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PHARMACEUTICAL STANDARDIZATION
Year : 2015  |  Volume : 36  |  Issue : 2  |  Page : 188-195  

High performance thin layer chromatography fingerprinting, phytochemical and physico-chemical studies of anti-diabetic herbal extracts


University Department of Pharmaceutical Sciences, Department of Pharmacognosy and Phytochemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India

Date of Web Publication3-Feb-2016

Correspondence Address:
Dr. Prakash R Itankar
Asst. Prof., University Dept. of Pharmaceutical Sciences, Dept. of Pharmacognosy and Phytochemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Amravati Road, Nagpur - 440 033, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-8520.175546

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   Abstract 

Introduction: Herbal medicines have gained increasing popularity in the last few decades, and this global resurgence of herbal medicines increases their commercial value. However, this increasing demand has resulted in a decline in their quality, primarily due to a lack of adequate regulations pertaining to herbal medicines. Aim: To develop an optimized methodology for the standardization of herbal raw materials. Materials and Methods: The present study has been designed to examine each of the five herbal anti-diabetic drugs, Gymnema sylvester R. Br., Pterocarpus marsupium Roxburgh., Enicostema littorale Blume., Syzygium cumini (L.) Skeels. and Emblica officinalis Gaertn. The in-house extracts and marketed extracts were evaluated using physicochemical parameters, preliminary phytochemical screening, quantification of polyphenols (Folin–Ciocalteu colorimetric method) and high performance thin layer chromatography (HPTLC) fingerprint profiling with reference to marker compounds in plant extracts. Results: All the plants mainly contain polyphenolic compounds and are quantified in the range of 3.6–21.72% w/w. E. officinalis contain the highest and E. littorale contain the lowest content of polyphenol among plant extracts analyzed. HPTLC fingerprinting showed that the in-house extracts were of better quality than marketed extracts. Conclusion: The results obtained from the study could be utilized for setting limits for the reference phytoconstituents (biomarker) for the quality control and quality assurance of these anti-diabetic drugs.

Keywords: Anti-diabetic, high performance thin layer chromatography fingerprint, physicochemical parameters, standardization


How to cite this article:
Itankar PR, Sawant DB, Tauqeer M, Charde SS. High performance thin layer chromatography fingerprinting, phytochemical and physico-chemical studies of anti-diabetic herbal extracts. AYU 2015;36:188-95

How to cite this URL:
Itankar PR, Sawant DB, Tauqeer M, Charde SS. High performance thin layer chromatography fingerprinting, phytochemical and physico-chemical studies of anti-diabetic herbal extracts. AYU [serial online] 2015 [cited 2020 Oct 20];36:188-95. Available from: https://www.ayujournal.org/text.asp?2015/36/2/188/175546


   Introduction Top


Nature has been a source of medicinal agents for thousands of years, and an impressive number of modern drugs have been isolated from natural sources. Many of these isolations were based on the uses of the agents in traditional medicine.[1] According to World Health Organization (WHO), about three-quarter of the world population relies upon traditional remedies for the health care of its people. In fact, plants are the oldest friends of mankind. They not only provided food and shelter, but also served the humanity to cure different ailments.[2] The plants and their extracts are a common elements in Indian systems of medicine. Major pharmaceutical companies are currently conducting extensive research on plant materials gathered from the rain forests and other places for their potential medicinal value. The plant-based, traditional medicine systems continue to play an essential role in health care.[3] The global market for herbal medicines currently stands at over $60 billion annually.[4]

In the world, diabetes is a serious disease due to irrational food habits. Most of the hypoglycemic agents used in allopathic practice to treat diabetes mellitus are reported to have side effects in long term use.[5] Hence, there is a need to search for effective and safe drugs for these ailments. Pharmaceutical research across the world shows that natural products are potential sources of novel molecules for drug development.

The recent global resurgence in herbal medicines has led to an increase in the demand for them. Commercialization of these medicines to meet this increasing demand has resulted in a decline in their quality, primarily due to a lack of adequate regulations pertaining to this sector of medicine. The need of the hour is to evolve a systematic approach and to develop well-designed methodologies for the standardization of herbal raw materials and their formulations. Various methods of phytochemical standardization, such as preliminary phytochemical screening, fingerprint profiling and quantification of the marker compound with reference to plant extracts and polyherbal formulations are used.[6] Standardization is necessary to make sure the availability of an uniform product in all parts of the world. It assures a consistently stronger product with guaranteed constituents.[7]


   Materials and Methods Top


Plant material and market extracts

The five herbal anti-diabetic drugs and their extracts, chosen for this study were Gymnema sylvestre R. Br. - Asclepiadaceae (Madhunashini - leaves), Pterocarpus marsupium Roxb. - Fabaceae (Vijaysara - heart-wood), Enicostema littorale Blume. - Gentianaceae (Mamejaka - whole plant), Syzygium cumini (L.) Skeels. - Myrtaceae (Jambu - seeds) and Emblica officinalis Gaertn. - Euphorbiaceae (Amalaki - whole fruits). All the authenticated crude drugs and market extracts of Madhunashini, Mamejaka, Jambu were procured from Plantex Agro Products (P) Ltd., Vashi, Navi Mumbai. Amalaki and Vijaysara market extracts were procured from Kisalaya herbals, Ratlam Kothi, Indore and Amruta Herbals Private Ltd., Sanwer Road, Indore respectively.

Chemicals and standards

Gallic acid, gymnemagenin (Sigma-Aldrich Co. LLC.), pterostilbine (Wuxi Cima Science Co. Ltd.), vanillin, Folin–Ciocalteu phenol reagent, ortho-phosphoric acid, sodium acetate, formic acid (Merck chemicals Ltd.), anisaldehyde, acetic acid, anhydrous sodium carbonate, sulfuric acid, methanol, ethanol, acetone, ethyl acetate, acetonitrile (SD Fine-Chem. Ltd.).

Preparation of in-house plant extracts

About 100 g of well-dried crude powder of Amalaki and Jambu were macerated with water while Madhunashini, Vijaysara and Mamejaka were macerated with hydro-ethanolic (1:1 v/v) solvents respectively. Each extract was then concentrated using rotary vacuum evaporator (25°C) and finally lyophilized (Maro Scientific Works, 10A/UA).

Physicochemical properties

Physical characteristics such as extractive values, moisture content, ash values, were determined according to the standard test procedures.[8],[9],[10],[11]

Preliminary phytochemical screening of extracts

All plant extracts were subjected to phytochemical screening for checking presence of chemical constituents such as alkaloids, tannins, flavonoids, saponins, sterols, proteins and carbohydrates.[12]

Total polyphenol estimation of extracts

Total polyphenol content was measured using Folin–Ciocalteu colorimetric method, in which 2 ml of test and standard sample was taken in 25 ml volumetric flask, 10 ml of demineralized water was added, then mixed with 2 ml of diluted Folin–Ciocalteu's phenol reagent (1:5 with distill water; 0.4 N). Then, volume was adjusted to 25 ml with 29% sodium carbonate solution. The reaction was kept in dark for 30 min and the absorbance was read at 760 nm (Shimadzu UV–VIS spectrophotometer 1600) against the corresponding test and standard blanks prepared in the same way without the extract. Gallic acid was used as a reference standard.

Standard solution was prepared by accurately weighing about 50 mg of gallic acid and adding it to 100 ml of volumetric flask. Made up the volume with demineralized water. Diluted 2 ml of resulting solution to 25 ml with demineralized water. Used the resulting solution as a standard solution.[13],[14]

Test solution was prepared by accurately weighing extract and adding to 100 ml volumetric flask. Added 80 ml of demineralized water and heated for 60 min on a water bath at 100°C. Cooled and made up the volume with demineralized water. Filtered the content of volumetric flask and diluted 5 ml of resulting the solution to 25 ml with demineralized water. Used the resultant solution as the test solution.

Total polyphenol as gallic acid (% w/w) = AT/AS × WS/DS × DT/WT × P/100 × 100

Where, AT = Absorbance of test solution at 760 nm; AS = Absorbance of standard solution at 760 nm; WS = Weight of standard; DS = Dilution of standard solution; DT = Dilution of test solution; WT = Weight of test extract;P = Potency of standard.

Development of high performance thin-layer chromatography (HPTLC) methods for extracts

Qualitative estimation of biologically active compounds like gallic acid from Amalaki and Jambu, gymnemagenin from Madhunashini, and pterostilbine from Vijaysara extract was performed by using HPTLC (CAMAG Linomat V applicator and CAMAG TLC SCANNER-III equipped with Win-CAT software).[15],[16]

Preparation of test solution

Accurately weighed 100 mg extracts were dissolved in 15 ml methanol and sonicated for 10 min. It was then diluted with methanol up to 20 ml (5 mg/ml). The solution was filtered through Whatman filter paper no. 1.

Preparation of standard solution

Accurately weighed 5 mg of standard was dissolved in 5 ml methanol, sonicated and diluted 1 ml of this solution to 10 ml with methanol (100 μg/ml).

Selection of mobile phase

It is a fact that mobile phase optimization is among the important steps that affect the quality of a separation in TLC method development. The results show the optimized solvent system for each extract.

Chromatographic condition

The samples were spotted in the form of bands, width 6 mm with a Camag 100 µl sample syringe (Hamilton, Bonaduz, Switzerland) on silica gel precoated aluminum plate 60 F254 plates, (20 cm × 10 cm with 250 μm thickness; E. Merck, Darmstadt, Germany) using a Camag Linomat V (Switzerland) sample applicator. The plates were developed using optimized mobile phase in twin trough development chamber. The plates were removed, dried at 105°C and sprayed with spraying reagents (Anisaldehyde: Sulphuric acid solution for Madhunashini; vanillin: Sulphuric acid solution for Vijaysara and 5% ferric chloride solution for Amalaki, Jambu and Mamejaka. Madhunashini (500 nm), Vijaysara plates (550 nm) were scanned at visible light and Amalaki, Jambu and Mamejaka were scanned under UV-254 nm.


   Results and Discussion Top


Physicochemical properties

Physicochemical properties play an important role in the evaluation of crude drugs. The less extractive value indicates addition of exhausted material, adulteration or incorrect processing during drying or storage. The extractive value also signifies the presence of chemical constituents extracted by a specific solvent. All the individual drugs were found to have water-soluble extractive and alcohol-soluble extractive in the range 18.90–23.04% and 12.20–23.18% w/w, respectively. Moisture is one of the major factors responsible for the deterioration of the crude drugs and formulations. Low moisture content is always desirable for higher stability of drugs. Moisture contents of the individual drug were found in the range of 1.35–5.39% w/w. A high ash value is indicative of contamination or carelessness in preparing the drug or drug combinations for marketing. All the individual drugs were found to have total ash and acid insoluble ash values in the range of 3.06–12.59% and 0.85–2.04% w/w, respectively [Table 1].
Table 1: Physicochemical parameters of crude drugs

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Percent yield of in-house plant extract

The extraction of crude material was carried out with appropriate solvent. Aqueous extracts of Amalaki and Jambu were prepared and hydro-alcoholic extracts of Madhunashini, Vijaysara and Mamejaka were prepared [Table 2].
Table 2: Extraction details and percent yield of in-house plant extract

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Preliminary phytochemical screening of extracts

In the preliminary phytochemical screening, Madhunashini, Mamejaka and Jambu extracts were found to contain alkaloids, tannins, flavonoids, saponins, sterols, proteins and carbohydrates. The Amalaki and Vijaysara contain tannins, flavonoids, saponins, sterols, proteins and carbohydrates. All the plants mainly found to contain polyphenolic compounds like tannins and flavonoids in their extracts.

Total polyphenol estimation of extracts

All the in-house extracts and market extracts were subjected to total polyphenol content estimation. It was found that polyphenol content of the extract was in the range of 3.6–21.72%. Amalaki contains the highest and Mamejaka contain the lowest content of polyphenol among the five plant extracts [Table 3].
Table 3: Total polyphenol content of extracts

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Development of HPTLC methods for extract

The standards like, gymnemagenin for Madhunashini [Figure 1]a and [Table 4], pterostilbine for Vijaysara [Figure 2]a and [Table 5], gallic acid for Amalaki and Jambu [Figure 3]a and [Figure 4]a [Table 6] and [Table 7] were used for checking quality of extracts. As per the HPTLC observations, the market extract of Madhunashini 9 peaks [Figure 1]c and [Table 4] and Mamejaka 11 peaks [Figure 5]b and [Table 8] shown more number of peaks but as per the height and area in comparison with marker compounds [Figure 1]b,[Figure 4]b the in-house extracts of all the plants [Figure 1]d,[Figure 4]d, [Figure 5]c were found to be of better quality than market extracts [Figure 1]c,[Figure 5]c. Typical HPTLC fingerprinting of particular plant species will not only help in the identification and quality control of a particular species, but also provide basic information useful for the isolation, purification, characterization, and identification of marker chemical compounds of the species and also employed for taxonomic categorization. Thus, the present study will provide sufficient information about therapeutic efficacy of the extracts and also in the identification, standardization, and quality control of medicinal plant.
Figure 1: High performance thin layer chromatography study of Madhunashini (Gymnema sylvestre); (a-d) represent as Madhunashini plate at visible light, fingerprint of standard gymnemagenin, Madhunashini market extracts (new extract) and Madhunashini in-house extract at 500 nm respectively

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Table 4: Rf values by densitometric scan of Madhunashini at 500 nm

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Figure 2: High performance thin layer chromatography study of Vijaysara (Pterocarpus marsupium); (a-d) represent as Vijaysara plate at visible light, fingerprint of standard pterostilbine, Vijaysara market extracts (new extract) and Vijaysara in-house extract at 550 nm respectively

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Figure 3: High performance thin layer chromatography study of Amalaki (Emblica officinalis); (a-d) represent as Amalaki plate, fingerprint of standard gallic acid, Amalaki Market extracts (new extract) and Amalaki in-house extract at 254 nm respectively

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Figure 4: High performance thin layer chromatography study of Jambu (Syzygium cumini); (a-d) represent as Jambu plate, fingerprint of standard gallic acid, Jambu market extracts (new extract) and Jambu in-house extract at 254 nm respectively

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Table 5: Rf values by densitometric scan of Vijaysara at 550 nm

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Figure 5: High performance thin layer chromatography study of Mamejaka (Enicostema littorale); (a-c) represent as Mamejaka plate, fingerprint of Mamejaka market extract and Mamejaka in-house extract at 254 nm respectively

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Table 6: Rf values by densitometric scan of Amalaki at 254 nm

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Table 7: Rf values by densitometric scan of Jambu at 254 nm

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Table 8: Rf values by densitometric scan of Mamejaka at 254 nm

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   Conclusion Top


The results obtained from the study could be utilized for the preliminary quality control and quality assurance of these anti-diabetic drugs. This component of research may also be useful in estimating the precise shelf life of the respective extract or formulations having these crude drugs as constituents.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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