INTRODUCTION:

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. 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.

At the same time added sugars are responsible for 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 and HD CARB composition. Notoriously CARB are 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 evaluated⁸. 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.

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 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 5% Glu solution – to the 5-th point at the starting line of the second chromatographic plate (CP-2).

After passing 14 cm through the front plate the CPs were removed, CPs were dried at air and treated with CARB detecting agents: anthrone reagent (AR), resorcinol reagent (RR) and diphenylamine reagent (DR)²⁷. CP-1 was submerged in AR No 1, then air dried and sprayed with AR No 2, heated at 108°C for 6 minutes.

Reagents for CARB TLC.

A solution of the Fru standard sample (SS). 0.1g of Fru was placed in a 100ml volumetric flask, dissolved in 85 ml of purified water, then same solvent was added until the solution reaches the mark and mixed.

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.

Diphenylamine reagent (DR). 12ml of n-butanol was mixed with 12ml of methanol, 1.2g of trichloroacetic acid; then 0.48 g of diphenylamine (CAS № 122-39-4) was dissolved in resulting solution.

Reagents for TCARB determination.:

Preparation of the Glu reference solution (RS). 50µg (accurately weighed) of Glu was dissolved in purified water in a 25ml volumetric flask, then same solvent was added until the solution reaches the mark and stirred. Array with Glu, based on anhydrous Glu in grams (m₁) is calculated by the formula:

where: m - Glu mass, g; W - moisture%.

Preparation of sodium hydroxide solution 40%. 40g of sodium hydroxide was dissolved in purified water in a 100ml volumetric flask, after was cooled, then same solvent was added until the solution reaches the mark and stirred. The solution was allowed to settle for 24 hours and the clear resulting liquid was decanted from the precipitate (reagent for analysis).

Preparation of 2,4,6-trinitrophenol (TNP) solution 1%. 1 g of TNP was dissolved in 90ml of purified water in a 100ml volumetric flask under heating, then same solvent was added until the solution reaches the mark and stirred.

Preparation of the sodium carbonate solution 20%. 20g of anhydrous sodium carbonate was dissolved in purified water in a 100m volumetric flask, then same solvent was added until the solution reaches the mark and stirred.

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:

where D- absorbance of the sample solution,

- specific absorption (Abs) index of the interaction products (Fru with resorcinol) in an acidic solution was equal to 298,

m - mass of the CHD, grams

W - loss on drying of CHD, %.

TCARB determination in terms of Fru in HMT:.

Quantification of TCARB in the snowdrops HMT in terms of Fru was performed according to the pharmacopoeial monograph “Burdock roots”²⁸. HMT 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. 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 (X) calculated by the formula:

where D - absorbance of the sample solution,

- specific Abs index of the interaction products (Fru with resorcinol) in an acidic solution was equal to 298,

a – HMT volume, ml.

TCARB determination in terms of Glu in CHD:

Quantification of TCARB in snowdrops CHD in terms of Glu was performed according to pharmacopoeial monograph “Flax seeds” ²⁹. The CHD was extracted with hot purified water at a boiling water bath under reflux, 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 determined under similar conditions.

TCARB content (percentage) in terms of Glu and absolute dry CHD (X) is calculated by the formula:

where D₁ - absorbance of the sample solution; D₀ - 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 determined under similar conditions.

TCARB content (percentage) in terms of Glu (X) was calculated by the formula:

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

RESULTS AND DISCUSSION:

Free reducing CARB presence was proved in a test extract. The qualitative reaction was carried out with Fehling reagent: the orange-red precipitate – cuprous oxide (copper (I) oxide) sedimentated. The Bertrand test was positive both for the CHD extract (1.2 A) and HMT (1.2 B).

There was loss of orange-red precipitate in all 4 extracts after the hydrolysis when heated. The volume of precipitate in extracts A 1.2 'and 1.2 D' exceeded the other ones obtained with 1.2 A and 1.2 B extracts.

Thus, at the study preliminary stage the presence of free and bound CARB in CHD and HMT of G. nivalis and G. woronowii was approved.

The obtained data was confirmed by TLC method. AR is specific reagent for keto- and pentoses. Ketopentoses possess purple color, ketohexoses – yellow color, ketoheptoses - yellow-orange color. The best separation 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 shape. The results are shown at Figure 1.

Figure 1: TLC free CARB in CHD and HMT of GW and GN. 1 - 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 GN. Mobile phase: isopropanol: water (4: 1). Developer: anthrone reagent (AR).

Figure 2: TLC bounded CARB in CHD and HMT of GW and GN. 1 - 5% Glu solution; 2 - 1 A ' an extract from CHD GW after hydrolysis; 3 - 1 B ', an extract from CHD of GN after hydrolysis; 4 - 2 A' HMT of GW after hydrolysis; 5 - B 2', HMT of GN after hydrolysis. Mobile phase: isopropanol: water (4: 1) Developer: diphenylamine reagent (DR).

Free CARB are presented in CHD and HMT of GW and GN. CP-2 was sprayed by DR (specific for aldose, aldohexoses gave a brown color, aldopentoses – purple color), 1,2A' and 1,2 B' extracts possessed a gray-brown color spot (with Rf value of Glu – 0.61). Figure 2 shows that the free CARB contained in the CHD and HMT of two snowdrops species.

TCARB determination in terms of Fru:

One method of quantitative CARB determination is standardized methodology. It is based on the measurement of the optical density of colored interaction products of Fru with resorcinol after heatingin an acid media ³⁰^,³¹^,³².

The Abs spectra of the reaction products and CHD extracts and HMT with resorcinol in an acidic solution, were characterized by two distinct Abs maxima at wavelengths of 420 ± 2; 482 ± 2 nm. The 482 ± 2 nm was used as an analytical wavelength. The Abs maxima of the reaction products of Fru with resorcinol were at 420 ± 2; 482 ± 2 nm. It allows to recommend the Fru as a standard sample in the calculation of the CARB amount in the CHD and HMT of GW and GN (Figure 3).

Figure 3: The Abs spectra of the reaction products with resorcinol in the acidic environment of the standard working sample of Fru (1), extracts from CHD of GN (2) and GW (3) HMT of GN (4) and GW (5).

The maximum optical density of the interaction products of Fru with resorcinol in an acidic media was achieved after 20 minutes when heated on a water bath at 80 ° C and did not change in 3.5 hours after followed heating. Specific Abs index of the reaction products of Fru with resorcinol in an acidic solution at a wavelength of 483 nm is equal to 298.

In order to establish the completeness of CARB extraction from snowdrops CHD studied the effect of the factors: the type of extragent, ratio of CHD and extragent, temperature, extraction time, extraction multiplicity.

Table 1: Relation between completeness of CARB extraction and extraction conditions in snowdrops CHD.

Extraction conditions	Total carbohydrate content in terms of Fru,%
Extraction conditions	CHD of GW	CHD of GN
Extragent type
Purified water	0.34	0.41
25 ethanol	0.34	0.41
40 % ethanol	0.33	0.41
70 % ethanol	0.25	0.32
96% ethanol	0.14	0.21
CHD-extragent ratio
1:10	0.33	0.39
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
Extraction multiplicity
1	0.29	0.37
2	0.35	0.42
3	0.36	0.43
4	0.36	0.43

Table 1 shows the optimal conditions for the extraction. They are: extragent – purified water; the ratio CHD and the extragent – 1:20; temperature – boiling water bath; extraction multiplicity – 3, 70 ml of purified water for 45 min, then twice 40 ml of purified water for 45 min, and 40 ml of purified water for 20 min.

The research data were used in TCARB determination of snowdrops CHD recalculation in terms of Fru, described in research methods.

TCARB determination in terms of Glu:.

Another way to quantify the CARB is a technique based on the measurement of the optical density of the colored interaction products of Glu with TNP in an alkaline solution by heating ³³^,³⁴.

The Abs spectra of the reaction products and CHD, HMT extracts with TNP in an alkaline solution, characterized by a clearly defined maximum Abs at a wavelength of 470 ± 2 nm. The maximum Abs of the reaction products of Glu with TNP is at the range of 470 ± 2 nm. It allows to recommend the Glu as a standard sample in the calculation of the CARB amount in CHD and HMT of GW and GN (Figure 4).

Figure 4: The Abs spectra of the reaction products with TNP in an alkaline solution of the standard working sample Glu (1), extracts from CHD of GN (2) and GW (3) HMT of GN (4) and GW (5)

Table 2: Metrological characteristics of quantitative TCARB determination in GW and GN CHD and HMT (n = 5, f = 4, P = 95%, T (f, P) = 2.7764)

Sample						E, %
TCARB content in terms of Fru
CHD of GW	0.36	1.0	1.00	4.47	0.01	3.5
CHD of GN	0.43	1.1	1.06	4.74	0.01	3.1
HMT of GW	0.46	1.1	1.03	4.59	0.01	2.8
HMT of GN	0.55	1.7	1.29	5.77	0.02	2.9
TCARB content in terms of Glu
CHD of GW	0.54	1.5	1.23	5.51	0.02	2.8
CHD of GN	1.39	1.1	1.05	4.67	0.01	0.9
HMT of GW	0.76	1.3	1.14	5.10	0.01	1.9
HMT of GN	0.95	1.6	1.25	5.57	0.02	1.6

Note. n – number of repeat tests, f – number of degrees of freedom, P % – confidence figure, T(f,P) – Student's coefficient, – mean value, S² – dispersion, S – standard deviation, – the standard deviation of the mean value, ΔX –confidence interval, E,% – relative error.

The maximum optical density of the interaction products of Glu with TNP in the alkaline solution was achieved after 10 minutes of heating on a boiling water bath (100 °C) and did not change in the few hours followed heating.

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.

CONFLICT OF INTEREST:

None.

ACKNOWLEDGMENT:

This paper was financially supported by “Russian Academic Excellence Project 5-100” (Sechenov 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 professor Popov Dmitry Matveyevich for the given technical possibilities and significant advice.

REFERENCES:

1. Popov DM, Levitskaya TG. Carbohydrates of Callisia fragrans. Farmaciya (Pharmacy). 2011; 8: 20-22 [in Russian].

2. Popova TS, Tereshina NS. Carbohydrates in the blackcurrant (Ribes nigrum L.) buds and leaves. Farmaciya (Pharmacy). 2013; 8: 10-13 [in Russian].

3. Naumova OA, Popov DM. Qualitative and quantitative determination of carbohydrates in fruits of Phellodendron amurense Rupt. Questions of Biological, Medical and Pharmaceutical Chemistry. 2011; 9 (8): 29-32 [in Russian].

4. Popov DM, Zarubina NV. Comparative quantitative and qualitative analysis of carbohydrates in blossoms and leaves of Tilia cordata. The development and registration of drugs. 2013; 3 (3): 50-53 [in Russian].

5. Kretovich VL. Phytochemistry. 2nd ed., Rev. and add. Moscow: “Higher School”. 1986, 503 p [in Russian].

6. Kumar SV, Praveen D. Sugar and Chronic Diseases. Research Journal of Pharmacy and Technology. 2016; 9(6): 650-654.

7. Dawson R., Elliot D. Elliot W. Handbook of biochemist. Moscow: “Mir”. 1991, 396

8. Artyushenko Z.T. Amaryllidaceae of USSR. Morphology, systematics and use. USSR Academy of Sciences. Nerd. Inst them. V.L. Komarov. Leningrad: “Science”. 1970, 41-83 [in Russian].

9. Radha R, Sivakumar T, Arokiyaraj S. Pharmacognostical evaluation of Plumeria alba Linn. Research Journal of Pharmacy and Technology. 2008; 1(4): 496-501.

10. Saravanan J, Shariff WR, Joshi NH, Varatharajan R, Joshi VG, Karigar AA. Preliminary pharmacognostical and phytochemical studies of leaves of Hemigraphis colorata. Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(1): 15-17.

11. Shah UD, Shah MB, Saluja AK. A Pharmacognostical study on Albizzia lebbeck bark. Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(3): 241-245.

12. Khan W, Mujum A, Shaikh T, Katekar SM, Tambe R, Rub RA. Pharmacognostic and preliminary phytochemical investigation of Salvadora persica Linn (Salvadoraceae). Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(4): 319-323.

13. Kumar PR, Vaidhyalingam V. Pharmacognostical and preliminary phytochemical studies on the aerial parts of Rubus racemosus (Roxb). Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(5): 381-385.

14. Rungsung W, Dutta S, Mondal DN, Hazra J. Pharmacognostical characterization on the rhizome of Ginger. Research Journal of Pharmacognosy and Phytochemistry. 2014; 6(2): 88-91.

15. Pawar KP, Bhitre MJ, Kalamkar PV, Kale MK. Pharmacognostical studies on leaves of Allamanda cathartica with detail physicochemical and phytochemical evaluation. Research Journal of Pharmacognosy and Phytochemistry. 2015; 7(2): 69-72.

16. Prasad MS, Madhu CH, Venkateshwalu G, Sabath M. Quantitative evaluation of carbohydrate levels in different natural foodstuffs by UV-visible spectrophometer. Asian Journal of Pharmaceutical Analysis. 2012; 2(1): 10-11.

17. Madhu C, Swapna J, Spandana RS, Niharika I, Raj KR, Kalyan B, Prathyusha T. Quantitative evaluation of carbohydrate levels in seeds for home use by UV-visible spectrophotometer. Asian Journal of Pharmaceutical Analysis. 2012; 2(4): 104-105.

18. Asha VS, Madhu C, Mannem K, Kumar VP. Quantitative Evaluation of Carbohydrate Levels in Seeds for Home Use by UV-Visible Spectrophotometer. Asian Journal of Pharmaceutical Analysis. 2012; 2(3): 71-72.

19. Madhu C, Swapna J, Spandana RS, Niharika I, Raj KR, Kalyan B, Rajarajeshwari M, Prathyusha T. Quantitative evaluation of carbohydrate levels in seeds for home use by UV-visible Spectrophotometer. Asian Journal of Pharmacy and Technology. 2012; 2 (3): 110-111.

20. Mannem K, Madhu C, Asha VS, Kumar VP. Quantitative evaluation of carbohydrate levels in green leafy vegetables for home use by UV-visible spectrophotometer. Asian Journal of Pharmaceutical Analysis. 2012; 2(3): 79-80.

21. Kumar VP, Madhu C, Mannem K, Asha VS, Rao AS, Prasad MS. Quantitative evaluation of carbohydrate levels in fruits by UV-visible spectrophotometer. Asian Journal of Pharmacy and Technology. 2012; 2(3): 99-100.

22. Garway SD, Garway DG, Gaikwad YT, Azad RM, Dhokey YB, Patel SN, Pandya GH. (2012). A Study of chemical compositional characteristics of Citrus fruits for D-limonene, organic acids, minerals and sugars. Asian Journal of Research in Chemistry. 2012; 5(2): 299-304.

23. Phale MD, Korgaonkar D. Current advance analytical techniques: a review. Asian Journal of Research in Chemistry. 2009; 2(3): 235-238.

24. Eslavath R, Harikrishna V, Kosuru N, Venkateshwarlu G, Sabat M, Kanakaiah K. Phytochemical screening and TLC, UV-spectrophotometer study of Bougainvillea glabra. Asian Journal of Pharmaceutical Analysis. 2013; 3(3): 83-85.

25. Pharmacopoeial monograph of State Pharmacopoeia of Russian Federation XIII edition «Homeopathic mother tinctures» [in Russian].

26. Kumar C, Mythily R, Venkatachalapathy R, Chandraju S. Bio-mimic conversion of Maida (polysaccharides) to reducing sugars by acid hydrolysis and its estimation using standard methods. International Food Research Journal. 2014; 21(2): 523-526.