INTRODUCTION:

Evaluation of drug means “confirmation of its identity and determination of its quality and purity and detection of nature of adulteration”. The evaluation of crude drug is necessary because of biochemical variation of the drug, deterioration due to treatment and storage and substitutions and adulterations, as results of carelessness, ignorance, fraud. Morphological or organoleptic evaluation refers to evaluation of drugs by color, taste, size, shape and special features like touch, texture etc. Organoleptic evaluation means conclusions drawn from studies resulted due to impressions on organs of senses. The study of form of a crude drug is morphology while description of the form is morphography. Chemical evaluation comprise of different chemical tests and chemical assay.

The isolation, purification and identification of active constituents are chemical method of evaluation. The chemical evaluation includes systemic investigation of plant material for its phytochemical behavior involves four different stages like procurement of raw material and quality control, purification and characterization of the constituents of pharmaceutical interest in process quality control, investigation of biosynthetic pathway to particular compounds. Physical evaluation includes ash values, extractive values, moisture content etc. The percentage of active chemical constituents in crude drug is mentioned on air dried basis. The moisture content of a drug should be minimizing in ordered to prevent decomposition of crude drug either due to chemical changes or microbial contamination. Ash value and extractives may be defined as the process in which the crude drug is treated with specific solvents whereby the medicinally active constituents are dissolved out, cell tissue and most of inactive component remain undissolved. Aqueous solvent, water is used as solvent for extraction of many types of active constituents such as proteins, gum, coloring agent, alkaloids, glycoside, sugars and tannins.

1Blumea lacera var lacera:

Synonym: BENGALI- Kukursunga, Kuksung, Barasukung; GUJRATI- Kalara, Chancharamari; HINDI- Kakranda, kukurbanda, divalimuli, janglimuli, kakronda; KARNATKA- Gandharigidda; MARATHI- Burando, Bhanurda; OIIYA- Pokasunga; SANSKRIT- Kukuradru; TAMIL- Narakakarandai, Kattumulangi; TELGU- Karrupogaaku, Adavimullangi.

Botonical description: Present throughout plains of india.Annual, with strong odor of turpentine; stem erect, 0.3- 0.9 m; Ash colored, dencely glandular-pubescent. Leaves are2.5 by 2.2- 6.3 cm. lower petiolated, upper subsessile, elliptic oblang or ovovate,silky pubescent on both side, sharply serrate dentate, base tappered Flower are heads 6- 8 mm in diameter numerous in short axillary and terminal spiciform panicles; flower yellow. Corolla-lobes hermaphrodite flowers nearly glabrous. Involucral bracts densely, villous, outer bracts somewhat herbacious, linear- lanceolate. Fruits are Achenes not ribbed, small, oblong,sub-4-gouns. Leaves of Blumea lacera are used to get rid of from the worm of the anal region; particularly the children of upto age of three year, leaves are applied on cuts water extract reported to taken orally as anthelmintic. ^(1A) A combination of Blumea lacera, Echinops echinatus Roxb and Datura stramonium juice or paste used to avoid premature ejaculation. ^(1B)Whole plant of blumea lacera possessing wound healing activity. ⁽²⁾Leaf extract of Blumea lacera was most active against test fungus.⁽³⁾Whole plant of Blumea lacera used in skin inflammation.⁽⁴⁾Water extract of Blumea lacera posses a broad spectrum of antimicrobial activity and antifungal activity. ⁽⁵⁾Root paste taken with honey three times a day to cure diarrhea. ⁽⁶⁾Root of blumea lacera used as blood purifier while leaves used to rectify urinary complaints.⁽⁷⁾Blumea lacera an ingredient of an ayurvedic formulation “Pilex” used to treat piles.⁽⁸⁾Plant used as antipyretic, stimulant, astringent, anthelmintic, febrifuge, diuretic and in hemorrhoids^(9), bronchitis ⁽¹⁰⁾sores and wound healing ⁽¹¹⁾The leaves are used as anthelmintic, astringent, diuretic ^{(12, 13, 14),}antidysentric ⁽¹⁵⁾Antimicrobial, for healing cuts ⁽¹⁶⁾in urinary troubles ⁽¹⁷⁾in irritation, as haemostatic⁽¹⁸⁾and anti-inflammatory.⁽¹⁹⁾ The root used in cholera ^{(12, 14)}. The plant was reported to yield compesterol.⁽²⁰⁾,two new glycoside, a triterpenoid glycoside 19 alpha- hydrooxy-12-ene-24,28-dioate 3-o-beta-D-xyloprranoside ⁽²¹⁾A preliminary screening of the plant showed presence of steroids, terpenoids and alkaloids, flavonoids, saponin and absence of tannin and phnolics.⁽²²⁾The essential oil isolated from the leaves yielded cineol, (+)-fenchone and citral.⁽²³⁾, 5-hydroxy-3, 6, 7, 3’, 4’-pentamethoxy flavones; 5, 3’, 4’-trimethoxyflavone and very small quantity of an unidentified flavones along with beta-setosterol were reported from the leaves ⁽²⁴⁾free vitamin c (30 mg/100g) was reported from the leaves ant its content increased significantly (50mg/10g) after frying in oil.^{(25) .}The 50% Ethanolic extract showed effect on guinea pig heart. LD50 of extract was found to be> 1000 mg/kg i.p in mise.⁽²⁶⁾Seeds were reported to exhibit antibacterial activity.⁽²⁷⁾Essential oil obtained from plant leaves showing insecticidal.⁽²⁸⁾

2. Blumea eriantha DC :

Synonym, MARATHI- Nimurdi SANSKRIT: Kukundar, kukundara HINDI: Kukraonda. Dichotomously branched, covered with white and silky hair, distributed in U.P., M.P., Bihar, Karnataka, Maharashtra, and Orissa. Perennial herb up to 1m in height. It is herbaceous; Stem is erect, slender about 30 to 45 cm high, dichotomously branched. Leaves: 2.5-7.5 by 1.5-3.8 cms, alternate and sessile. Leaves are irregularly teethed; these teeth are alternately long and short especially in upper leaves. Lower obovate, short petiolated, upper elliptic- ovate to oblanceolate, cordate clasping. Inflorescence: ¹Heads are 6-8 mm diameter. Corolla lobes of hermaphrodite flowers are hairy. capitula axillary or terminal, with numerous yellow florets, rarely bisexual marginal female, Fruit: achenes brown, shining, sparsely pilos with white pappus. ^(1A)Paste of leaf of Blumea eriantha used for cuts and wounds.⁽²⁹⁾ Ethanolic extract showing adulticidal and repellant activity against Musca domestica .⁽³⁰⁾Ethanolic extract of plant blumea eriantha showing good . larvicidal and adulticidal activity and extract is very good. Insect repeiiant.⁽³¹⁾Potent Antimicrobial activity of volatile oil obtained from fresh leaves of Blumia eriantha D C leaves reported. Ethanolic extract of Blumea lacera and Blumea eriantha showing significant In-vitro antimicrobial activity. ⁽³²⁾Blumea eriantha plant is used in Rheumatic pain.⁽³³⁾Blumea eriantha plant leaves used in treatment of cough and common cold. ⁽³⁴⁾Blumea eriantha Juice of herb used as ‘carminative’. Leaves along Vitex negundo Linn and Careya arborea Boxb as Fomentation, sudorific, diuretic and emmenagogue. Warm infusion is given as sudorific in catarrhal affections .Leaves contain essential oils which have antibacterial, antifungal properties and are also used in dermatological medicaments.⁽³⁵⁾The volatile oil from blumea eriantha and powdered seed showed insecticidal activity against Callosobruchus chinesis ans Sitophillus oryzae two stored grain pests. ⁽³⁶⁾An essential oil possessing camphor like smell and consisting chiefly of α-carvotanacetone and erianthin was obtained from seeds. ⁽³⁷⁾The vapors of leaf oil were found to interfere predominantly with some component of mating behavior of males in boll worms Earias vitella . ^{(38, 39,
40, 41)}LD₅₀ of 50% Ethanolic extract of plant was found to be >1000mg/kg intra-peritoneal in mice. ⁽²⁶⁾Essential oil extracted from leaves of blumea eriantha and stems showed potent antibacterial activity against E.coli, Bacillus subtilis, Vibrio cholera, Staphlococcus aureus, Salmonella typhii. Oil from plant was found to be active against Trichophyton terrestre, Candida albicans, Aspergillus flavus, Aspergillus niger, Aspergillus oryzae.⁽⁴²⁾The structure of erianthin was revised and assigned as 5-hydroxy-3.3’,4’6,7 pentamethoxy flavones.The erianthin (C₂₀H₂₀O₈) m.p.-161 ^oC is identical with artemetin isolated from Artemesia absinthium.^(43).

MATERIAL AND METHODS:

The plants were collected from rural area of Sultanpur (Dwarikganj of KNIMT-Faridipur campus) in the month of March-April 2009, and authentification done by Director and Head of Department Pharmacognosy KNIMT- Faculty of Pharmacy and received Authentification certificate, and for

further references and uses well prepared ‘Herbarium File’ submitted in Department of Pharmacy- under voucher specimen- KNIMT-FOP 2009- 2A and KNIMT-FOP 2009- 2B respectively. Microscopical evaluation as per the schedule given by Sass.⁽⁴⁴⁾, Sectioning as per standard procedure.⁽⁴⁵⁾. Photographs of different magnifications were taken with Nikon Labphot 2 Microscopic Unit. For normal observations bright field was used. For the study of trichomes, crystals, starch grains and lignified cells, polarized light was employed. Since these structures have birefringent property, under polarized light they appear bright against dark background. Magnifications of the figures are indicated by the scale–bars. Descriptive terms of the anatomical features are as given in the standard Anatomy books^{(46a, 46b)}, Extractive value⁽⁴⁷⁾, Ash values^(48), loss on drying⁽⁴⁹⁾ were determined by using standard method. Microchemical evaluation done as per Kay LA (1938) ⁽⁵⁰⁾Fluorescence analysis of the powder and extracts were carried out.⁽⁵¹⁾

Extraction:

The collected, cleaned and powdered samples were used for the extraction purpose. 150 grams of powdered sample was evenly packed in the soxhlet apparatus. It was extracted with 70% ethanol in adequate quantity. The solvent used was purified before use. After solvent extraction, the extract was filtered while hot through Whatman filter paper to remove any impurities if present. The extract was concentrated by vacuum distillation to reduce the volume to 1/10. The concentrated extract was transferred to 100 ml beaker and the remaining solvent was evaporated on the water bath. Then they were collected and placed in desiccators to remove the excessive moisture. The dried extract were packed in air tight container and used for further studies such as phytochemical screening⁽⁴⁷⁾ and toxicological studies as per OECD guidelines.⁽⁵²⁾

RESULTS:

1. Result for microscopical evaluation. TS of Root

Fig. 1: Blumea lacera root without staining

Fig.2:Blume alacera root staining with phloroglucinol: HCl (1:1)

Description-yellowish-green cortex inner cells distinct, elongated thin walled containg oil glands, cotex-narrow parenchymatous rod shaped crystal of calcium oxalate, phloem-parenchymatous containg intercellular spaces, cells present in inner part (towards the cambium) are smaller without intercellular spaces, phloem fibres present, cambium prominent with tangentially elongated typical cells. xylem consist of xylem vessel, xylem parenchyma and medullary rays, xylem vessel lignified, stones cells are present, medullary rays prominent pith present.

Fig. 3: Blumea eriantha DC root without staining

Fig. 4: Blumea eriantha DC staining with phloroglucinol: HCl (1:1)

Table 1: Result for Microchemical evaluation:

Parts	Treatment	Test for	*Reaction B.eriantha*	*ReactionB.lacera*	Results B.eriantha	Results B.lacera
Root	Iodine solution	Starch	Blue color	Blue color (cortex)	++	++
	Ferric chloride solution	Tannin	No color	No color	_	_
	Sudan III solution	Oil globules	No change	No change	_	_
	Con. HCl	Crystals	Effervescence	Effervescence	+	++
	Phloroglucinol :HCl (1:1)	Lignin	Trichomes and vessels turn red	Trichomes ,xylem and vessels turn red	++	++
Stem	Iodine solution	Starch	Blue color (cortex+ pith)	Blue color (cortex+ pith)	++	++
	Ferric chloride solution	Tannin	No color	No color	_	_
	Sudan III solution	Oil globules	Red color	Red color	+	+
	Con.HCl	Crystals	Effervescence	Effervescence	+	++
	Phloroglucinol :HCl (1:1)	Lignin	Trichomes, Vessels, fibres and stone cells turn light pink	Trichomes Vessels, fibres and stone cells turn light pink	+	+
Leaves	Iodine solution	Starch	Blue color cortex+ pith	Blue color cortex+ pith	+	+
	Ferric chloride solution	Tannin	No color	No color	_	_
	Sudan III solution	Oil globules	Red color	Red color	+	+
	Con.HCl	Crystals	No change	Effervescence	_	++
	Phloroglucinol :HCl (1:1)	Lignin	Vessels turn pink.	Xylem .Trichomes turn pink	++	++

Abundantly present= (++); scantly present=( +) ; absent=( -)

Table 2: Result for successive extraction values:

Plant species

Pet. ether

Benzene

Chloroform

Acetone

Alcohol

Water

Blumea eriantha DC (% )

1. Leaves

2. Stem

3. Root

3.10

2.38

2.35

1.32

1.24

1.02

0.82

0.65

0.44

1.12

1.04

1.02

8.8

8.7

8.5

12.9

12.8

12.5

Blumea lacera var lacera (% )

1. Leaves

2. Stem

3. Root

3.11

2.39

2.34

1.34

1.22

1.04

0.93

0.69

053

1.14

1.05

1.03

9.2

9.1

8.9

13.2

13.6

12.4

Table 3: Result for ash values, extractive values and moisture content evaluation:

Values

Blumea eriantha DC

Blumea lacera var lacera

Total ash (% w/w) -

1. Leaves

2. Stem

3. Root

8.5

8.8

7.8

8.4

8.9

7.8

Acid insoluble ash (% w/w) -

1. Leaves

2. Stem

3. Root

1.3

1.1

1.2

1.1

0.99

1.2

Water soluble ash. ( % w/w)-

1. Leaves

2. Stem

3. Root

7.2

7.7

6.6

7.3

7.6

6.8

Alcohol soluble extractive value (%)

1. Leaves

2. Stem

3. Root

12.9

12.8

12.5

13.2

13.6

12.4

Water soluble extractive value. ( %).

1.Leaves

2. Stem

3. Root

8.8

8.7

8.5

9.2

9.1

8.9

Ether soluble extractive value. ( %).

1.Leaves

2. Stem

3. Root

3.0

2.8

2.5

3.1

2.9

2.4

Moisture content ( % )

1.Leaves

2. Stem

3. Root

8.26

13.02

8.48

8.31

12.7

8.21

Description- Brownish -green cortex inner cells distinct, elongated thinwalled containg oil glands, cotex-narrow parenchymatous rod shaped crystal of calcium oxalate, phloem-parenchymatous containg intercellular spaces, cells present in inner part (towards the cambium) are smaller without intercellular spaces, phloem fibres present, cambium prominent with tangentially elongated typical cells. xylem consist of xylem vessel, xylem parenchyma and medullary rays, xylem vessel lignified, stones cells are present. medullary rays prominent. pith present. patches of sieve tubes.

2. TS of leaf

Description-wavy wall, ractangular arrangement, palisade parenchyma elongated, compactly arranged, calcium oxalate crystal present rod shaped, starch grain present. covering trichomes unlignified and uniseriate, multicellular with acute apex, glandular trichomes biseriate multicellular.

Fig. 5: Blumea lacera Staining with phloroglucinol: HCl (1:1

Fig. 6: Blumea lacera covering trichomes without staining

Fig. 7: Blumea eriantha showing covering trichomes

Fig. 8: Blumea eriantha showing glandula trichomes without staining

Description- wavy wall, ractangular arrangement, palisade parenchyma elongated, compactly arranged,collenchyma present, xylem- endarch, calcium oxalate crystal present rod shaped, starch grain present. covering trichomes unlignified and uniseriate, multicellular with acute apex, glandular trichomes biseriate multicellular club shaped..

3. TS of Stem

Fig. 9: Blumea lacera staining with phloroglucinol: HCl (1:1)

Fig. 10: Blumea eriantha staining with phloroglucinol: HCl (1:1)

Description-epidermis single layered with thick cuticle, shunken stomata between vertical ridges present, cortex3-4 layered parenchmatous below epidermis loosely arranged palisade like cells are present, non lignified uniseriate multicellular covering trichomes are present, glandular trichomes are biseriate, multicellular and club shaped, pericyclic fibres are lignified, covering phloem on its outer side, vascular bundles-colateral, conjoint open and arranged in ring, xylem well developed.

Table 4: Result for fluorescence evaluation:

Treatment

Observed under visible ray

Observed under UV ray (254 nm)

Observed under UV ray (365 nm).

B.eriantha

B.lacera

B.eriantha

B.lacera

B.eriantha

B.lacera

Powder as such 1. Leaves

2. Stem

3. Root

DSG

DGG

SYG

DSYG

SYG

BYG

DYG

DYR

DYG

In methanol 1. Leaves

2. Stem

3. Root

DGG

SYG

DGG

SGG

DGG

DYG

SYG

BYG

SYG

DYR

SYR

In methanolic NaOH 1. Leaves

2. Stem

3. Root

DGG

SYG

DYG

SYG

BYG

SBG

BSB

LSB

BSB

In ethanol 1. Leaves

2. Stem

3. Root

DGG

SYG

DYG

SYG

BYG

DCR

BCR

DCR

BCR

In Ethanolic NaOH 1. Leaves

2. Stem

3. Root

DGG

SYG

DGY

SYG

BYG

DSG

BSG

BSB

In dill HCl 1. Leaves

2. Stem

3. Root

DGG

SYG

DYG

SYG

SGG

SYG

DYG

SGG

SBG

DGG

SGG

DGG

SGG

Abbreviations: DSG= Dull Straw Green, GG= Grayish Green, DSYG= Dull Straw Yellowish Green, BYG=Bright Yellowish Green, DYG= Dull Yellowish Green, YG= Yellowish Green, DGG= Dull Grayish Green, DYR= Dull Yellowish Red, SYG= Straw Yellowish Green, BGY= Bright Greenish Yellow, BSB= Bright Sky Blue, DG= Dirty Blue, DCR=Dull Crimson Red,

Table 5: Result for preliminary phytochemical evaluation: Ethanolic extracts (70 % v/v)

Extracts

Constituents

Blumea eriantha DC

Blumea lacera var lacera

1. Leaves

2. Stem

3. Root

Alkaloid

1. Leaves

2. Stem

3. Root

Carbohydrate

1. Leaves

2. Stem

3. Root

Phytosterol

1. Leaves

2. Stem

3. Root

Phenolic compounds

1. Leaves

2. Stem

3. Root

Tannins

1. Leaves

2. Stem

3. Root

Saponin

1. Leaves

2. Stem

3. Root

Glycoside

1. Leaves

2. Stem

3. Root

Terpenoids

1. Leaves

2. Stem

3. Root

Flavenoids

Table 6: Acute toxicity study:

Group

Extracts

No. of animal

Dose in mg/kg

Result

Blumea lacera

Blumea eriantha DC

1.Leaves

2. Stem

3. Root

No death

1. Leaves

2. Stem

3. Root

No death

1. Leaves

2. Stem

3. Root

300

No death

10.

11.

12.

1. Leaves

2. Stem

3. Root

2000

2 death

1 death

Description-Epidermis single layered with thick cuticle, shunken stomata between vertical ridges present, cortex3-4 layered parenchmatous below epidermis loosely arranged palisade like cells are present, non lignified uniseriate multicellular covering trichomes are present, glandular trichomes are biseriate, multicellular and club shaped, pericyclic fibres are lignified,covering phloem on its outer side, vascular bundles-colateral, conjoint open and arranged in ring, xylem well developed.Pith thinwalled, lignified, parenchymatous with intercellular space.

Fig. 11: Blumea lacera leaf staining with phloroglucinol: HCl (1:1)

Fig. 12:Blumea eriantha without staining

DISCUSSION:

Microscopical evaluation basically targeted on diagnostic characters. In Blumea lacera characteristic arrangement of xylem in radial row in the stem, presence of uniserrate, multicelluler covering trichomes and biseriate multicelluler club shaped glandular trichomes which terns pinkish when stained with phloroglucinol+ HCl. Oil glands and ring of stone cells present in the stem of both plants.Thin section of root of both plants showing blue color when treated with weak iodine solution. Stomata are very common in epidermis of leaf in both species and it is diacytic type.

Other diagnostic characters which are analyzed are resinous cells in root, spindle shaped fibres in root, pericycle made up of 1-2 layer of parenchyma in the stem. Physicochemical analysis like total ash, water soluble ash, acid insoluble ash values, different extractive values, fluorescence studies, successive solvent extraction values of both species are presented in table 1- 6. Acute toxicological study result presented in table which clears the LD ₅₀ value of both plant is more than 2000 mg/kg body weight (LD ₅₀> 2000mg/kg body weight.) Therefore ED ₅₀ or dose for pharmacological activity can be easily calculated.

ACKNOWLEDGEMENT:

The author is very thankful to the Director Kamla Nehru Institute of Management and Technology -Faculty of Pharmacy, Sultanpur , for providing the necessary facilities and guidelines to carry out the research work in respect of NIMS University Jaipur Rajasthan India.

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Received on 28.05.2012 Modified on 07.05.2012

Research J. Pharm. and Tech. 5(6): June 2012; Page 834-841