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PHYTO PHARMACOGNOSTICAL AND ISOLATION OF CHEMICAL CONSTITUENTS FROM BAUHINIA VARIEGATA LEAF EXTRACT

 

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ABOUT AUTHOR:
Sudheer kumar Kandibanda
Biju Patnaik University of Technology & Sciences(BPUT)
Orissa
sudheer.y2k8@gmail.com

ABSTRACT
The pharmaceutical sector is focused on development of new drugs and plant based drugs through investigation of leads from traditional system of medicine Ayurveda which is being practiced for thousands of years. Bauhinia variegata is a species of flowering plant in the family Fabaceae. Bauhinia variegata is native to southeastern Asia, from southern China west to Pakistan and India. The major chemical constituents of the plant was found to beflavone, 5,7-dimethoxy-30, 40-methylenedioxy flavone and a new dihydrodibenzoxepin,5,6-dihydro-1,7-dihydroxy-3,4-dimethoxy-methyldibenzoxepin, flavonolglycoside, triterpene saponin, phenanthraquinone, flavonoids.

Bauhinia variegatais found as chemoprotective antitumor activity, hepatoprotectivity,anti-inflammatory, anti-mutagenicity, anti-diabetic,antinociceptive, antioxidant and antihyperlipidemicetc.,In our present investigation we propose to isolate the major phytochemical constituents from Bauhinia variegate to carry out structural characterization by analytical methods for the isolated phytochemical constituents.


Reference ID: PHARMATUTOR-ART-1956

1. TAXONOMICAL CLASSIFICATION


TABLE 1

Kingdom

planate

Sub-kingdom

trachembionta

Super division

spermatophyte

Division

magnoliophyta

Class

Magnoliopsida.

Sub class

rosidae.

Order

roseales

Family

fabaceae

Sub family

caesalpinaceae

Genus

bauhinia

Species

variegata.L.

Botanical name

Bauhinia variegata Linn

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2. VERNACULAR NAMES

TABLE 2

Sanskrit

phalgu

Hindi

Kachanar.

Telugu

adavimandaramu, devakanchanamu

Tamil

Kattaki, kanjani.

Odia

kachan,borada, kosonara, kenjpni, rongakonjono, debokanjoro

Malayalam

chuvannamandaram,   mandaramu

Punjabi

Kanchanal,kovidara, kolar

Kannada

kanchavala,bilimandar

3. PLANT DESCRIPTION

Botanical description:
Botanically Raktakanchanara is identified as Bauhinia variegata and is a member of caesalpinaceae family. Bauhinia which is a large genus belongs to caesalpiniaceous subfamily under which 250 species have been described. In India the family is represented by 16genera. In bauhinia about 15 species occur in India.

Morphology:

Distribution:
Distributed throughout India in areas about 1800 meters in elevation. It is also distributed throughout tropical regions of the world.

Habitat:
Distributed in Punjab, central and south India, Assam, sikkim, chotanagpur, western peninsula, kumaon pakisthan, nepal, bhutan, burma, myanmar and china. It occurs in tropical regions and found throughout India. It is found wild in sub Himalayan tract and outer Himalaya’s upto1300 meters.

Habit:
A medium sized deciduous tree ascending to an altitude of 1300metresin Himalayas found in deciduous forests. Also grown as ornamental tree for its beautiful appearance in flowers.

Bark:
Gray with longitudinal cracks, pale pink inside.Wood moderately hard.Grayish brown with irregular darker patches.

Leaves:
Leaves are 10-15 cm long, petiolate divide into2 lobes. Base is usually deeply cordate, 11-15 nerved. Leaflets 11-13 cm, adnate to about 2/3rds up, ovate- oblong or rounded, nerves 11 from base, transverse nervules.-Prominent, connate for about 2/3rds up, leaflets ovate, rounded apex, upper side glabous.

Flowers:
White/pink, large, fragrant .The uppermost petal darker and variegated usually appearing before the leaves in short axillary or terminal racemes. Pedicels short or sessile. Calyx. Grey tomentose, tube slender 1.3 to2.5 cm long, limbspathaceous, as long as the tube 5 toothed at the apex.Calyx pulses cent outside. Petals.5-6.3 cm

Longobvate or oblanceolate with long rather broad claws all white or 4petals pale purple and the 5 the darker with dark purple veins. Stamens 5fertile, rarely less, staminodesabsent.B.purpurea usually 3fertile stamens, so easily differentiated from B. variegata. B.purpurea Stigma large, oblique.

Fruit:
15-20 /1.2 cmflat,hard dehiscent, dark brown pod glabous. 10-15 seeded.

Substitutes and adulterants:
Bauhinia purpurea, Bauhinia tormentosa,
Bauhinia racemosaare used as substitutes and adulterant.

Varieties:
B.purpureaB.tomentosa.

Synonyms:-
Asphotala,
Kovidasa,
Kundali,
Kuddala,
Asmantaka,
Marika,
Tamrapuspa,
Mahayamalapatraka.

RESEARCH:

  • Plant was found to have anti-tumour, anti-inflammatory, anti-ulcer, anti-helminthic, anti-microbial, amphetamine, hyperactivity, hypothermic activities, chemoprevention, and cytotoxic effect.
  • It is found due to its potency septic activity.it controls internal bleeding from intestines in raktapitta.
  • It is also an anti-fat remedy and therefore valuable for corpulent persons.

Anti-tumour activity:1&2&3
The anti-tumor activity of ethanol extract of Bauhinia variegata has been evaluated against Dalton’s ascetic lymphoma in Swiss albino mice.

Chemoprevention and cytotoxic effect:1&2&5
Chemoprevention and cytotoxic effect of ethanol extract of Bauhinia variegata was evaluated in N-nitrosodiethylamine (200mg/kg) induced experimental liver tumor in rats and human cancer cell lines.

Serine protease inhibitor:1&8&10

  • In this report the properties of two highly homologous inhibitors respectively isolated for Bauhinia variegata and Bauhinia urgulate seeds both inactivate plasma kallikreina.
  • Bark decoction of Bauhinia variegata inhibited the activities of protopectinase and polygalacturonase.
  • The decoctions inhibiting the enzyme activity show the presence of tannins or phenolic Compounds in their tissues.

Anti-microbial activity:1&2&9
The anti-microbial activity of ethanolic extract of total seed proteins, globulins, albumins, fractions of albumins, lectins of Bauhinia variegata was investigated. Ethanol extracts and globulins of different species have no anti-microbial activity the alcoholic extract showed CNS activity.

The clinical studies have revealed that preparation of stem bark of kanchanara enhance the effect of anti-tuberculosis drugs used in cases of tubercular cervical lymphadenitis.

4. Aim & Objective of the Study

  • To carry out the pharmacognostical studies and isolation ofmajor phytochemical constituents fromBauhinia variegata leaf extract.
  • Structural characterization of the isolated compounds by analytical methods

Need of study:
* Bauhinia variegatais a species of flowering plant in the family Fabaceae.Bauhinia variegatais native to southeastern Asia, from southern China west to Pakistan and India.

* The major chemical constituents of the plant was found to beflavanone1, 5,7-dimethoxy-30,40-methylenedioxy flavones and a new dihydrodibenzoxepin, 5,6-dihydro-1, 7-dihydroxy-3, 4-dimethoxy-methyldibenz doxepin, flavonolglycoside2, Triterpene saponin3, phenanthraquinone4, flavonoids.

* Bauhinia variegata is found as chemoprotective6 antitumour activity7, hepatoprotectivity8, anti inflammatory2, antimutagenicity9, anti diabetic10, ant nociceptive, antioxidant and antihyperlipidemic12etc.

* In our present investigation I propose to isolate the major phytochemical constituents from Bauhinia variegate to carry out structural characterisation by analytical methods for the isolated phytochemical constituents.

A through survey of literature indicates that there is a no work done on the pharmacognosy of the drug. The present study is therefore undertaken to study the pharmacognostical and phytochemical screening of Bauhinia variegata Linn leaves which could be used as one of the parameter for the standardization of the crude drug.

4.Plan of Work
1. Pharmacognostical Studies.
a. Collection of plant material.
b. Authentication.
c. Microscopical Study.

2. Stomatal Index & Stomatal Number.

3. Vien islet & Vien termination Number.

4. Physical Evaluation
a. Determination of Moisture Content.
b. Determination of Extractive Value.
c. Determination of Ash Value.

5. Extraction of Plant material and Isolation of chemical constituents

6. To carry out isolation and characterization of major constituents fromBauhinia    variegata leaf extract.

7. Preparation of extracts fromBauhinia variegata.

8. To study the TLC of the prepared extracts Bauhinia variegata.

9. Isolation of phytochemical constituents.

10. Isolation of  markers from extract of Bauhinia variegata by column chromatography / HPLC, Identification and characterization of the isolated phytochemical markers from analytical data’s – IR, NMR.

Materials:

  • The collected plant material was ofter removal of soil and adhering material dried at room temperature for 5-6 days.
  • Powdered to 60 # and were used for the powder study.

5. Methodology:

Pharmacognostical Evaluation:
After decades of serious obsession with the modern medicinal system, people have started looking at the ancient systems like Ayurveda, Siddha and Unani. This is because of the adverse effects associated with synthetic drugs (Thomas et al. 2008). Plants have been associated with the health of mankind from time immemorial

In the past sickness was viewed as a punishment from the god’s hence was treated with prayers and rituals which included “magic proportion” prepared from local herbs (Sandhya et al . 2010).

Herbal drugs play an important role in health care programs especially in developing countries However a key obstacle, which has hindered the acceptance of the alternative medicines in the developed countries, is the lack of documentation of research work carried out on traditional medicines and stringent quality control that have been suitably prepared and standardized (Donald kar et al. 2000) .

With this backdrop, it becomes extremely important to make an effort to wards standardization of the plant material to be used as medicine Pharmacognostical parameters like microscopy, quantitative leaf microscopy, fluorescence, physicochemical and phytochemical studies are few of the basic protocol for standardization of herbals

Hence, in the present work establishment of the pharmacognostical profile of the leaves of Bauhinia variegatais carried out.

Which will assist in standardization, can guarantee quality, purity and it can also be used to prepare a monograph for the proper identification of the plant

5.1 Organoleptic Evaluation:
In organoleptic evaluation, various sensory parameters of the plant material, such as color, odour, taste, shape and texture of the powder were recorded.

TABLE 4

1

Color

Greyish-Brown

2

Odor

Characteristic

3

Taste

Astringent

4

Shape

Curved

5

Texture

Rough

5.2 Microscopical Study.

Leaf constants of Bauhinia variegata
Free hand transverse sections of fresh leaf were taken. Leaf constant such as Stomatal index, palisade ratio, vein islet and vein termination number were determined.
Stomatal index: Stomatal index is the percentage which the numbers of stomata form to the total number of epidermal cells each stoma being counted as one cell.
Stomatal number: is average number of stomata per sq.mm of epidermis of the leaf.

Procedure:
Small piece of Bauhiniavariegataleaf were taken in a test tube with chloral hydrate solution and boiled till chlorophyll is removed and from that small portion of leaf taken and kept on the glass slide covered with cover slip with a drop of glycerin and observed under compound microscope using 10x eye piece and camera Lucida and number of stomata were counted.

Palisade ratio: is defined as average number of palisade cells beneath each epidermal cell.

Procedure:
Small piece of Bauhiniavariegataleaf were taken in a test tube with chloral hydrate solution and boiled till chlorophyll is removed and from that small portion of leaf taken and kept on the glass slide covered with cover slip with a drop of glycerin and observed under compound microscope using high magnification.

Vein-islet number: is defined as the number ofvein-isletsper sq.mm of the leaf surface mid-way between mid-rib and the margin.

Vein-termination number: is defined as the number ofvein-let terminationsper sq.mm of the leaf surface mid-way between mid-rib and the margin

Procedure:
Small piece of Bauhiniavariegataleaf were taken in a test tube with chloral hydrate solution and boiled till chlorophyll is removed and from that small portion of leaf taken and kept on the glass slide covered with cover slip with a drop of glycerin and observed under compound microscope.

Microscopical Investigation:

  • Bauhinia variegata leaf shows single layered epidermis covered with thin cuticle.
  • Epidermis is made up of thin walled, rectangular cells, is followed by 2-3 layers of collenchymatous tissues which are arranged compactly and this is followed by parenchymatous cells which are thin walled and 2-4 layered.
  • Some of the cells Show abundant solitary calcium oxalate crystals.
  • Xylem and phloem are well developed.

Lamina:

  • Bauhinia variegata shows well developed upper and lower epidermis covered by thin cuticle and made up of thin walled rectangular Cells.
  • Palisade tissue is 2 layered, cells are columnar and sponge tissues, are loosely arranged and each nerve of the leaf shows well developed vascular bundle with xylem and phloem.

* Midrib:

  • Bauhinia variegata shows upper and lower epidermis with well-developed thin cuticle. Both the epidermal cells are rectangular.
  • Epidermis is followed by 2-3 layers of collenchymatous tissue and 2-4 layers of thin walled parenchymatous cells.
  • Almost all cells are loaded with abundant solitary crystals of calcium oxalate

* Powder microscopy:
The powder microscopy of B. variegata leaves showed the presence of parenchyma cells with calcium oxalate crystals which are solitary and prism shaped,

Epidermal cells with anomocytic stomata.(The stomata are anomocytic in which the guard cells are not surrounded with any subsidiary cell)

5.3 Physicochemical investigation Bauhinia variegata:
Physical Evaluation: Following parameters were used for physicochemical investigation of plant material

FOREIGN MATTER ANALYSIS
A 100 g of the plant material was spread in a thin layer and the foreign matter was sorted into groups by visual inspection and using a hand lens. The remainder of the sample was sifted through a no. 250 sieve; dust was regarded as mineral admixture. The sorted foreign matter was weighed. The content of each group was calculated in grams per 100 g of air dried sample. The observations were recorded.

LOSS ON DRYING (LOD)
About 2-3 gm. of powder was accurately weighed in a Petridish and kept in a hot-air oven maintained at 1100C for four hours. 

After cooling in a desiccator, the loss in weight was recorded.

This procedure was repeated till constant weight was obtained.

The moisture content of the sample was calculated with reference to air-dried drug and the results are noted

                                                Loss in weight
Loss on drying (%) (LOD)  =    -------------------------- X 100
                                         Weight of the drug (in gms)

Determination of ash values:
The ash remaining following ignition of medicinal plant materials is determined by 3 different methods which measure total ash, acid insoluble ash and water soluble ash.

The total ash is designed to measure the total amount of material remaining after ignition. This includes both “physiological ash” which is derived from the plant tissue itself and “non-physiological ash” which is the residue of the extraneous matter (sand and soil) adhering to the plant surface.

Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth.

Water-soluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water

Total ash
Accurately weighed 2 g of ground dried material was taken in a previously ignited and tarred crucible (usually of platinum and silica). The material was spreaded in an even layer and ignited by gradually increasing the heat to 500-6000C until it was white, indicating the absence of carbon. Then it was cool in a dessicator and weighed. The content of total ash was calculated in mg per g of air dried material.

                               (Z – X)
Total ash (% w/w) =  ---------- X 100
                                  Y

z = weight of the dish + ash (after complete incineration)
x = weight of the empty dish
y = weight of the material taken

Acid-insoluble ash
To the crucible containing total ash, 25ml of hydrochloric acid was added. Crucible was covered with a watch glass and boiled gently for 5 minutes. The watch glass was rinsed with 5 ml of hot water and this liquid was added to the crucible. The insoluble matter was collected on an ash less filter paper (Whatman No. 41) and was washed with hot water until the filtrate was neutral. The filter paper containing the insoluble matter was transferred to the original crucible, dried on hot plate and ignited to constant weight. The residue was allowed

To cool in suitable dessicator for 30 minutes, and was weighed. The content of acid-insoluble ash was calculated in mg per gm. of air dried material.

                                                a
Acid-insoluble ash (% w/w) =   ---------- X 100
                                                y

a = weight of the residue
y = weight of material taken (WHO, 1998)

Water-soluble ash
Another 2 gm. of powder was boiled for 5 minutes with 25 ml of water; the insoluble matter was collected on an ash less filter paper.

Washed with hot water, and ignited for 15 min at a temperature not exceeding 450ºC.

(w-a)

Water soluble ash (%w/w) = ------- X 100

Y
w = Weight of total ash
a = Weight of the residue
y = Weight of the material taken

Extractive values:
This method determines the amount of active constituents extracted with solvents from a given amount of medicinal plant material. It is employed for materials for which as yet no suitable chemical or biological assay exists.

Determination of water soluble extractive
Accurately weighed 4 gm. (Bauhinia variegata) of coarsely powdered air dried material was taken, in a glass stoppered conical flask. The material was macerated with 100 ml of water specified for the plant material concerned for 6 hours, shaking frequently, and then it was allowed to stand for 18 hours. The contents were filtered rapidly. 25 ml of the filtrate was

Transferred to the tarred bottom dish and was evaporated to dryness on water bath. Then it was dried at 1050 C for 6 hours, cooled in a dessicator for 30 minutes and was weighed. The content of extractable matter was calculated in mg per gm. of air dried material.   

Determination ofAcetone-Water (70:30) soluble extractive value:
Accurately weighed (5 gm.) of powder of B. variegata was macerated with 100 ml of Acetone-Water (70:30) in a closed flask, Shaken frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precaution against loss of Acetone-Water (70:30). Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105°C and weighed. Percentage Acetone-Water (70:30) soluble extractive was calculated with reference to the air-dried drug.

Determination of Chloroform soluble extractive value:
Accurately weighed (5 gm.) of powder of B. variegata was macerated with 100 ml of chloroform in a closed flask, Shaken frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precaution against loss of Chloroform. Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105°C and weighed. Percentage chloroform soluble extractive was calculated with reference to the air-dried drug.

Determination of Petroleum ether (40-60°C) soluble extractive value:
Accurately weighed (5 gm.) of powder of B. variegata was macerated with 100 ml of Petroleum ether in a closed flask. Shaken frequently during the first 6 hours and allowed to stand for 18 hours. Thereafter, it was filtered rapidly taking precaution against loss of Petroleum ether. Evaporated 25ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 105°C and weighed. Percentage Petroleum ether soluble extractive was calculated with reference to the powder
1. Extraction
2. Isolation&Purification
3. Identification
4. Characterization

Extraction with chloroform:
200gm of bauhinia variegata leaves were dried and powdered and that powder was extracted with 300ml of chloroform of 100% chloroform for 2hrs at 55-60O C for three times and filter the extract by using  Buchner funnel and filtrate was concentrated by using rota vapour and got the 5gms of powder.

Extraction with Methanol:
200gm of bauhinia variegata leaves were dried and powdered and that powder was extracted with 30ml of Methanol of 100%chloroform for 2hrs at 65-75O C for three times and filter the extract by using  Buchner funnel and filtrate was concentrated by using rota vapour and got the 3gms of powder.

Extraction with Ethyl acetate:
200gm of bauhinia variegata leaves were dried and powdered and that powder was extracted with 300ml of Ethyl acetate of 100% chloroform for 2hrs at 80-85O C for three times and filter the extract by using  Buchner funnel and filtrate was concentrated by using rota vapour and got the 7.5gms of powder.

Due to more yield in ethyl acetate it is used for extraction of bauhinia variegata.

Phytochemical studies:
Phytochemical studies have attracted the attention of plant scientists due to the development of new and sophisticated technique use.

These techniques played a significant role in giving the solution to systematic problems on the one hand, and in the search for additional resources of raw materials for pharmaceutical industry  on the other hand Plant  synthesizes a wide variety of chemical compounds,  which can  be  sorted by  their  chemical  class, biosynthetic origin and  functional  groups  into  primary and  secondary  metabolites.

Primary metabolites make up the physical integrity of the plant cell and are involved with the primary metabolic process of building and maintaining of living cells. Secondary metabolites do not seen to be vital to the immediate survival of the organism that produces them and are not an essential Part of the process of building and maintaining living cells.

With the development of natural product chemistry, the potential of chemotaxonomy is now b becoming increasingly obvious the application of chemical data to systematics has received serious attention of a large number of biochemists and botanists during the last three decades.

The plant may be subjected to preliminary phytochemical screening for detection of various bioactive chemical constituents.  The important steps involved in the phytochemical screening of a plant include extraction of constituents using suitable solvents followed by screening with various chemical tests.

In the process of phytochemical screening, the crude extracts or isolated constituents are subjected to qualitative and quantitative chemical analyses. Qualitative chemical analysis includes the determination of nature of the constituents in an extract or its fractions which lead to the isolation of the active lead compound.

Quantitative chemical analysis includes the determination of the purity of isolated substances or group of substances in a mixture by finger printing and different analytical techniques.

Phytoconstituents of different extracts of Bauhiniavariegata
The coarse powder of leaves of Bauhinia variegata (25 g) was subjected to successive extraction with different solvents in their increasing order of polarity from petroleum ether (60-800C), chloroform, methanol and water. The extracts were concentrated and subjected to various chemical tests to detect the presence of different phytoconstituents with diff chemical tests.

Qualitative tests:

A) Test for carbohydrates;

1. Molisch Test:
It consists of treating the compounds of a-naphthol and concentrated sulphuric acid along the sides of the test tube. Purple color or reddish violet color at the junction between two liquids.

2. Fehling’s Test:
Equal quantity of Fehling’s solution A and B is added. Heat gently, brick red precipitate is obtained.

3. Benedict’s test:
To the 5ml of Benedict’s reagent, add 8 drops of solution under examination. Mix well, boiling the mixture vigorously for two minutes and then cool. Red precipitate is obtained.

4. Barfoed’s test:
To the 5ml of the Barfoed’s solution add 0.5ml of solution under examination, heat to boiling, formation of red precipitate of copper oxide is obtained.

B) Test for Alkaloids

1.Dragendroff’s Test:
To the extract, add 1ml of Dragendroff’s reagent Orange red precipitate is produced.

2.Wagner’s test:
To the extract add Wagner reagent. Reddish brown precipitate is produced.

3. Mayer’s Test:
To the extract add 1ml or 2ml of Mayer’s reagent. Dull white precipitate is produced.

4. Hager’s Test:
To the extract add 3ml of Hager’s reagent, yellow precipitate is produced.

C) Test for Steroids and Sterols

1.Libermann Burchard test:
Dissolve the test sample in 2ml of chloroform in a dry test tube. Now add 10 drops of acetic anhydride and 2 drops of concentrated sulphuric acid. The solution becomes red, then blue and finally bluish green in colour.

2.Salkowski test:
Dissolve the sample of test solution in chloroform and add equal volume of conc. sulphuric acid. Bluish red cherry red and purple color is noted in chloroform layer, whereas acid assumes marked green fluorescence.

D) Test for Glycosides

1.Legal’s test:
Sample is dissolved in pyridine; sodium nitropruside solution is added to it and made alkaline. Pink red colour is produced.

2.Baljet test:
To the drug sample, sodium picrate solution is added. Yellow to orange colour is produced.

3. Borntrager test:
Add a few ml of dilute sulphuric acid to the test solution. Boil, filter and extract the filtrate with ether or chloroform. Then organic layer is separated to which ammonia is added, pink, red or violet colour is produced in organic layer.

4. Killer Killani test:
Sample is dissolved in acetic acid containing trace of ferric chloride and transferred to the surface of concentrated sulphuric acid. At the junction of liquid reddish brown color is produced which gradually becomes blue.

E) Test for Saponins

Foam test:
About 1ml of alcoholic sample is diluted separately with distilled water to 20ml, and shaken in graduated cylinder for 15 minutes.1 cm layer of foam indicates the presence of saponins.

F) Test for Flavonoids
Shinoda test:

To the sample, magnesium turnings and then concentrated hydrochloric acid is added. Red colour is produced.

Lead acetate test:
The extracts were treated with few drops of lead acetate solution; formation of yellow precipitate indicates the presence of flavonoids.

Alkaline reagent test:
The extracts were treated with few drops of sodium hydroxide separately. Formation of intense yellow color, which becomes colorless on addition of few drops of dilute acid, indicates the presence of flavonoids

G) Test for Triterpenoid
In the test tube, 2 or 3 granules of tin was added, and dissolved in a 2ml of thinly chloride solution and test solution is added. Pink colour is produced which indicates the presence of triterpenoids.

H) Test for Protein and Amino acid

1. Biuret test:
Add 1 ml of 40% sodium hydroxide and 2 drops of 1% copper sulphate to the extract, a violet colour indicates the presence of proteins.

2.Ninhydrin test:
Add 2 drops of freshly prepared 0.2% ninhydrin    reagent to the extract and heat. A blue colour develops indicating the presence of proteins, peptides or amino acids.

3.Xanthoprotein test:
To the extract, add 20% of sodium hydroxide or ammonia. Orange colour indicates presence of aromatic amino acid. (Evans W.C 1996, Kandelwal, 2004.)

6. CHARACTERIZATION
HPTLC FINGERPRINT OF BAUHINIAVARIEGATA LEAF EXTRACT

INSTRUMENT:

CAMAG High Performance Thin Layer Chromatography (HPTLC)

Comprising of:
Applicator –Linomat 5, digistore-2, Multiwavelength scanner. Transparent chromatographic tank and HPTLC pre coated silica gel plates, silica gel 60F254, 10X10 cm (Merck)

Sample preparation A
Extract 500mg of Bauhiniavariegata leaf extract with 50ml methanol for 10 minutes. Filter and concentrate to 5 ml. proceed for spotting.

Sample preparation B
Extract 5 mg of BLE/RD/01 isolated compound with 10 ml methanol for 10 minutes filter and concentrate to 5 ml.proceed for spotting.

Sample preparation C
Extract 5 mg of BLE/RD/01 reference STD with 10 ml methanol for 10 minutes filter and concentrate to 5 ml.proceed for spotting.

Mobile phase
Benzene: Ethyl acetate