A Pharmacognostical Study on Fumaria parviflora Lamk

Ipomoea reniformis Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and neurological disorders. In the present study, pretreatment effect of methanolic extract of Ipomoea reniformis on epilepsy and psychosis was evaluated in rodents using standard procedures. Besides evaluating epileptic and behavioral parameters, neurotransmitters such as Gamma-Amino Butyric Acid (GABA) in epilepsy and in psychosis dopamine, noradrenaline and serotonin contents in the rodent brain were estimated. The extract pretreatment reduced maximal electro shock; Isoniazid (INH) and Pentylenetetrazole (PTZ) induced seizures and also significantly inhibited the attenuation of brain GABA levels by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central dopaminergic, noradrenergic and serotonergic pathways or by enhancing the GABAergic system. The results obtained in present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents.


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
Fumaria parviflora Lamk., (Syn. Fumaria indica (Haussk.) Pugsley, Fam. Fumariaceae commonly known as Parpata, is a valued herb in Ayurvedic system of medicine but falls under the category of controversial drugs. It is diffuse, annual weed growing through out India from Indo-Gangetic plains to down Nilgiris in South 1 .
Entire herb is traditionally used in leprosy, fever 2 , detoxification, and as laxative, diuretic and diaphoretic 3 .

Abstract
Fumaria parviflora Lamk., is a valued herb in Ayurvedic medicine and is used as Parpata by majority of Ayurvedic practitioners amongst the other plant sources mentioned under the same common name. It is found in many parts of India from Indo-Gangetic plain and Nepal down to the Nilgiri Mountains. The whole plant is diuretic, diaphoretic, aperient, laxative and anthelmintic. It is used as antipyretic, blood purifier and in skin disorders. In the present study, physico-chemical parameters were established for identification of the drug. Protopine and β-sitosterol were quantified by validated HPTLC method, developed using precoated silica gel plates as a stationary phase and toluene:ethyl acetate:diethyl amine (7:2:1) and toluene:methanol (9.4:0.6) as a mobile phase respectively. It is a diffuse, annual herb with thin winged stem; alternate leaf finely divided into small, linear lanceolate segments, small white or pink flowers with purplish tips. Microscopically root can be characterized by the presence of centrally located diarch primary xylem encircled by wide secondary xylem occupying major area and a narrow cork; stem by collenchymatous hypodermis, vascular bundle capped with lignified pericyclic fibres and hollow pith; leaf by vascular bundles with groups of sclerenchyma underneath the phloem and narrow spongy parenchymatous lamina. Powder can be typified by xylem vessels with varied thickening, lignified and thick walled testa and spherical pollen grains. The plant was found to be rich in alkaloids. The amount of protopine and β-sitosterol were found to be 0.47-0.50% w/w and 0.23-0.26% w/w. The quality parameters and HPTLC method developed would serve as useful gauge in standardization of Fumaria parviflora.

Plant Material
Fresh, fully-grown, flowering plants of F. parviflora were collected from Jammu in the month of January 2012. The plants collected were authenticated by taxonomist of Gujarat University, Ahmedabad, Gujarat. Voucher specimen sample (LM 632) was deposited at the Department of Pharmacognosy, L. M. College of Pharmacy, Ahmedabad, Gujarat. The plant material was cleaned, dried, powdered to 60 # and used for the present study.

Chemicals
Standards protopine and β-sitosterol were procured from Extra synthese, France and Natural Remedies, India respectively. All the solvents used were of chromatography grade and other chemicals used were of analytical (AR) grade.

Pharmacognostical Studies
Whole plant was studied for morphological characters.
Microscopical study was performed for both entire (free hand transverse sections) and powdered material. Moisture content 16 , ash values and extractive values were determined 17 . Phytochemical screening was performed and alkaloids 18 , flavanoids and phenolics 19 were estimated. The method was validated in terms of linearity, interday precision, intraday precision, repeatability, accuracy, specificity, limit of detection and limit of quantification.

Calibration Curve
A stock solution (1 mg/ml) was prepared by dissolving accurately weighed each of 5 mg of protopine and β-sitosterol in 5 ml methanol in a volumetric flask separately. Standard solutions for calibration were prepared by dilution of the stock solution with methanol; the concentrations were such that amounts of protopine between 1000-5000 ng and that of β-sitosterol 500-2500 ng. The correlation coefficient, slope intercepts and regression equation were also calculated to provide mathematical estimate degree of linearity. A calibration curve was derived by plotting peak area (Y axis) versus concentration (X axis).

Quantification of Protopine and β-sitosterol in Extract
5 g crude drug powder was exhaustively extracted by refluxing with ethanol (3x50ml). Extract was passed through charcoal, concentrated and vacuum dried to yield 7.52%w/w of residue. It was dissolved in methanol in a volumetric flask to get the test solution of 5 mg/ml. 10 µl of this solution was used for protopine estimation. After developing the plates, densitometric scanning was done at 293 nm and visualization of spots was achieved using dragendorff 's reagent for photodocumentation. β-sitosterol was quantified using 5 µl of the test solution and densitometric scanning was performed at 545 nm after derivatizing with anisaldehyde sulphuric acid reagent followed by heating at 110°C. The peak area values of standard and sample were used to calculate the amount of protopine and β-sitosterol present in extract. bearing lateral wiry rootlets; erect, longitudinally wrinkled, often branched 20-30 cm long stem with 4 to 5 winged projections; alternate, exstipulate leaf that is finely divided into narrow flat segments, each segment being broad oblong or linear lanceolate, 2 to 3 cm in length and 1 to 2 mm in width with acute or subacute apex, 2 to 4 cm long twisted petiole, sheathing at base; small white or pink flowers with purplish tips, in terminal inflorescence; indehiscent, tiny, sub-globose and externally faintly rugose fruits and globose minute seeds.

Microscopical Evaluation
Transverse section of the root is almost circular with slightly irregular margin, shows a diarch centrally  located primary xylem in the centre encircled by wide secondary xylem composed of wide lumened xylem vessels (xyv) radially running at places and associated with tracheids, fibres and parenchyma that are traversed with multiseriate medullary rays (mr); narrow band of parenchymatous phloem (ph) and cortex (ct) encircled by cork (ck). Transverse section of the stem is pentagonal to irregularly circular in outline and shows epidermis (e) covered with thick cuticle; 3 to 4 rows of collenchymatous hypodermis (hyp); narrow parenchymatous cortex (ct); indistinct endodermis; wedge shaped collateral vascular bundle (vb) arranged at the ridges and capped with lignified pericyclic fibres (per) and wide centrally hollow pith (pi).
Transverse  and lower epidermis (le) covered with thin cuticle; three conjoint collateral vascular bundles (vb) in the midrib the central one being biggest in size and with groups of sclerenchyma (scl) underneath the phloem and narrow lamina enclosing 5 to 7 rows of spongy parenchyma, embedded with rows of small sized meristeles.

Powder
It shows testa in surface view (a); large, spherical pollen grains having more than six pores with raised areas on the exine (b); longitudinally cut fragments of bordered pitted (c), annular (d) and spiral xylem vessels; polygonal cells of cork in surface view (e) and fibres with phloem parenchyma (f).

Physicochemical Evaluation
Data of Physico-chemical parameters including moisture content, ash and extractive values are given in Table 1.
The plant was found to be rich in alkaloids and phenolics ( Table 2).

Estimation of Protopine and β-Sitosterol by HPTLC Method
TLC studies of extract indicated presence of both protopine and β-sitosterol at R f 0.88 and R f 0.39 respectively exactly matching with reference standards. The content of protopine and β-sitosterol were found to be 0.47 -0.50% w/w and 0.23 -0.26% w/w.