|RESEARCH IN PROGRESS
|Ahead of print publication
Phytochemical screening and high-performance thin-layer chromatography fingerprint profile of three species of leucas (Lamiaceae)
K Geethika, P Sunojkumar
Department of Botany, University of Calicut, Malappuram, Kerala, India
Department of Botany, Angiosperm Taxonomy and Floristic Division, University of Calicut, Malappuram - 673 635, Kerala
Source of Support: None, Conflict of Interest: None
Background: The members of genus Leucas possess high economic potential. As medicinal herbs these were well known as 'Droṇapuhṣpī' in Ayurveda literature. The present study aims to carry out the phytochemical screening as well as the HPTLC fingerprint profiling of three species of Leucas. Materials and Methods: Aqueous, methanol, ethanol and chloroform extracts of each plant were subjected to qualitative phytochemical screening. The total phenols, flavonoids and tannins were quantified in the methanolic extract by standard spectrophotometric methods. HPTLC method for the separation of the active constituents in extracts has been developed and TLC of the methanolic extracts on silica gel pre-coated aluminum plates of Merck by automatic TLC applicator and using solvent system Toluene: ethyl acetate:7:3 was performed. Results: Preliminary phytochemical screening of different extracts showed the presence of different phytoconstituents such as flavonoids, terpenes, tannins, carbohydrates, glycosides, phenolic compounds, proteins and amino acids. Among all the three extracts studied, L. ciliata had the highest concentration of phenolics (34.5 ± 0.35), flavonoids (48.25 ± 1.06) and tannins (6.45 ± 0.5). A HPTLC fingerprint scanned at 550 nm for methanol leaf extracts revealed 12 peaks for L. stelligera, 15 peaks for L. eriostoma and 13 peaks for L. ciliata with Rf values in the range of 0.06 to 0.98.
Keywords: Extracts, high-performance thin-layer chromatography fingerprinting, Leucas, phytochemical screening, phytoconstituents
| Introduction|| |
Aromatic plants and their medicinal constituents provide raw material in pharmaceutical cosmetics and drug industries. A large variety of chemical compounds are found in plants and quite often the biosynthetic plants producing these compounds differ in various plant groups. Primary metabolites such as amino acids, carbohydrates and proteins are vital for the maintenance of life processes, while secondary metabolites such as alkaloids, phenolics, steroids, terpenoids have toxicological, pharmacological and ecological importance.
Chromatographic techniques are generally carried out for the separation and purification of phytoconstituents based on their charge, shape, or size. Thin layer chromatography (TLC) is generally considered as an authentic and reproducible method for the analysis of different drugs. This technique is extensively adopted for the rapid analysis of drugs and drug preparations. It provides a chromatographic drug fingerprint in a short time. A chromatographic fingerprint of herbal medicine is a chromatographic pattern of the extract of some common chemical components of pharmacologically active or chemical characteristics. By using the chromatographic fingerprints the identification and authentication of herbal medicines can be precisely conducted, even if the amount and concentration of the chemically characteristic constituents is not exactly the same for different samples of the drug. Hence it is very important to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic components of the herbal drug.,,, Moreover, it is suitable for as certaining the identity and purity of drugs, and for detecting adulterations as well as substitutions.
High-Performance Thin-Layer Chromatography (HPTLC) is the most advanced form of TLC and comprises the use of chromatographic layers of utmost separation efficiency and the employment of state-of-the-art instrumentation for all steps in the procedure. These include precise sample application, standardized reproducible chromatogram development and software controlled evaluation. HPTLC is an entire concept that includes a widely standardized methodology based on scientific facts as well as the use of validated methods for qualitative and quantitative analysis. Major advantage of HPTLC is its ability to analyse several samples simultaneously using a small quantity of mobile phase.
The genus Leucas belongs to the subfamily Lamioideae that are not chemically explored much, probably due to lesser quantity of volatile oils in them. The plants are known as 'Tumba' in regional languages in Southern India and used widely as an essential ingredient in many folk medicines. Medicinal and therapeutic effects of Leucas are clearly described in the Bhāvaprakāśa Nighaṇṭu, written based on Indian Materia Medica of Bhāva Miśra (c 1600-1600 CE) and in Śodhala Nighaṇṭu. The name 'Kutumbikā' used in the Charaka Saṃhitā is identified as Leucas. The plants are known as Droṇapuṣhpī in Ayurveda literature and are recorded as having sweet, salty, pungent, bitter taste along with heavy and dry properties. It is known to have antibiotic, antipyretic, antiseptic, anthelmintic, germicidal and insecticidal properties., Its penetrating character and hot potency aggravates pitta and alleviates vata-kapha stages of the diseases. It promotes taste sensation and increases the digestive fire and also increases the intellectual capacity. Leucas is capable of expelling out waste products from the body and it also helps in subsiding edemas in the body and also it relieves piles, diabetes and it is beneficial against diseases such as fever, jaundice and in respiratory diseases. A paste made of Leucas leaves and coconut oil is found effective against skin diseases such as psoriasis.
The present research deals with the phytochemical investigation and development of HPTLC fingerprints of methanol leaf extract of three species of Leucas, viz. Leucas stelligera, Leucas eriostoma and Leucas ciliata which can be used for identification, authentication and characterization.
| Materials and Methods|| |
Fresh leaves of Leucas stelligera, Leucas eriostoma and Leucas ciliata [Figure 1] were collected from different regions of Western Ghats and identified. Voucher specimens were deposited in Calicut University Herbarium.
|Figure 1: (a) Leucas stelligera; (b) Leucas eriostoma; (c) Leucas ciliata|
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Preparation of crude extracts
The collected leaves were washed with distilled water, shade dried and powdered. These dried samples were powdered and stored at 4°C until further use. Crude extracts (10% w/v) were made using four solvents viz. water, methanol, ethanol and chloroform. The extracts were filtered through a fine muslin cloth and the clear filtrate was evaporated to dryness to form the crude extract.
The chemical tests were carried out with the crude extracts of each plant i.e. aqueous extract (AE), methanol extract (ME), ethanol extract (EE) and chloroform extract (CE).
Qualitative analysis was done to identify the presence of the following phytoconstituents - alkaloids, flavonoids, phenols, tannins, terpenoids, saponins, carbohydrates, glycosides, proteins and amino acids using standard procedures.,
Detection of alkaloids
- Hager's test: To one ml of extract, two drops of Hager's reagent was added. Formation of yellow colored precipitate indicates positive test
- Dragendroff's test: To one ml of extract, two drops of Dragendroff's reagent was added. Formation of reddish orange precipitate indicates positive test
- Wagner's Test: To one ml of extract, two drops of Wagner's reagent was added. Formation of reddish brown precipitate indicates the presence of alkaloids
- Mayer's Test: To one ml of extract, two drops of Mayer's reagent was added along the sides of the test tube. Appearance of whitish yellow precipitate indicates the presence of alkaloids.
Detection of flavonoids
- Alkaline Reagent Test: Extracts were treated with few drops of NaOH solution. Formation of an intense yellow color which becomes colorless on addition of few drops of dilute acid indicates the presence of flavonoids
- Lead acetate Test: Extracts were treated with few drops of lead acetate solution. Formation of yellow colored precipitate indicates the presence of flavonoids.
Detection of phenols
- Ferric chloride test: One ml of extract was treated with 5% Ferric chloride solution. A dark bluish green color indicates the presence of phenolic compounds [Figure 1].
Detection of tannins
- Gelatin Test: To the extract 1% gelatin solution containing 10% sodium chloride was added. Formation of white precipitate indicates the presence of tannins
- About 1 g of extract was dissolved into 10 ml of 10% KOH in a beaker and shaken to dissolve. A dirty precipitate indicates the presence of tannins.
Detection of terpenoids
- Salkowski's Test: 2 ml of extracts were dissolved in 3 ml chloroform and filtered and two drops of concentrated Sulphuric acid was added along the sides of test tube. The formation of reddish brown ring at the interface indicates the presence of terpenoids.
Detection of saponins
- Foam test: 100 mg of extract was diluted with distilled water to 20 ml. The suspension was shaken in a graduated cylinder for 15 min. Formation of 2 cm foam layer indicates the presence of saponins.
Detection of carbohydrates
- Molisch's test: Two ml of extract was added with two drops of alcoholic solution of α-naphthol. The mixture was shaken well and one ml of concentrated sulphuric acid was added along the sides of the test tube and allowed to stand. A violet or purple ring indicates the presence of carbohydrates
- Fehling's test: One ml of extract was boiled on the water bath with one ml of each of Fehling's solution A and B. A red precipitate indicates the presence of carbohydrates.
Detection of proteins and amino acids
- Biuret test: An aliquot of two ml of extract was treated with one drop of 2% copper sulphate solution. To this, one ml of (95%) ethanol was added, followed by excess of potassium hydroxide pellets. Pink color in the ethanolic layer indicates the presence of proteins
- Ninhydrin test: Two drops of Ninhydrin solution was added to two ml of aqueous extract. A characteristic purple color indicates the presence of amino acids
- Xanthoproteic test: Extracts were treated with few drops of concentrated HNO3. Formation of yellow color indicates the presence of proteins.
Detection of glycosides
Keller Killiani Test: 0.5 g of the extract was treated with 2 ml of glacial acetic acid and a drop of 5% (w/v) FeCl3 was added to it. 1 ml of concentrated H2 SO4 was added to it. Presence of brown ring at interface indicates the presence of glycosides.
Quantitative analysis of phytochemicals
Total phenolic content
The total phenolic content was estimated by Folin-Ciocalteu method. An aliquot (1 ml) of extracts or standard solution of gallic acid (20, 40, 60, 80 and 100 μg/ml) was added to a 25 ml volumetric flask, containing 9 ml of distilled water. A reagent blank was prepared using distilled water. One milliliter of Folin-Ciocalteu phenol reagent was added to the mixture and shaken. After 5 min, 10 ml of 7% Na2 CO3 solution was added to the mixture. The volume was then made up to the mark. After incubation for 90 min at room temperature, the absorbance against the reagent blank was determined at 550 nm with an UV/Vis spectrophotometer. Total phenolics content was expressed as mg gallic acid equivalents (GAE).
Total flavonoid content was measured by the Aluminium chloride colorimetric assay. An aliquot (1 ml) of the extracts or standard solutions of quercetin (50, 100, 150, 200 and 250 μg/ml) was added to a 10 ml volumetric flask containing 4 ml of distilled water. To the flask, 0.30 ml of 5% NaNO2 was added and after 5 min, 0.3 ml of 10% AlCl3 was added. After 5 min, 2 ml of 1M NaOH was added and the volume was made up to 10 ml with distilled water. The solution was mixed and absorbance was measured against a blank at 510 nm. The total flavonoid content was expressed as mg quercetin equivalents (QE).
Determination of tannins
In this method, an aliquot (1 ml) of the extracts or standard solutions of tannic acid (1, 2.5, 5.0,10 μg/ml) was added to a 10 ml volumetric flask containing a 4 ml of distilled water. A reagent blank was prepared using distilled water. 500 μl of Folin-Denis reagent was added to the mixture and shaken. After 5 min, 1 ml of 7% Na2 CO3 was added and the volume was made up to 10 ml with distilled water. The solution was mixed and absorbance was measured against a blank at 775 nm. The concentration of tannins was expressed as tannic acid equivalents in milligram per gram (TAE mg/g) of crude extract.
The samples were analyzed in triplicates (n = 3) and the results were expressed as mean ± standard deviation values.
HPTLC quantification was performed using CAMAG Switzerland. Stationary phase was aluminium backed precoated silica gel plates, Merck 60 F254 (0.2 mm thickness). Samples were applied to the plate as bands at 10 mm from the bottom of the plate and 15 mm from the sides using CAMAG ATS 4. Development of the plate was carried out by CAMAG ADC 2. The plates were run up to 80 mm in ascending mode with solvent system Toluene: Ethyl acetate (7:3 v/v) at room temperature (28 ± 2°C) in a Twin Trough Chamber which was previously saturated with mobile phase. After development, the airdried plate was scanned at 254 nm, 366 nm and 550 nm in CAMAG TLC SCANNER 3 using Deuterium lamp with Camag winCATS software. Under conditions of 60% Relative Humidity.
| Results and Discussion|| |
Preliminary phytochemical screening of methanol, ethanol, chloroform and aqueous extracts of the leaves of Leucas stelligera, Leucas eriostoma and Leucas ciliata showed the presence of various phytoconstituents [Table 1]. Leucas members are rich in phenolics such as flavonoids. All the three species showed the presence of a minimum quantity of tannins in methanolic extracts only. The results show that Leucas species are poor in alkaloid composition. All species show a high degree of carbohydrate composition in their respective extracts. The presence of proteins and amino acids were clearly distinguished in ethanolic extracts. Methanol extracts showed the presence of most of the phytochemicals analysed compared to other solvents.
|Table 1: Phytochemical composition of the leaves of Leucas stelligera, Leucas eriostoma and Leucas ciliata in aqueous, methanol, ethanol and chloroform extracts|
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Methanolic extracts of leaves of L. stelligera, L eriostoma and L. ciliata were prepared to examine the total phenolic content, flavonoids and tannins. The results are tabulated in [Table 2]. Among all the three extracts studied, L. ciliata had the highest concentration of phenolics (34.5 ± 0.35), flavonoids (48.25 ± 1.06) and tannins (6.45 ± 0.5). The concentration of total tannins is significantly lesser compared to total phenolics and flavonoids. L. stelligera shows lesser quantity of phenolics (13.75 ± 0.35) and tannins (1.63 ± 0.11) whereas L. eriostoma shows lesser quantity of flavonoids (32.50 ± 3.18). Higher solubility of phenolic compounds in polar solvents provides higher concentration of these compounds in the extracts obtained using polar solvents for the extraction., Flavonoids can modulate the various enzymatic activities due to their interaction with various biomolecules since they have potent antioxidant properties.
|Table 2: Quantitative analysis for total phenolics, flavonoids and tannins|
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The HPTLC fingerprint study revealed that all the three species of Leucas showed best results in Toluene: Ethyl Acetate: 7:3 solvent system for methanolic extracts. After scanning and visualizing the plates in absorbance mode at 254nm, 366 nm and 550 nm (after spraying with anisaldehyde sulphuric acid reagent) best results were shown at 550 nm.
The results from HPTLC fingerprint scanned at wavelength 550 nm for methanol extract of Leucas stelligera, Leucas eriostoma and Leucas ciliata leaves revealed the presence of different polyvalent phytoconstituents [Table 3]. The Rf values ranged from 0.06 to 0.98 for the three species. It is also clear from [Table 3] and the chromatogram as shown in [Figure 2] that out of 12 components in Leucas stelligera, the component with Rf value 0.59 was found to be more predominant as the percentage area is 18.98%. Similarly out of 15 polyvalent compounds in Leucas eriostoma, and 13 polyvalent compounds in Leucas ciliata the compounds with Rf value 0.42 and 0.06% were found to be more predominant as the percentage area is 31.96% and 11.72% respectively. TLC plates showed different color phytoconstituents of methanolic extracts L. stelligera, L. eriostoma and L. ciliata. [Figure 3]. HPTLC fingerprint analysis not only gives the idea for the authentication of the plant extracts and its constituents but also provides the parameters for quality of herbal formulations.
|Table 3: Data pertaining to high-performance thin-layer chromatography fingerprint of methanolic extract of leucas stelligera, leucas eriostoma and leucas ciliate at 550 nm|
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|Figure 2: Chromatogram of methanol extract at 550 nm. (a) Leucas stelligera; (b) Leucas eriostoma; (c) Leucas ciliata|
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|Figure 3: High-performance thin-layer chromatography plate seen at visible light after spraying with anisaldehyde sulphuric acid reagent. Track 1: Leucas stelligera; Track 2: Leucas eriostoma; Track 3: Leucas ciliata|
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The members of the genus Leucas have valuable medicinal properties and hence possess high economic potential. Traditionally some Leucas species were used as antipyretics and insecticides. Flowers are valued as stimulant, expectorant, aperient, diaphoretic, insecticide and emmenagogue. Leaves of some species are considered useful in chronic rheumatism, psoriasis and other chronic skin eruptions. Crushed leaves are applied locally in snake bites. Further studies on separation and purification of individual compounds will lead to identification of new phytochemicals.
| Conclusion|| |
HPTLC studies show that the methanolic leaf extracts of three Leucas species contain a mixture of compounds. Further works have to be carried out for the characterization of other chemical constituents and for quantitative estimation with marker compounds. Even though these data can be considered along with the other values for fixing standards to these plants.
The first author express her sincere gratitude to Kerala State Council for Science, Technology and Environment for financial support and also thankful to Phytochemistry Division, Centre for Medicinal Plants Research, Arya Vaidya Sala for Technical assistance. The second author thank the Department of Biotechnology, Government of India (Grant No. BT/PR5423/BCE/8/907/2012; dated 19/6/2013).
Financial support and sponsorship
This study was financially supported by Kerala State Council for Science, Technology and Environment.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Bhattacharjee SK. Hand Book of Medicinal Plants. Vol. 5. Jaipur, India: Pointer Publishers; 2008. p. 56-77.
Sharma RK, Bhagwan D. Samhita C. Vol. 2. Varanasi, India: Chowkhamba Sanskrit Series; 1996. p. 17-101.
Helftmann F. Chromatography: Fundamental and Applications of Chromatographic and Electrophoretic Techniques. Vol. 5. Orinda, USA: Elsevier; 1992. p. 520-2.
Wagner H, Bladt S, Zgainski EM. Plant Drug Analysis: A Thin Layer Chromatography Altlas. Munich, Germany: Springer-Verlag; 1984. p. 5-21.
Patil PS, Shettigar R. An advancement of analytical techniques in herbal research. J Adv Sci Res 2010;1:8-14.
Liang YZ, Xie P, Chan K. Quality control of herbal medicines. J Chromatogr B 2014;812:53-70.
Ong ES. Chemical assay of glycyrrhizin in medicinal plants by pressurized liquid extraction (PLE) with capillary zone electrophoresis (CZE). J Sep Sci 2002;25:825-31.
Xie PS. A feasible strategy for applying chromatography fingerprint to assess quality of Chinese herbal medicine. Trad Chin Drug Res Clin Pharm 2001;12:141-69.
Kiritikar KR, Basu BD. Indian Medicinal Plants. Vol. 3. Reprint in 1975. Dehra Dun: Bishen Singh Mahebdra Pal Singh; 1918.
Rastogi RP, Mahrotra BN. Compendium of Indian Medicinal Plants III. Lucknow: CDRI; 1993.
Tiwari P, Kumar B, Kaur N, Kaur G, Kaur H. Phytochemical screening and extraction: A review. Int Pharm Sci 2011;1:98-106.
Harborne JB. Phytochemical methods-A guide to modern techniques of plant analysis. Springer Sci Bus Med 1998;3:5-30.
Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic phoshotungstic acid reagents. Am J Enol Vitic 1965;16:144-58.
Zhishen J, Mengcheng T, Jianming W. The determination of flavonoids contents in mulberry and their scavenging effect on superoxide radicals. Food Chem 1999;64:555-9.
Geethika K, Sunojkumar P. Preliminary phytochemical screening of 6 members of Leucas
). Int J Pharm Sci Rev Res 2017;47:60-4.
Mohsen MS, Ammar SM. Total phenolic contents and antioxidant activity of corn tassel extracts. Food Chem 2008;112:595-8.
Zhou K, Yu L. Effects of extraction solvent on wheat bran antioxidant activity estimation. LWT Food Sci Technol 2004;37:717-21.
Catapano AL. Antioxidant effect of flavonoids. Angiology 1997;48:39-44.
Prajapati MS, Patel JB, Modi K, Shah MB. Leucas aspera
: A review. Pharmacogn Rev 2010;4:85-7.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]