INTRODUCTION:

Herbal plants are natural source for obtaining naturalmedicines. It has low side effect and cheap medicaments as compare to the synthetic medicines. According to World Health Organization the 80% population of India has relies on Herbal medicines, and used for treatment of various diseases. The herbalism divided into different regions such as Traditional chinese herbalism and Ayurvedic herbalism. The Traditional drugs have that much importance as the herbal drugs but, the Ayurvedic system is the most important system in India.

There are various sources for obtaining natural products such as microorganism, animals, plants and marine sources.

· Plant Name: Caralluma fimbriata wall.

· Family: Asclepiadaceae

· Synonym: Caralluma fimbriata wall.

MATERIALS AND METHODS:

Drugs, chemicals and solvents:

Petroleum ether (60/80°), Methanol, Isopropyl alcohol, Benzene, Glacial acetic acid, Silica gel

Extracts used:

a) Methanolic extract of whole plant of Caralluma fimfriata wall.

b) Aqueous extract of whole plant of carallumafimbriata wall.

Instruments used:

a) Soxhlet continuous extraction apparatus (Borosil Mumbai)

b) IR Spectrophotometer Jasco FTIR- 8400

c) UBSET Jasco V-550 UV/Vis- spectrophotometer

d) Melting point apparatus-Veego VMP-1

Collection of plant:

Caralluma fimbriata plant material were collected from Dandoba Hill, Pandharpur road, Miraj (Maharashtra)

Authentification of the plant material:

Mr. M. D. Wadmare Dept. of Botony, Smt. Kasturbai Walchand College Sangli, authenticated the plant material.

Drying of Whole plant Material:

In the present study the collected plant material were sorted carefully and washed thoroughly to remove unwanted and debris. The material was dried in air. After complete drying the plant material were powdered by mixer grinder to obtain coarse powder.

Extraction of C. fimbriata Wall.:

The extaction of whole plant material was done by soxhlet extraction Method

Preparation of Methanolic extract of C. fimbriata Wall:

The aerial part of plant wasdefatted with the Petroleum ether to remove fatty material from plant. The Defatted material further extracted with pet ether by using methanol as a solvent, at the temperature 40-60 degree the extraction was continue until the extract or liquid of thimble became clear. After completion of extraction, the extract was concentrated at low temperature and also by using rotavapour. The extract was kept for drying in dessicator. The extract was used for further phytochemical analysis.

Phytochemical Screening of C. fimbriata Wall.

Table No. 1 Phytochemical Test for identification of chemical constituent

Sr. No.	Test	Flavanoid	Alkaloid	Steroid	Glycoside
1	Mayer test	-	+	-	-
2	Hagers test	-	+	-	-
3	Lieberman test	-	-	+	-
4	Lieberman Burcherd test	-	-	+	-
5	Keller killani test	-	-	-	+
6.	Saponin test	-	-	-	+
7.	Shinoda test	+	-	-	-

Isolation of Chemical Constituent by Using Chromatographic Technique

Thin layer chromatography

Different constituent of extract of Plant material were separated by thin layer chromatographic plates containing silica gel in the Isopropyl alcohol: Benzene: Glacial acetic acid (2:8:1)

Table no. 2 Number of spot and Rf value

Sr. No.	Sample	R_Fvalue
1.	Spot No 1	0.90
2.	Spot No 2	0.72
3.	Spot No 3	0.63

Fig No1 TLC

Isolation of Chemical Costituent by using column Chromatography:

The Different chemical constituent were separated by column chromatography by using mobile phase such as Isopropyl alcohol: Benzene: Glacial acetic acid (2:8:1) and all the chemical constituent were collected. And compared with standard drug, Physical and Chemical properties were evaluated. These isolated constituent were subjected for spectral analysis.

Physical and Chemical test for isolated fraction

Table No.3 Physical and chemical test of Isolated fraction

Test	Observation	Inference
Physical evaluation Melting point	130-132°c	Phytosterol (B-sitesterol) might be present.
Chemical evaluation a) Isolated fraction + chloroform+ conc. H₂SO₄	Reddish color in upper layer	Sterol present
b) Isolated fraction +H₂SO₄+ drops of acetic anhydride	First red, then blue and finally green color appears.	Sterol present
c) Isolated fraction + acetic anhydride. Heat and cool. Add few drops of H₂SO₄	Blue color appears	Sterol present

Structural Elucidation:

The structural elucidation were done by UV-Visible Spectrophotometer, Infrared Spectrocopy, NMR, Mass Spectrophotometer.

Table No.6 Major ion fragment of Isolated compound

Probable molecular fragments	Peak (m/z)
	413
	371
	302
	218
	114

Pharmacological Study:

Experimental The Methanolic extract of C. fimbriata Wall and The dose was determined by depending on oral acute toxicity study.

Neutrophil Adhesion Test:

Rats were divided into four groups of five animals each. The rats were divided into grps such as control, test 1 and test 2 and standard. The dose and treatment were showed in the following chart.

Table No 7 Number of grps and treatment

Sr. No	Groups	Treatment, Dose and Route
1.	Control	Distilled water
2.	Test 1	C. fimbriata Wall 100mg/kg(oral)
3.	Test 2	C. fimbriata Wall 150mg/kg(oral)
4.	Standard	Levamisole 50mg/kg(oral)

The blood samples from all the groups were collected by puncturing retro orbital plexus under mild ether anesthesia. Blood was collected in vials pre-treated by disodium EDTA and analyzed for total leukocyte count (TLC) and differential leukocyte count (DLC) by fixing blood smears and staining with Leishman’s stain. Percent neutrophil adhesion was calculated as follows,^[3]

Neutrophil adhesion (%) = NIu – NIt × 100/NI

RESULT AND DISCUSSION:

· The Extraction of whole plant material of Themethanolic extract of C. fimbriata Wall wallwas done by soxhlet extraction process. The plant extract was dried under dessicator and the extract further subjected to Phytochemical analysis.

· The extract were subjected to chromatographic analysis by thin layer chromatography, the mobile phase was Isopropyl alcohol: Benzene: Glacial acetic acid (2:8:1), from the thin layer chromatography it was showed 3 spots This spot were isolated by using Column chromatography.

· The isolated spot were identified by the physical and chemical analysis

· The isolated spot were subjected to spectral analysis for structural elucidation.

Spectroscopic study:

UV analysis of isolated sterol compound found maximum at 220 nm which was comparable with wavelength of standard sterol (b- sitesterol)

IR spectrum of sterol compound

Exhibitated characterstick peaks at 3370.96 (O-H str.), 2922.29 (aliphatic CH), 1072.23 (Cycloalkane), 3234.04 (cyclic olefinic, -HC=CH), 1590.02 (C-C), 1650.77 (C=C).

H¹NMR Spectrum of sterol exhibited peaks at 0.805(angular methyl group), 1019 (terminal CH₃), 2.23(OH group), 7.3 (aromatic group).

Mass Spectrum of sterol compound showed molecular mass at 413 having characteristic fragments observed at m/z 371,302,218,114.

Pharmacological Activity:

Neutrophil adhesion in control group animals was 30.68, in extract treated group animals at dose 100mg/kg body weight was 43.05, while treated animals group 150mg/kg body weight was 47.83. Standard drug treated animal group was 72.71.

There was a significant increase in % neutrohil adhesion for 100mg/kg 150mg/kg body weight and the standard Levamisole 50mg/kg body weight as compared to the control group increases% neutrophil adhesion value to 43.05±0.37, 47.83±0.51, 72.71±0.38 respectively.

The %neutrophil adhesion was significantly increased by C. fimbriata Wall. (100mg/kg/oral) and (150 mg/kg/oral) when compared with the control group, showed possible immunostimulant effect

Table no.8 Observation table of Neutrophil adhesion

Sr. No	Groups	Treatment, Dose and Route	Neutrophil adhesion (Mean ±S.E.M.)
1.	Control	Distilled water	30.684±0.47
2.	Test 1	C. fimbriata Wall 100mg/kg(oral)	43.054±0.37
3.	Test 2	C. fimbriata Wall 150mg/kg(oral)	47.832±0.51
4.	Standard	Levamisole 50mg/kg(oral)	72.71±0.38

Statistical Analysis:

All the results were expressed as mean ± Standard Error (SEM). Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Tukey-Kramer multiple comparison test

Fig no. Graph of %Neutrophil Adhesion Test

CONCLUSION:

From above observation, it was concluded that, the C. fimbriata Wall plant material contain different chemical constituent such as alkaloid, flavonoid, glycoside, steroid. In the immunomodulatory activity, from the observation it was showed that the C. fimbriata Wall plant shows increase in immunomodulatory activity as compared with control group, it may be due to presence of steroid in Plant material.

ACKNOWLEDGMENT:

Author Thankful and grateful to the Appasaheb Birnale College of Pharmacy, Sangli, for providing all necessary requirement for the completion of researchwork.and also thankful to the Principal Dr. D. D. Chougule Sir and Research Guide Dr. P. J. Shirote.

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Received on 04.06.2019 Modified on 25.06.2019

Res. J. Pharmacognosy and Phytochem. 2019; 11(4):217-221.

DOI: 10.5958/0975-4385.2019.00037.2

Sr No.	δ ppm	Indication
1	0.805(S)	Angular Methyl group
2	1.19(d)	terminal CH3
3	2.23(s)	OH group
4	7.3(s)	Aromatic Group

Sr No.	Wave no. (cm^-1)	Functional group
1	3370.96	O-H str.
2	2922.29	aliphatic C-H str.
3	1650.77	C=C str.
4	1072.23	cycloalkane
5	3234.04	Cyclic oiefin
6	1590.02	C-C