RESEARCH ARTICLE Preliminary Phytochemical ...

RESEARCH ARTICLE

Preliminary Phytochemical Analysis and Screening of Whole Plant of Calotropis gigantean Linn. Mrunal K.Shirsat1*, Gupta S.K1., Ashwat M.S.1, Singhvi I.J.1, Mahatma O.P.2 Abstracts: The genus “Calotropis” includes about 2000 species under the family “Asclepideaceae” (Apocynaeace). There are nearly 280 species in India, especially, in the all dirty place an altitude of more than 500-1300 m. scientific information on their Pharmacognosy, Phytochemistry and Pharmacology are very scant. Hence, the current study describes whole plant of one of those species namely Calotropis gigantean Linn The samples for research were collected from Pratap nagar, Udaipur, India and authentificated by depts. botany, Nagpur University Nagpur and then subjected for morphological, and physicochemical analysis. The parameters from the above were recorded with an objective of drawing an attention on those populations as well as a reference for further scientific investigations. Key Words: Asclepideaceae, Pharmacognosy, Preliminary Phytochemical, Calotropis gigantean Linn. INTRODUCTION The search for anti-inflammatory and analgesic activity in modern times was marked by the introduction of salicin for the treatment of inflammatory swelling due to rheumatic fever and rheumatoid arthritis1. Herbal drugs are being proved as effective as synthetic drug with lesser side effects. Herbal medicines are in line with nature with no hazardous reactions2. Calotropis gigantean Linn (Asclepideaceae) is a small spreading, evergreen tree with short thick crooked trunk commonly known as rui (madar) some time reaching the height of 12 m. native to topical in India and naturalized in warmer part of specially near the water dump. Various part of this plant are in the traditional system of medicine, latex is used as anti-spasmodic, antiasthmic, piles, boils, ulcer, scabies, eczema, leprosy 3, flower as asthma ,cough, cold, catarrh, digestive, stomatic, tonic,4 root and leaves is used for anti-cancer activity5, intermittent fever, paralised part of body, painful joint, swelling, heal wounds.6 Literature survey revealed that latex gives promising anti-inflammatory activity to presence of calotropin, calactin, and βsitisterol. The latex also contain cardiac glycoside, calotropin, calotoxin, uscharin, βamyrin, β–sitosterol 7 have been already investigated and reported in literatures for their medicinal values. It may best suit to state that the phytochemical based pharmacology of those species is very inconsistent or obscure. Hence, it may an absolute necessity to create a profile in regards to their identification and then standardization which may lead to further scientific investigations. This paper encompasses some of the pharmacognostical

1Pacific

college of Pharmacy, Pacific hills, airport road, Pratap nagar extension, Udaipur313003(raj.) India. Email: [email protected] 2B.N.girls

college of Pharmacy, Udaipur Raj. India

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investigations carried out on the whole plant of one of the species namely Calotropis gigantean Linn The assignment such as macroscopy, micro measurements and preliminary Phytochemical screening were performed since the species was not noted for its Pharmacognosy and bioactivity in the past. MATERIALS AND METHODS Plant materials The whole plants specimens for the proposed study were collected in month of January 10, from Pratap nagar area of Udaipur, Rajasthan, India and authenticated as Calotropis gigantean linn by Depts. Botany, Nagpur University Nagpur, voucher specimen no.9111.has been deposited to Botany Department. Physico-chemical and pharmacognostic studies The research specimens were morphologically and organoleptically screened and subjected to physicochemical and parameters such as Extractive values 8, Ash values 9, 10 Histochemical studies11, 12, Fluorescence analysis13 and Micrometrics including leaf constants 14, 15 (Table no 1, 2) Powder microscopy The Whole plants dried in shade were finely powdered separately and screened for the presence of its own and foreign vegetative matters (other than the organ selected for the research studies). The powder was passed through a sieve No.180 and a sieve No.125, separately, to obtain fine and very fine powder respectively and then subjected for microscopic examination. The sample was treated with following reagents and studied for their components of diagnostic value (50% glycerin as temporary mounting; phloroglucinol (2%W/V) in ethanol (90%) and Conc. HCl (1:1) for lignin; 5% W/V of alcoholic ferric chloride for phenolic compounds; 3% Iodine solution for starch grains; and Ruthenium red (0.08%) in 10% lead acetate

for mucilage).also used for specific reagent its Conc. H2SO4, Conc. HCL, Acetic acid, Benzene, Distilled water respectively .for identification for specific colour reaction. Preliminary Phyto-chemical screening The whole plant were dried in shade at room temperature and screened for the presence of foreign matter. The whole plants were ground to a moderately coarse powder in a Mechanical grinder. About 200g of the powder was extracted successively with petroleum ether (60 - 80º C), chloroform, ethanol (95%) and distilled water using soxhlet apparatus. The extraction with each solvent was carried for 24 h. Finally, the marc left was extracted with water by digesting on a boiling water bath. The extraction was continued till a few drops of the last portion of the extract left no residue on drying. The extracts were taken in a tarred porcelain dishes and evaporated to dryness on a water bath and dried at 105º C to a constant weight. The percentage extractives were calculated with reference to air dried drug .16 Powder characteristics Microscopic components of significant diagnostic value were studied under different magnifications; polarized light was subjected to study the starch grains and crystals. The powder was pale green in colour; with no any specific odour; slightly bitter; smooth and slippery in texture. The stomata were of anamocytic in nature. The epidermal cells were lobed with anticlinal walls. The scales were thin plates with a central stalk the scales were in average 80 μm in diameter. The calcium oxalate crystals were of drugs ranging from 17–24 μm in diameter Fragments vascular tissue appeared pinkish to phloroglucinol and HCl (1:1); cortical parenchyma fragment appeared blackish revealed the presence of lignin and phenolic compounds respectively. However, starch grains and mucilage were not evident against addition of Iodine solution and 0.2% Sudan red–III respectively. Physico-chemical analysis Histochemical analysis revealed the presence of phenolic compounds, mucilage and saponin. However, presence of alkaloids and starch grains were not evident .The aqueous extractive value was higher than the alcoholic extractive value revealing presence of larger amount of water soluble constituents in the leaves such as plant acids, carbohydrates and phenolic compounds. The total ash value was higher than that of the acid insoluble and water soluble ash value and a decrease in the acid insoluble ash value may be due to presence of smaller quantity of siliceous matters. The stomatal number is not a reliable parameter for a species. Hence the

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RESEARCH ARTICLE Table No.1: Percentage extractive, fluorescence analysis, and preliminary phytochemical screening of Whole plant of Calotropis gigantean Linn. Fluorescene Chemical constituents Solvent (% W/W) Observed Extracts Alkaloids Sterols Terpenoid Sugar Glycosides Phenolics (UV 366nm) Pet. Ether (602.75±0.076 Orange colour ---++ +++ ----------800C) Chloroform

1.20 ±0.060

Reddish orange colour

-----

++

+

++

++

++

Ethanol 95 %

3.25±0.070

Yellowish Orange

++

++

++

+

+++

++

----

+

++

++

+

Water 7.09±0.067 Greenish Blue ---+ Present, ++ Significantly Present, +++ Prominently Present,, ---- Absent

Table No. 2: Physicochemical analysis and micrometrics of leaves of Calotropis gigantean Linn

Physicochemical analysis Percentage of extractives a)Ethanol b)Water c)Pet. ether d)Acetone e)Chloroform Ash Value %W/W a)Total ash b)Water soluble ash c)Acid insoluble ash d)Sulphated ash Micrometric for leaf only a)Stomatal number b)Stomatal Index c)Vein islet number d)Vein termination number e)Palisade ratio f)Glandular trichomes

Root

Leaf

Flower

15.79 % 15.75 % 14.37 % 15.34 % 7.55 %

11.73 % 10.30 % 6.56 % 7.70 % 8.76 %

55.37 % 54.36 % 9.10 % 6.05 % ----

1.53% 1.45% 0.120% 1.20% min

19.80% 0.110% 10.17% ----mean

11.20% 0.20% 4.20% -----max

7 11.33 22.50 22 3.75 75.50

5.0 8.35 19 20.0 3.2 d 66

9.0 14.30 26 24 4.3 d85

 -Result presented as mean , Percentage of extractive & Ash value result in - % w/w, d- Dimension in -µm, - Range obtained from average of 10 measurements for micrometric analysis

Table No.3: Histochemical Analysis of whole plant Calotropis gigantean Linn A. Cold Test (well prepared reagent without heating) Specimen 2%Phloroglucinol and 3%Iodine Alcoholic FeCl3 conc.HCL solution Leaves Light Yellow Dark Red Dark yellow Flower Light Yellow Dark Red Dark yellow Stem Light Yellow Dark red Dark Yellow Root Light Yellow Dark red Dark Yellow Seed Light Yellow Dark Red Light green NA- No colour indicating in test tube B. Hot Test (well prepared reagent with heating) Specimen 2%Phloroglucinol and 3%Iodine solution Alcoholic FeCl3 conc.HCL Leaves Greenish Dark Brown Green Flower Light Yellow Dark Red Green Stem Light Green Dark red greenish Root NA Light brown Light Greenish Seed Light Yellow Brown Light green

0.2%Sudan RedIII in Alcohol Pink Pink Pink Pink Light Pink

Vannilin Conc.H2SO4 NA Light Pink NA NA NA

0.2% sudan RedIII in Alcohol Pink Light Pink Pale Yellow Pale Yellow Light Pink

Vanillin Conc.H2SO4 Brown Dark Pink NA NA NA

0.2%sudan Red-III in Acohol Light Yellow Light Pink NA NA Light Pink

Vannilin Conc.H2SO4 Red Brown Dark Pink Brown Light brown Dark Pink

NA- No colour indicating in test tube

C. Hot test after 24 hours in day light Specimen 2%Phloroglucinol and conc.HCL Leaves Dark Brown Flower Red Stem Dark brown Root Dark Brown Seed Dark Brown NA- No colour indicating in test tu


3%Iodine solution Light Brown Yellow Yellowish Colour Yellow Yellowish Colour

Alcoholic FeCl3 Green Green Green Green Green


RESEARCH ARTICLE

D. Cold Test (well prepared reagent without heating) Specimen Conc.H2SO4 4ML Leaves Red brown Flower Red Brown Stem Brown Black Root Red Brown Seed Red Brown NA- No colour indicating in test tube E. Hot Test (well prepared reagent with heating) Specimen Conc.H2SO4 4ML Leaves Black Brown Flower Black Brown Stem Black Brown Root Black Brown Seed Black Brown NA- No colour indicating in test tube F. Hot test after 24 hours in day light Specimen Conc.H2SO4 4ML Leaves Black Flower Black Stem Black Root Black Seed Black Brown NA- No colour indicating in test tube measurements were made at the base, midrib and apex of a leaf then the mean of the readings was accounted to render reproducibility. The peltate scale measured from 66-85 μm. Palisade ratio was determined to be not more than 4.3. The vein islets and termination numbers were comparable (Table no.2). Fluorescence analysis showed no any specific fluorescence. The percentage extractive of water was higher than the rest of the extractives. Petroleum ether, chloroform, ethanol and water extracts showed the presence of phytoconstituents such as triterpenoids and sterols where as ethanol (95%) and Aqueous extracts showed phenolic compounds, glycosides, flavonoids, saponins and reducing sugar RESULTS AND DISCUSSION The whole plant of Calotropis gigantean Linn collected from Pratap nagar district, Udaipur India were subjected for macroscopy, physicochemical and preliminary phytochemical analysis. The objective of


Conc.HCL 4ML Dark Yellow Dark Yellow Light Green Green Light red brown

Acetic Acid 4ML Light Yellow Pink Light Yellow NA Cream colour

Benzen 4ML Light Green NA Yellow NA NA

Water 4ML NA NA NA NA NA

Conc.HCL 4ML Dark Green Red Brown Brown Light Brown Dark red brown

AceticAcid 4ML Light Brown Pink Light Yellow NA Light Yellow

Benzen 4ML Green NA Light Yellow NA Light Yellow

Water 4ML Light green Light Yellow NA NA Whitish Colour

Conc.HCL 4ML Back Black Black Brown Black Dark red brown

AceticAcid 4ML Brown Pink Light Yellow NA Light Yellow

Benzen 4ML Green White Light Yellow NA Light Yellow

Water 4ML Light green Cream NA NA Precipitate

investigations was easing the identification of the species in whole and powdered form. The presence of valuable phytoconstituents such as triterpenoid, glycosides and phenolic compounds also demand further phytochemical studies of the species. REFERENCES AND NOTES 1. Harish V. Pharma Time, , 33, 14, (2001). 2. Rama Rao. A.V.and Gurjar, M.K., Pharma Times, 1990, 22, 19, (1990). 3. Indian J.exp, bio., 6,232, (1968) 4. Kirtikar, K.R., Basu B.D., Indian Medicinal Plant, Published by Lalit Mohan Basu, M.B. 49 Allahabad, 1607-1608, (1990) 5. Khare, C.P., Springer, Varlag, Berlin, “Medicinal plant of India An encyclopedia”, New York, 119121, (2004). 6. Medicinal plant, Part-I vol-A Published by center Govt. Press, 49,(1996) 7. Khare C.P., Springer, Varlag, Berlin, “Medicinal plant of India An encyclopedia”, New York, , 119. (2004). 8. Anonymous. Pharmacopoeia of India, Ministry of Health and Family Welfare, The Controller of Publications, New Delhi, 2, A47 - A89. (1996).

9. Anonymous. British Pharmacopoeia, Ministry of Health and Social Services for Northern Ireland, 2, A139 -A140. (1988) 10. WHO/PHARM/92.559/rev.1.Quality Control Methods for Medicinal Plant Materials, Organization Mondiale De La Sante, Geneva, 9, 2234,( 1992). 11. Khandelwal, K.R. Practical Pharmacognosy Techniques and Experiments. Nirali Prakashan, India, 16, 15–163 ( 2006). 12. Lala, P.K. Practical Pharmacognosy, Lina Guha Publication, India, 1, 136-153, (1981). 13. Wahi, A.K., Khosa, R.L. and Mohan, Y. Bot Research. 3,205 ( 1981). 14. Trease, G.E. and Evans, W.C. Pharmacognosy, 10th ed, Berilliee, Tindal, London, 519 – 547(2002). 15. Wallis, T.E. Text Book of Pharmacognosy, 5th ed, CBS Publishers and Distributors New Delhi, India, 559–618. (2005). 16. Harbone J.B., Photochemical methods, 3rd Ed, Chapman and Hall, London, , 49-52( 2005). ACKNOWLEDGEMENTS: We thank to B.N. PG. College of Pharmacy, Udaipur for providing the facilities to carry out the research work.
