Comparative Physico-chemical, Phytochemical and L-dopavariations of Kapikachhu (Mucuna pruriens) seed and rootby UV - Spectrophotometer

INTRODUCTION

Ayurveda is a science of life that has a history of over 5000years. It gives prime importance to drug which is one of the tools of treatment. Many treatment modalities are using in the management of CNS disorders one such modality and drug is Kapikachhu, which contains L-dopa that is mainly used in Parkinson’s disease (kampavata). The main seat of L-Dopa in kapikachu is seed but there are reports which suggest that this chemical even present in traces at leaves also but no reports are exist on roots thus present study was planned. Kapikachhu (Mucuna pruriens) is one where its seed contains about 3.1– 6.1% L-dopa, with trace amounts of serotonin, nicotine, dimethyl tryptamine-n-oxide, bufotenine, 5-MeO-DMT-n-oxide and beta-carboline is used for the management of vatavyadhi, kampavata (Parkinson’s disease). Similarly its root do contain L-Dopa and other chemicals but comparative study on its phyto-chemicals with respect to quantification and standardization of its seed and root is yet to be done thus present study was undertaken.

Chemo profile of Ayurvedic drugs or herbal medicines is limited. Development of this would certainly help to widen the standard of herbal medicine and also to predict the action of drugs and drug interaction if used in clinically.¹

Many methods are reported for the determination of L-dopa which include HPLC,² fluorimetry,³ ionchromatography,⁴ coulometry,⁵ gas chromatography,6 and electro chromatography.⁷ Spectrophotometric method is known for its simplicity, sensitivity, and rapid determination. Thus in present study this method was adopted.

Methodology

Kapikachu (Mucuna pruriens) seeds were genuinely collected from authenticated venders and certified in Dravyaguna and Research Analytical Lab RGES AMC Ron. Then these were cultivated according to approved protocol for cultivation in controlled natural habitat (garden). After the full maturation, seeds and roots were collected and stored. A herbarium prepared and stored in RAL. Thus collected seeds and roots were further assessed on its morphology, Physicochemical, phytochemical and microscopic study according to the API protocol.

TLC Study: Precoated silica gel 60F 254 plate (E. Merck) of uniform thickness 0.2mm were used with the solvent system of toluene: ethyl acetate (7:3). The both seed and root water and alcoholic extracts were subjected to TLC.

UV-Spectrophotometric analysis: Standard L-dopa (assigned purity, 99.8 %) was purchased from Natural remedies Bangalore, India. All the chemicals used in the experiments are of analytical grade. UV-Spectrophotometer UV-1800 (Infotech) equipped with UV lamp and 1cm quartz cell was used for analysis.

Preparation of Extract : The dried seeds and roots of M. pruriens were powdered in a mechanical grinder. These powders were initially defatted with petroleum ether (60-800C) then aqueous extract was prepared by cold maceration method. After seven days, the extract was filtered using Whatman filter paper (No. 1) and then concentrated in vacuum and dried.

Calibration Curve of Standard L-dopa: A stock solution of L-dopa (1µg/ml) was prepared by dissolving an accurately weighed 10 mg of L-dopa standard in 5 ml of 0.1N formic acid and volume was made up to 100 ml with 0.1N formic acid in a volumetric flask. Again 1 ml of this solution was transferred in 100 ml volumetric flask and diluted up to the mark with 0.1 N formic acid. Standard working solutions were prepared by diluting stock solution with 0.1N formic acid in the concentration range 30-800 ng/ml. absorption was measured for all the solutions at 279 1100 nm wavelength.

Estimation of L-dopa in Herbal Extract: To determine the content of L-dopa in herbal extracts, an accurately weighed 100mg of dry aqueous extract was transferred into 100 ml volumetric flask and 10 ml of 0.1N formic acid was added, sonicated for 10 min and volume was made up to 100 ml with 0.1N formic acid. The extract was filtered on a Whatman no.1 filter paper, from which 1.0 ml of the solution was diluted to 1000 ml with 0.1N formic acid in volumetric flask. Finally solution was diluted to get concentration of 500 ng/ml and Spectrophotometric measurement done. [8]The analysis was repeated for five times.

Results

The study provide the various differences in organoleptic charecters between seed and roots of M. pruriens as seed has length varies from 1.5- 2cm, 0.5-1cm breadth with 2.5-2.6gm per weight. With black kidney to rectangular in shape, it has smooth surface and sweet tasted. Similarly roots are 5-17cm long, 0.5 to 1cm breadth and 0.15gm weight. These have black colored irregularly curved rough surfaced branches. Each root has rough surface with faint characteristic odour and shart fractured and bitter tasted.

Microscopic structure: The T S of the seed is oval in outline with testa has a) Epidermis: Single layer, polygonal tabular cells with thin anticlinal walls filled with mucilage. b) Sub-epidermis : One or two layers of cylindrical collenchymas and Inner Coat (Inner Integument) has a) Sclerenchymatous layer: Longitudinally elongated, lignified scleroids, 120-190μ long and 14-17 μ wide, thick walled, pitted, very small lumen. b) Parenchymatous layer: One or two layers, thin, tangentially elongated, collapsed parenchymatous cells, and c) Pigment layer: Single layer of flattened polygonal pigment cells with reddish brown. The whole content of endosperm is Polyhedral, cellulosic parenchyma with oil globules. In image no 1. Similarly root microscopy showed that outer surface is Cork, It has 3 - 4 layers of yellowish brown colour thin walled cork cells. Cortex is middle part which is a wide zone it contains pitted Stone cells and sclerenchymatous patches and extends to wards interior part. Phloem tissue found next to the cortex. The Medullary rays extends to the cortex region from pith and biseriate, the ray cells are thin walled elongated with with starch grains in it. The wood occupies about 40% composed of Vessels, Parenchyma, Fibers and Medullary rays. The pith occupies of the center with the parenchyma cells. Powder microscopy showed that seed has lignified sclerids, mucilage cells, aluerone grains and oil globules but has vascular bundles, sclerenchymatous fibers and starch.

Phytochemical assessment showed in table no 1.

Physicochemical study showed that ash value, acid insoluble ash, alcoholic extractive value, water soluble extractive value and moisture content of root and seed are 2.45% and 3.75%, 0.48% and 1.9%, 8.5% and 5.5%, 1.85% and 2.83% respectively. TLC study showed maximum spots were seen in seed than root presented in table no 2.

The spectrophotometric study showed that maximum absorption was seen in seed extract than root as presented in table 3 and image no 2.

Conclusion

The comparative chemo-profile variation of seed and root of Kapikacchu showed there is vast difference exist and chemically seeds have more potensive than root because of high percentage of chemicals. The Spectrophotometric study showed L-Dopa was more 1.9% in seed compare to root of 0.81%.