INTRODUCTION:

Alkaloid based on the steroid nucleus is not very widely distributed being restricted to plants of the Holarrhena, Solanum and Veratrum species. Among the bases there is relatively little variation in structural type, the following sub-group may be distinguished¹.

Solasodine is the aglycone of solasonine (solancarpine, purapurine) of soladamine, and of solamargine and is prepared from these by hydrolysis with 3% hydrochloric acid at 100°C. The cooled reaction mixture deposits the sparingly soluble hydrochloride from which the free base may be regenerated. It is dimorphic but both forms melt at 198°-200°C²

Solasodine is a weaker base with molecular formula C₁₂H₄₃O₂N and pKb 6.30³. One of the oxygen atom present as a secondary hydroxyl in position 3 as shown by the formation of a sparingly soluble digitonide. The functional nature of the second oxygen was more difficult to determine. More recent investigations have shown that the second oxygen is present as cyclic ether and the second active hydrogen is present on secondary nitrogen.

¹³C-NMR spectra of solasodine and their assignments have been reported. Mass spectroscopic fragmentation pattern of solasodine has also been reported⁴.The steroid bases of solasodine group occur naturally as the glycosides usually containing three sugars. On hydrolysis the glycosides yield the steroid in the aglycone form.

The alkaloid content is usually determined by extraction apparatus, removal of the solvent and precipitation of bases with ammonia and weighing of the crude base. From the literature review of analytical methods for the determination of alkaloid, a number of methods have been reported, like potentiometeric titration using HCl Hamid and Bakeshi⁵ colorimetric method using bromothymol blue, antimony trichloride⁶. Among these the methods of Gupta and Basu⁷acetic resorcinol-alkaloid complex, methyl orange-alkaloid phase-transfer complex method of Birner⁸were mostly developed suitable HPLC methods are also developed⁹. The HPLC methods were most developed for glycoalkaloids and too sensitive to assay crude sample.

Materials and Methods:

Plant material:

Lawsonia inermis, geographical source: a shrub or a small tree, cultivated throughout India. Family: Lythraceae Linn. syn. Alba Lam, Henna, Family – Lythraceae ) is the eaty species of the monotypic family). It is a glabrous, much branched shrub or small tree with opposite, whorled or spiral, simple and entire leaves and grayish brown bark. The plant is cultivated in many tropical and warm temperature regions as an ornamental and commercial dye crop ^{10, 11}. The plant has been identified by Dr. Stephen Dept. of Botany, American College, Madurai.

Pharmacognostical studies:

The macro and microscopical structure of the leaf, petiole and leaf powder of Lawsonia inermis have been described. The leaves are simple with aromatic smell when crushed and are sweet, mucilaginous and slightly astringent in taste, petiole is concavo- convex in transaction with a stele consisting of an intra xylary phloem and continuous xylem cylinder within. The transverse section of leaf presents an isobilateral structure with ranunculaceous type of stomata on both the surfaces. Some of the epidermal cells are specialized into bigger elliptical mucilage sacs. The parenchyma of leaf contains oil globules and monoclinic prisms of calcium oxalate crystals. Histochemically, the presence of tannins, oil globules and mucilage was confirmed in the leaves. The leaf powder of Lawsonia inermis fluoresced green under ultraviolet light, whereas the powder mounted in nitro-cellulose gave light green with violet tinge, while the powder treated with sodium hydroxide in methanol gave a dark green fluorescence. A method for determining the purity of henna powder has been described. It is based on the determination of mucilaginous sacs present in the epidermal tissue of henna leaves. Spectrophotometric and chromatographic studies of henna leaf and powder extracts have also been used for the appraisal of product quality and detection of adulterants like extraneous dyes. The bark is said to be useful in jaundice and enlargement of spleen, calculous affections, as alternative and in skin diseases and leprosy. The leaves are useful in menorrhagia, leucorrhoea and are also applied externally in headache^12-16. A decoction of leaves is used as an astringent. The leaf juice mixed with water and sugar is given in spermatorrhoea. The leaves are prophylactic against skin diseases. The paste is used in case of the burning sensation of fact. It is also said to have some action in typhoid and haemorrhagic conditions. The plant is considered to be useful by the tribals (Santals) in headache and muscle pains. The flowers are refrigerant and soporific. The leaves are used for colouring palms of hands, soles, nails and for dyeing hair, beard and eye brows^17-21.

Collection of plant materials:

The description of the plant is already given in the preceding pages. The fruits of Lawsonia inermis were collected from six trees near Yanamalai, othaakadai, located on the Madurai-Chennai highway during the months of September and October. T Stalkse removed and the fruits were shade dried for 25 days. The dried fruits were made into a coarse powder and used for different investigate. The fresh fruits were used for certain preliminary experiments enzyme inhibition studies and alkaloid content studies.

Quantitative estimation of alkaloid content:

Method:

Preparation of standard curve A:

Reagents:

1. standard solution of Solasodine was prepared by weighing exactly 10 mg of pure solasodine and dissolve in 25ml of 20% acetic acid A.R., dilutean aliquot a further 10 times with 20% acetic acid. This solution contains 40mcg/ml.

2. Acetate Buffer pH 4.7 – Dissolve 5.44g sodium acetate A.R. in water and 2.40 ml of acetic acid and adjust volume to 100ml.with water.

3. Methyl orange – 0.05 % solution in water.

Procedure A:

Preparation of Standard Curve:

Into four suitable separators are pipette 0,1,2 and 3ml of 40 mcg/ml standard solutions and the volume of each is made up to 5ml with 20% acetic acid, to each separator 5ml of acetate buffer and1ml of methyl orange are added. After shaking for 10 sec. 5ml of Chloroform is added .The separators are stoppered and shaken for 3 min after standing for a few minutes chloroform layers are withdrawn into dry test tubes, dried with small amount of anhydrous Na₂SO₄ and absorbance’s were read on a Spectrophotometer at a420nm using 10mm cells. From the readings the standard curve was constructed Graph 1.

Graph 1

B. ESTIMATION OF CRUDE ALKALOID IN FRESH FRUITS:

One hundred grams of fresh fruits is homogenized with 100ml 2% acetic acid, transferred into two 500ml conical flasks and shaken for 3 hrs. The volume is measured and the suspension is centrifuged from the supernatant an amount equal to one tenth o the measured volume is transferred into a 150ml beaker, heated till boiling and the alkaloid precipitated by addition of 1:2 ammonia in water until the pH reaches 9.5-10.0, the content is transferred into a 100ml conical centrifuge tube and spun for 15min at 2,000 r.p.m. the supernatant is removed by suction or decanting and the precipitate dissolved in 1N HCl. The solution is transferred to a 100ml volumetric flask and adjusted to he mark with 1N HCl. It is then filtered through filter paper into a dry vessel and 5ml pipette into a small flask for hydrolysis on a water bath by refluxing for 2 hrs.

To the flask is then added 5ml of 1N NaOH and 20 ml of acetic acid. The contents transferred into a 100ml volumetric flask and to the mark with water. Each ml of this solution is equivalent to 5 mgs of fresh berries.

1 to 3 ml of this solution is transferred into a separator and the procedure followed as described in A.

C. ESTIMATION OF CRUDE ALKALOID IN DRY FRUITS:

One hundred mgs of dried fruits finely powdered and 40 ml of 95% ethanol are refluxed in a 100-ml flask for 30min. the extract is then filtered. The residue is washed twice with 2ml of ethanol. The washings are added to the original filtrate and transferred into a 50-ml standard flask, the volume being adjusted to the mark with 95% ethanol and 5ml of this solution is pipette into a test tube and ethanol completely removed by evaporation on a water bath. The residue is treated with 3ml of 1N HCl and refluxed for two hours for hydrolysis. The acid is neutralized by adding 3ml of 1N NaOH, to ml of concentrated acetic acid is then added and the content transferred to a 10 ml standard flask, the volume being adjusted to the mark with water. One ml of this solution is equivalent to 1mg of dry material. One to three ml is then pipetted into a separator and the procedure followed as for A. Based on the quantitative estimation and TLC using the system chloroform-methanol 19:1, the alkaloid content was established. The quantitative estimation results were described along with description of isolated compound table-1.

Table- 1 Determination of alkaloid as solasodine content

S. No.	Sample	Total alkaloid	% of total alkaloid based on dry weight
1	Fresh fruit	58	0.58
2	Dried fruit	142	1.42

ISOLATION AND IDENTIFICATION OF PHYTOCONSTITUENTS:

COLUMN CHROMATOGRAPHY:

500 gms of dried fruit powder was placed in 5 liter round bottom flask with 4 litre of distilled methanol for almost 35 days. This solvent was concentrated and the extract is called as methanolic crude extract. The marc was again extracted with 2 liter of hot ethanol. This solvent was evaporated and the residue added to the crude extract. The resultant methanol extract was collected and concentrated to yield dark brown color viscous residue (23 grams). The residue was subjected to column chromatography, for isolation of some compounds.

Preparation of the column:

A glass column of 3.2 cm diameter was packed with activated silica gel (100–200 mesh) in the form of petroleum ether slurry. The column was packed to a height of 45 cms in order to establish a column volume of 375ml.The column was developed according to the following lines, 2 volumes of petroleum ether were allowed to pass through the activated silica gel before the residue was loaded. The solvent was kept above 5 cm above the bed and the residue was carefully loaded. The column was then developed with the series of solvents starting with petroleum ether benzene, chloroform, ethyl acetate and methanol. The different ratios with succeeding solvents were fixed and fractions of 50ml were collected up to ethyl acetate there after methanolic fractions were collected in smaller volumes and checked with TLC and pooled.

Experimental

Melting points were taken in open capillaries and are uncorrected. IR spectra were recorded on a Perkin –Elmer FTIR using KBr discs. PMR on Bruker spectrospeir 200MHz NMR instrument using CDCl₃ as solvent and TMS as internal reference (Chemical shifts in δ, ppm) Elemental analysis of all the synthesized compounds were performed on a Perkin Elmer 2400. Series–II Elemental CHNS analyzer

Phytochemical investigation^22-24

These plant parts were dried, crushed into a coarse form and extracted. Melting points were recorded on a Veego-Vmp-I apparatus. Infra-red-Spectra were recorded on a shimadzu and FT-IR Perkin Elmer spectrometer by using potassium bromide pellet and nujol mul for a solid and semisolid compound respectively. NMR spectra were recorded on a H¹ NMR dueteriated chloroform (CDCl₃) 500 MHz Tetra methylsilane as internal standard.

IDENTIFICATION OF ISOLATED COMPOUNDS:

Compound A

From dried fruits after chromatography from chloroform : ethylacetate 7:3 fraction; amount 27mgs, Brownish shining crystals. Solubility in chloroform. Melting point 251 °c. TLC: Chloroform : methanol 95 : 5 . R_f = 0.49. Spraying reagent is Iodine

IR spectrum 3371(Cm-1)-O-H Stretching, 2945(Cm-1) and 2841(Cm-1)-C-H Stretching,

4680(Cm-1)-C=C Stretching, 1480 C-O Stretching, 139 0 Angular methyl group. NMR Spectrum 0.8 – 1.8 (ppm)-due to CH₃and CH₂ proton in rings. 2.2 (ppm)-CH₂ adjacent to C=C. 4.2 (ppm) – O-H proton. 5.2 (ppm) – may be C=CH proton.

Compound B:

The compound was isolated from benzene; chloroform 7:3 fraction. Amount – 27 mgs, Appearance – Greenish, Solubility – Soluble in ether, Chloroform and absolute methanol, M.P.-218°C. T.L.C:Chloroform; Ethylc acetate (98:2), R_f= . 51.It is detected in UV lamp – Bright Fluorescent. IR Spectra was taken in KBr pellet about 10mgs. On Perkin Elmer IR spectrophotometer ( spectrum attached). The IR peaks and the groups assigned are as flollows.3250 (Cm^-1) - -O-H, 1720 (Cm^-1) – C=O, 1640 C=O, 1100,995 (Cm^-1) – Bending of Aromatic C-H.

Compound C

Isolated from ethylacetate; methanol 7:3 fraction, amount; 40mgs, appearance:brownish shining crystals, Soluble in ethyl acetate and methanol, Melting point: 222°C.

T.L.C: Butanol; Acetic acid; water (4: 1: 5 ), R_f= 0.71, It is detected in UV Detection and Color Spray (1%) Alcoholic ferric chloride, light red colour.

Compound D

Isolated from ethylacetate; methanol 7:3 fraction, amount;40mgs, appearance:brownish shining crystals, Soluble in ethyl acetate and methanol, Melting point: 215°C.

T.L.C: Butanol; Acetic acid; water (4: 1: 5 ), R_f= 0.71, It is detected in UV Detection and Color Spray (1%) Alcoholic ferric chloride, light red colour.

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Received on 25.09.2009 Modified on 11.03.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 736-739