Sharique A. Ali et al.

INTRODUCTION

Since the beginning of human civilization, medicinal plants have been used by mankind for their thera- peutic values. Nature has been a source of medicine 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. This plant-based tradi- tional system of medicine continues to play an essen- tial role in health care, with about 80% of the world’s inhabitants relying mainly on traditional medicines for their primary health care and safety, (Kirtikar and Basu, 1989; Hassan, 2012, Singh et al., 2014 and Miraj et al., 2014).

In India, the Ayurvedic system has described a large number of such medicines based on plants or plant products and the determination of their morphologi- cal, pharmacological or pharmacognostical charac- teristics has provided a better understanding of their active principles and mode of action. Although there are thousands of plant species around the globe, only a small proportion has been investigated both phyto- chemically and pharmacologically. When one consid- ers that a single plant may contain up to thousands of constituents, the possibilities of making new discover- ies become evident, (Baker et al., 1995; Agrawal, 2002; Singh et al., 2014).

In recent years, the need for quality assurance tools to ensure the identity, purity and quality of botanical materials has risen dramatically. The crucial factor for the ultimate success of an investigation into bioactive plant constituents is thus the selection of plant mate- rial and its qualities. In view of the large number of plant species potentially available for any study, it is essential to have efficient systems available for the rapid chemical and biological screening of the plant extracts selected for any investigation, (Mukerjee, 2002 and Ras- togi, 2009).

High-performance thin-layer chromate-graphy (HPTLC) is emerging as a versatile, high-throughput and cost effective technology that is uniquely suited to assessing the identity and quality of botanical materi- als. Basically HPTLC is a chromatographic technique that can separate a mixture of compounds and is used in phytochemical and analytical chemistry to identify, quantify and purify the individual components of the mixture, (Cannell, 1998).

In the light of above facts, the present study was designed where we focused various aspects regard- ing the characterization of plant extracts using HPTLC, which have tremendous medicinal and therapeutic prop- erties and are frequently used in India,

MATERIAL AND METHODS

We have used six commonly known medicinal plants as mentioned in table no. 1 for identification and scientific validation of their active constituents, which are present in them. We have also quantified and studied the rela- tive quantities of the active principles in plant extracts which can be further exploited for their medical effica- cies. The highly sensitive HPTLC method has been used to accomplish this goal in which chromatographic sepa- ration of plant active constituents was performed on 20 cm ×10 cm aluminium backed HPTLC plates coated with 200 μm layers of silica gel 60F254 (E. Merck, Darmstadt, Germany).

Before use, the plates were washed with methanol and activated at 110°C for 5 min. Both test and standard samples (5 μL each) were applied on to HPTLC plates as 6

mmwide bands and 12 mm apart from middle of bands by spray-on technique along with nitrogen gas supply for simultaneous drying of bands, by means of a Camag Linomat V auto sample applicator fitted with a 100 μL syringe (Hamilton, Bonaduz, Switzerland).

A constant spot application rate of 150 nL/s was used. Plates were developed to a distance of 165 mm, in the dark, with different combinations of various solvent systems in different ratio were used as mobile phase for effective separation of different compounds (Table no. 1). Before development the chamber was saturated with mobile phase for 15 min at room temperature (25 ± 2°C) and 50% relative humidity. Chromatography was per- formed in Camag’s twin-trough chamber.

Densitometric analysis of different active compounds was carried out in the different absorbance modes mentioned in table no. 1. Densitometric scanning was performed with a Camag TLC scanner 3 in reflectance- absorbance mode at wavelength 356 nm, under control of Camagwin CATS planar chromatography manager software version 1.4.4. The slit dimensions were 6 mm × 0.30 comm and the scanning speed was 100 nm/s.

RESULTS AND DISCUSSION

Chromatographic fingerprint analysis has shown to be a rational and feasible approach for the quality assess- ment and species authentication of traditional medicine. It utilizes chromatographic techniques to construct spe- cific patterns of recognition of medicinal plants. The developed fingerprint pattern of components can then be used to determine not only the absence or presence of markers of interest but the ratio of all detectable ana- lytes as well, (Kamboj and Saluja, 2013).

The present study was designed to identify and quantify some active constituents, which generally

have various medicinal values and have been used in Indian culture since long back. For this purpose HPTLC characterization has been employed for identification and quantification of active ingredients of six selected medicinal plants: Piper nigrum, Aloe vera, Arachis hypogea, Ocimum sanctum, Berberis vulgaris and Cur- cuma longa.

The method for quantitative analysis of different plant extracts was validated with regard to its specifi- city, precision, accuracy and linearity. The composition of the mobile phase for TLC was optimized by testing

Sharique A. Ali et al.

different solvent mixtures of varying polarity. The best results were obtained using specific solvent system (mobile phase) and detected under UV at specific wave- length as mentioned in Table no. 1, which showed good resolution of the compounds and visualization of the same with precision.

The specificity of the method was ascertained by analyzing standard and samples. The spots for different active ingredients in the sample were confirmed by com- paring the Rf value and the spectrum of the spot with that of standard which is showed in Table No. 1. Compara-

Sharique A. Ali et al.

tive study of different biomarker compounds of different plants has shown that they are present in higher amounts in the specific part of that particular plant species and the activity of a plant extract is always influenced by the quantity of active principles present in the extract.

anxiolytic, immuno-modulatory, antibacterial, anti- fungal, anti-thyroids, antiapoptotic, anti-metastatic, antimutagenic, anti-spermatogenic, anti- colon toxin, insecticidal and larvicidal activities, (Damanhouri and Ahmad, 2014) .

PIPER NIGRUM

HPTLC analysis of seed extract of P. nigrum against standard piperine revealed that best results were obtained using Benzene- Ethyle acetate in 8:4 ratio. Rf value of standard aloin was matched with the Rf value of the extract which was found about 0.51. The contents of piperine were quantified using TLC densitometric meth- ods and were found to be 5.29 % in seed extract of P. nigrum (Table-1).

Previous literature showed that P. nigrum is a valua- ble medicinal plant, which is one of the most commonly used spices and considered as “The King of spices’’ among various spices and is also used as an impor- tant component of many Ayurvedic treatments. Donata et al., (1990) have used P. nigrum fruits orally with other ayurvedic herbs including Psoralea corylifolia for the treatment of vitiligo where majority of subjects showed positive response.

Lin et al., (1999a) also reported that P. nigrum dried fruits and its pure active ingredient piperine induced melanogenesis in cultured mammalian melanocytes.

Later on, melanogenic properties of P. nigrum extract, piperine have been further rediscovered and scientifi- cally validated by Sajid and Ali, (2011) using piperine, being the main active ingredient of Piper nigrum, on animal melanocyte models. Their data clearly showed that in tadpoles and adults of the frog, Rana tigerina and the toad, Bufo melanostictus, the melanin disper- sion within the black pigment cells, the melanophores is stimulated by piperine-like receptors, similar to that of the cholinergic ones.

Sajid and Ali, (2011) have concluded that piperine can be used as a novel cellular moderator in activating the cholinergic or piperine-like receptors for their var- ied melanogenic actions. The pioneering study of Sajid and Ali, (2011) also signifies the evolutionary aspect of the receptors of the lower vertebrate melanophores, their phylogenetic development which is homologous to melanocytes in a more evolved mammalian-melanocyte receptor system.

Apart from its melanogenic activity, piperine also exhibits various other pharmacological activities like being an antidepressant (Li et al., 2007), hepato-protec- tive (Matsuda et al., 2008), antihypertensive (Taqviet al., 2008), antioxidant, antitumor (Manoharan et al., 2009), antiasthmatic, (Parganiha et al., 2011), antipyretic, anal- gesic, anti-inflammatory, anti-diarrheal, antispasmodic,

ALOE VERA

HPTLC analysis of leaf extract of A. vera against stand- ard aloin revealed that best results were obtained using ethylacetate - methanol - water in 10: 1.4: 1 (v/v) ratio. The Rf value of standard aloin was matched with the Rf value of extract which was found about 0.76. The con- tents of aloin quantified using TLC densitometric meth- ods were found to be 44.41 to 65.56 % in leaf extract of A. vera (Table-1).

Literature suggests that A. vera is an important medicinal plant belonging to family Liliaceae, of which there are about 360 species. The peripheral bundle sheath cells of A. vera produce an intensely bitter, yellow latex, commonly termed as aloe juice, or sap, or aloes which mainly contains aloin, responsible for their strong laxa- tive effects.

A. vera has huge demand and is traded in medicinal drug markets of the world for a wide range of therapeu- tic applications such as wound healing effect, reduction of blood sugar in diabetes, for soothing burns, for easing intestinal problems and for reducing arthritic swellings. Various cosmetic products are also made from the muci- laginous tissues of A. vera leaves, commonly called as aloe gel (Dal’Belo et al., 2006).

Tan et al., (2002) demonstrated that aloin can bind not only to the enzyme tyrosinase but also to the enzyme– substrate complex, leading to inactivation of the enzyme resulting lightening of skin. Similarly Cheng et al., (2002) have also reported that aloin is a potent inhibitor of tyro- sinase which plays an important role in melanogenesis.

Ali et al., (2012) have also reported anti melanogenic and melanin aggregatory potential of Aloe vera leaf gel extract containing aloin in which they found that the leaf extract of A. vera and its active ingredient aloin induced powerful, dose-dependent, physiologically sig- nificant melanin aggregating effects in the isolated tail melanophores of B. Melano-stictus leading to the light- ening of the skin.

Their data suggests that the active ingredients of A. vera leaves, particularly aloin, stimulate the abundantly present adrenergic receptors of α2 type in tadpole tail skin leading to skin lightening effect. These findings also point to the novel role of aloin as a new sympathomi- metic compound, which can have clinical application as nontoxic melanolytic agent, for the treatment of hyper pigmentation, or can be used as a skin fairness agent without any toxicological implications.

ARACHIS HYPOGAEA

HPTLC analysis of seed skin extract of A. hypogaea against standard resveratrol revealed that optimum results were obtained using Chloroform: Ethylacetate: Formic acidin in 2.5 : 1 : 0.1 ratio. The Rf value of standard aloin was matched with the Rf value of extract which was found about 0.31. The contents of quantified using TLC densitometric methods were found to be 26 % in seed skin extract of A. hypogaea (Table-1).

It is well known that A. hypogaea is an important plant which is widely used for the treatment of various ailments in different countries, and is a rich source of resveratrol. Because of the significant pharmacological activities exhibited by the resveratrol, several research- ers have focused on the development of various analyti- cal methods to determine resveratrol in different matri- ces such as plant extracts, wine and serum.

Resveratrol is a strong antioxidant and has been reported to have protective effects against atheroscle- rosis, coronary heart disease, postmenopausal problems, inhibits platelet aggregation and a broad spectrum of degenerative diseases and also possess cancer chemopre- ventive properties, (Jang et al.,1997 and Bagchi, 2000;).

Recently, Galgut and Ali, (2011) have also reported that the active ingredient of A. hypogaea such as res- veratrol can act as a sympathomimetic compound and can induce aggregation of melanophores of the tadpole, Bufo melanostictus via the induction of beta type of the adrenoceptors. Their study has suggested a novel way for the use of A. hypogaea and its active ingredient, res- veratrol for clinical application as a nontoxic melano- lytic compound for the treatment of hyperpigmentation or making the skin fairer in complexion.

In this regard the authors have also filed a patent for the quantification, specific use and its mechanism at the cellular level, through National Research Development Corporation, New Delhi, Patent No. 2895/ MUM / 2012 (Ali SA & J Galgut ).

OCIMUM SANCTUM

HPTLC analysis of leaf extract of O. sanctum against standard eugenol revealed that best results were obtained using Toluene: Ethyl acitate: Formic acid in 90:10:01 ratio. Rf value of standard was matched with the Rf value of extract which was found about 0.59. The contents of eugenol quantified using TLC densitometric methods were found to be 98.39 % in seed extract of O. sanctum (Table-1).

O. sanctum is a member of the Lamiaceae family, commonly known as ‘Tulsi’ in Hindi and ‘Holy Basil’ in English and is used as food seasoning. Tulsi has been well documented for its therapeutic potentials in

Sharique A. Ali et al.

Ayurveda described as Dashemani Shwasaharni (anti- asthmatic) and antikaphic drugs (Kaphaghna) (Gupta et al., 2002; Yanpallewar et al., 2004).

Species of tulsi are also valuable due to their phar- maceutical properties i.e. antipyretic, anti-inflammatory, cardio-protective, central nervous system (CNS) depres- sant, chemo preventive, antiulcer and anticancer proper- ties. Leaves of Tulsi contain a bright yellow volatile oil, which is useful against insects and bacteria. The prin- cipal constituent of this essential oil is eugenol (4-allyl 2-methoxyphenol) which is responsible for medicinal properties of tulsi (Prakesh and Gupta, 2005).

Many scientific studies have showed that sweet basil extract is a strong radical scavenger and can be consid- ered as a good source of natural antioxidants, (Pandey and Madhuri, 2010;Kumar et al., 2012). Recently, from our laboratory, the powerful melanolytic role of extract of O. sanctum has been demonstrated on B16 melanoma cell lines, thus describing the extract as a possible can- didate for its commercial use in making the skin fairer, Nargis and Ali, (2015).

BERBERIS VULGARIS

HPTLC analysis of whole plant extract of B. vulgaris against standard berberine revealed that best results were obtained using n-Propanol-Water-Formic Acid in 90:8.0:0.4 ratio. Rf value of standard aloin was matched with the Rf value of extract which was found about 0.56. The contents of eugenol quantified using TLC densito- metric methods were found to be 3.8 % in whole plant extract of B. vulgaris (Table-1).

Berberis vulgaris L. (barberry) belongs to the Berberi- daceae family and it is a small shrub which grows in Europe, but also in Africa and Asia, especially to the for- est edge, in shining places. It is cultivated for its valua- ble biological properties. The main bioactive compounds from B.vulgaris are alkaloids (berberine, berbamine, jatrorrhizine, columbamine, berberubine, oxicanthine, palmatine; figure 1), vitamin C, resin, and tannins, but also flavonoids like quercetin and kaempferol (Singh et al., 2011).

Literature showed that berberine possesses a wide range of medicinal property including antimicrobial, hepatoprotective, ionotropic, antiarrhythmic, hypoli- pidemic and anti inflammatory (Dehar et al., 2012). Various clinical studies have established the efficacy of hydrochloride of berberine in the treatment of oriental sore (Dhar, 1980), trachoma (Babbar et al., 1982; Mohan et al., 1982), CHF18 and Type 2 diabetes mellitus (Yanxia, 1995; Yin et al., 2008).

Chiou et al., (1991) reported that berberine vasodilates the rat mesenteric artery in part by indirectly releasing EDRF, but mainly by directly blocking the release of

Sharique A. Ali et al.

Ca2+ from internal stores. Recently it has been revealed that the extract of Berberis vulgaris exert skin darken- ing effect on pigment cells, i.e. melanohores of Bufo melanostictus (Ali et al., 2014).

Recently Ali et al., (2014) stated that extract of Ber- beris vulgaris exert skin darkening effects on pigment cells, i.e. melanohores of Bufo melanostictus. They reported berberine as a powerful melanogenic agent, as it induced a physiological melanophore dispersion effect, leading to darkening of the B. melanostictus skin.

Ali et al., (2014) have pharmacologically demonstrated that the berberine isolated from roots of B. vulgaris behaves like isoprenaline, the well known sympatho- mimetic agonist, in activating the dominantly present β2 adrenoceptors of the neuronmelanophore junction of this species, to induce distinct and marked melanin dis- persion of the B. melanostictus melanophores.

CURCUMA LONGA

HPTLC analysis of rhizome extract of C. longa against standard curcumin revealed that optimum results were obtained using chloroform and methanol in 95:5 ratio. The Rf value of standard aloin was matched with the Rf value of extract which was found about 0.47. The contents of curcumin quantified using TLC densitomet- ric methods were found to be 28.10% in rhizome extract of C. longa (Table-1).

The rhizome of turmeric (Curcuma longa L.) has a rich history in India as spice, food preservative, and coloring agent and has been used for centuries in the Ayurvedic system of medicine. Its use as a remedy for hypercho- lesterolemia, arthritis, indigestion and liver problem has been known since long back (Srimal, 1997).

The continuing research indicates that turmeric and its active principle curcumin have unique anti- oxidant, antimutagenic, antitumorigenic, and anticar- cinogenic, antiinflammatory, antiarthritic, antimicro- bial, and hypocholesterolemic properties as reviewed elsewhere (Majeed et al., 1995; Kapoor, 2001; Miquel et al., 2002). Recently from the authors laboratory, Miraj et al., 2014, Miraj and Ali (2014, 2015) have recently described the amelioration of metal toxic- ity using plant extracts and also have demonstrated for the first time anti rheumatic properties of C.longa via iontophoresis, a unique transdermal delivery sys- tem using low current for the treatment of rheumatic arthritis in a rat model.

Present outcomes gained from the study clearly dem- onstrate characteristic HPTLC fingerprint of particular active compound of a particular plant species which 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. Thus the present communication will provide sufficient information about therapeutic efficacy of cer- tain herbal based drugs and also in the identification, standardization and quality control of medicinal plants, from the Indian herbal treasure.

CONCLUSION

HPTLC is one of the sophisticated instrumental tech- niques which has been widely used for the qualitative and quantitative analysis of the herbs and herbal drugs. In the present investigation we have taken crude extracts of different parts of six plants namely P. nigrum, A.vera, A. hypogea, O. sanctum, B. vulgaris and C. longa for the quantitative determination of bioactive compounds present in them and have reported that the signifi- cant quantities of active ingredients are present in the extracts of these plants.

The pharmacological efficacies of these six plants have been further investigated using various animal models. It has been found that crude extracts of three plants i.e. A.vera, A. hypogea, and O. sanctum exert pow- erful melanolytic responses, hence these plant extracts can be exploited as novel skin lightening agents in cos- metic industry for the treatment of hyperpigmentation or dark skin which has considerable social implications. However, the other three plant extracts of P. nigrum, B. vulgaris and C. longa exhibited skin darkening effects, which suggest that active ingredients from these plant extracts can serve as promising therapeutic candidates for the treatment of hypopigmentary disorders like vitiligo, which again is a social evil and has no proper treatment till date. Similarly, curcumin can be used as a powerful anti rheumatic agent if properly applied as in the present case we have used iontophoretic transder- mal delivery system, envisaging maximum efficacy with a minimum dose. From these findings, it is concluded that bioactive components of these plants can be used as novel and safe candidates for the treatment of hyper as well as hypo pigmentary disorders, along with a host of other bio medical efficacies, like that for the untreatable dreadful rheumatic disease.

ACKNOWLEDGEMENTS

Authors thank the Principal and Secretary Saifia Science College Bhopal for providing necessary facilities. SAA acknowledges financial support in the form of major research projects from the MP Biotechnology Council Bhopal and MP Council of Science & Technology Govt of MP Bhopal.

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