 32. extraction conditions in snowdrops CHD.
Total carbohydrate content in terms
The Abs spectra of the reaction products and CHD Extraction conditions of Fru,%
extracts and HMT with resorcinol in an acidic solution, CHD of GW CHD of GN
Extragent type
were characterized by two distinct Abs maxima at
Purified water 0.34 0.41
wavelengths of 420 ± 2; 482 ± 2 nm. The 482 ± 2 nm 25 ethanol 0.34 0.41
was used as an analytical wavelength. The Abs maxima 40 % ethanol 0.33 0.41
of the reaction products of Fru with resorcinol were at 70 % ethanol 0.25 0.32
420 ± 2; 482 ± 2 nm. It allows to recommend the Fru as 96% ethanol 0.14 0.21
CHD-extragent ratio
a standard sample in the calculation of the CARB
1:10 0.33 0.39
amount in the CHD and HMT of GW and GN (Figure 3). 1:20 0.34 0.41
1:30 0.34 0.41
1:50 0.34 0.40
Temperature range
Without heating 0.21 0.31
Water bath 40°C 0.28 0.35
Water bath 60°C 0.32 0.37
Water bath 80°C 0.34 0.40
Water bath 100°C 0.35 0.42
In an open fire of gas burner 0.33 0.39
Extraction time
15 0.28 0.31
30 0.35 0.40
45 0.36 0.43
60 0.35 0.42
90 0.33 0.42
120 0.32 0.40

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 Research J. Pharm. and Tech. 13(1): January 2020

Extraction multiplicity Table 2: Metrological characteristics of quantitative TCARB

1 0.29 0.37 determination in GW and GN CHD and HMT (n = 5, f = 4, P =
2 0.35 0.42 95%, T (f, P) = 2.7764)
3 0.36 0.43 Sample E, %
4 0.36 0.43 TCARB content in terms of Fru
CHD 0.36 1.0 1.00 4.47 0.01 3.5
Table 1 shows the optimal conditions for the extraction. of GW
CHD 0.43 1.1 1.06 4.74 0.01 3.1
They are: extragent – purified water; the ratio CHD and of GN
the extragent – 1:20; temperature – boiling water bath; HMT 0.46 1.1 1.03 4.59 0.01 2.8
extraction multiplicity – 3, 70 ml of purified water for 45 of GW
min, then twice 40 ml of purified water for 45 min, and HMT 0.55 1.7 1.29 5.77 0.02 2.9
of GN
40 ml of purified water for 20 min.
TCARB content in terms of Glu
The research data were used in TCARB determination of CHD 0.54 1.5 1.23 5.51 0.02 2.8
of GW
snowdrops CHD recalculation in terms of Fru, described
CHD 1.39 1.1 1.05 4.67 0.01 0.9
in research methods. of GN
HMT 0.76 1.3 1.14 5.10 0.01 1.9
TCARB determination in terms of Glu:. of GW
Another way to quantify the CARB is a technique based HMT 0.95 1.6 1.25 5.57 0.02 1.6
on the measurement of the optical density of the colored of GN
Note. n – number of repeat tests, f – number of degrees of freedom, P
interaction products of Glu with TNP in an alkaline % – confidence figure, T(f,P) – Student's coefficient, – mean value,
solution by heating 33, 34. S2 – dispersion, S – standard deviation, – the standard deviation of
the mean value, ΔX –confidence interval, E,% – relative error.
The Abs spectra of the reaction products and CHD,
HMT extracts with TNP in an alkaline solution, The maximum optical density of the interaction products
characterized by a clearly defined maximum Abs at a of Glu with TNP in the alkaline solution was achieved
wavelength of 470 ± 2 nm. The maximum Abs of the after 10 minutes of heating on a boiling water bath (100
reaction products of Glu with TNP is at the range of 470 °C) and did not change in the few hours followed
± 2 nm. It allows to recommend the Glu as a standard heating.
sample in the calculation of the CARB amount in CHD
and HMT of GW and GN (Figure 4). The optimal conditions for CARB extraction in
snowdrops CHD were developed for determining the
TCARB in terms of Fru. Experimental data proved
quantitative methodology for TCARB determination in
terms of Glu in snowdrops CHD and HMT.

CONCLUSION:
The presence of free (fructose) and bounded (glucose)
CARB in GW and GN CHD and HMT is confirmed by
qualitative reactions and TLC. Conditions for the 2
methods of quantitative spectrophotometric determination
of TCARBS in terms of fructose and glucose in CHD and
HMT of GW and GN were selected experimentally.
TCARB content in terms of fructose is established, it is
amounted 0.36 ± 0.01% in CHD of GW, 0.43 ± 0.01% in
CHD of GN, 0.46 ± 0.01% in HMT of GW, 0.55 ± 0.02%
in HMT of GN. TCARB content in terms of glucose is
established, it is amounted 0.54 ± 0.02% in CHD of GW,
1.39 ± 0.01% in CHD of GN, 0.76 ± 0.01% in HMT of
GW, 0.95 ± 0.02% in HMT of GN.

AUTHOR’ CONTRIBUTIONS:
Bokov D.O., Kulaeva I.R. contributed equally to this
work.

Figure 4: The Abs spectra of the reaction products with TNP in an CONFLICT OF INTEREST:
alkaline solution of the standard working sample Glu (1), extracts None.
from CHD of GN (2) and GW (3) HMT of GN (4) and GW (5)
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 Research J. Pharm. and Tech. 13(1): January 2020
ACKNOWLEDGMENT:
This paper was financially supported by “Russian
Academic Excellence Project 5-100” (Sechenov 18.
University). The publication has been prepared with the
support of the “RUDN University Programm 5-100”.
Author would like to thank professor, corresponding
member of Russian Academy of Sciences Irina
Aleksandrovna Samylina for her useful communications
and constant help. Also, author would like to thank 20.
professor Popov Dmitry Matveyevich for the given
technical possibilities and significant advice.
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