Analysis of Crude Drugs Present In the Hepatoprotective Polyherbal Formulation by HPTLC Technique

 

C. S.  Kandasamy1*, Sai Lohit Ch.1, R. Siva Kumar1,V. Gopal2, Chandini, R. Nair3 and R. Venkatanarayanan1.

1Department of Pharmacognosy, R.V.S College of Pharmaceutical Sciences, Sulur, Coimbatore.

2Department of Pharmacognosy, College of Pharmacy, Mother Theresa Institute of Health Sciences and Research Institute, Pondicherry.

3Department of Biochemistry, SASTRA University, Tanjore.

 

ABSTRACT:

Extracts of crude drugs such as Ajowan, Mace, Cardamom, Cumin, Nutmeg and Clove used in the Polyherbal formulation - RVSPHF567 were taken in account and dissolved in their appropriate solvents. These extracts were analyzed individually by High Performance Thin Layer Chromatography (HPTLC) to detect the presence of their phytoconstituents. The instrument used for High Performance Thin Layer Chromatography (HPTLC) was CAMAG LINOMAT 5. These analytical investigations were highly useful for preparing Polyherbal formulation - RVSPHF567 which is highly effective as Hepatoprotective agent and can be used in-vivo as a potential therapeutic agent. The sample was prepared by solvent extraction. Then 2µl of test solution was loaded as 6mm band length on the 9 x 10 Silica gel 60F254   TLC plate. This was done by using Hamilton syringe. These plates were kept in respective mobile phase Chloroform: Methanol: Water (6.5: 2.5: 0.4) for spot development to 90mm. These plates were dried and kept in Photo-documentation chamber - CAMAG REPROSTAR 3. The images were captured at White light, UV 254nm and UV 366nm. The developed plates were sprayed with respective spray reagent and photo-documented in White light and UV 366nm mode. Then the same were scanned at 500nm. The Peak table, Peak display and Peak Densitogram show the terpenoids profile in all the crude drugs.

KEYWORDS: Polyherbal, Liver Protective.

 

 

The practice of herbal medicine dates back to the very earliest periods of known human history. There is evidence of herbs having been used in the treatment of disease and for revitalizing body systems in almost all ancient civilization like Indian, Chinese and even the Greek Roman civilization. India, with the varied heritage and geographical boundary has been as the botanical garden of the world1. The Polyherbal formulation consists of the crude drugs such as Ajowan, Mace, Cardamom, Cumin, Nutmeg and Clove. The extracts of each crude drug useful for preparing Polyherbal formulation - RVSPHF567, which is highly effective as Hepatoprotective agent2.

 

A simple, precise, accurate and rapid reverse phase high performance thin layer chromatographic method is developed3. HPTLC analysis of solvent extract of crude drugs can be carried out by using Linomat IV applicator and Camag scanner III with Cats 4 software4.

Table – I: SOLUBILITY OF THE CRUDE DRUGS IN DIFFERENT SOLVENTS

Drugs

Alcohol

Benzene

Chloro-form

Water

Ajowan

Partially soluble

Partially soluble

Partially soluble

Completely soluble

Cardamom

Completely soluble

Partially soluble

Partially soluble

Partially soluble

Clove

Partially soluble

Partially soluble

Partially soluble

Completely soluble

Cumin

Completely soluble

Partially soluble

Partially soluble

Partially soluble

Mace

Partially soluble

Partially soluble

Completely soluble

Partially soluble

Nutmeg

Completely soluble

Partially soluble

Partially soluble

Partially soluble

 

Drugs

Carbon tetra chloride

Dichloro- ethane

Dichloro-methane

Ajowan

Partially soluble

Partially soluble

Partially soluble

Cardamom

Partially soluble

Partially soluble

Partially soluble

Clove

Partially soluble

Partially soluble

Partially soluble

Cumin

Partially soluble

Partially soluble

Partially soluble

Mace

Partially soluble

Partially soluble

Partially soluble

Nutmeg

Partially soluble

Partially soluble

Partially soluble

 

 

 

Patients taking a pharmaceutical product should be safe and efficacious. Pharmaceutical regulatory agencies worldwide demand that the product remains its identity, quality, purity and potency for the identity, quality, purity and potency for the time the product is commercially available5.

 

High performance thin layer chromatography plays a vital role to determine the presence of phytoconstituents quantitatively and qualitatively. It helps in the phytochemical investigation of crude drugs.

 

MATERIAL AND METHODS:

EXPERIMENTAL DESIGN:

The crude drugs were powdered well by grinding and avoiding the moisture contamination. These powdered drug passed through sieve for obtaining uniform particle size Then these powdered crude drugs was dissolved in several solution to determine their optimum solubility. The powdered drugs were dissolved in their appropriate solvent. Table No. I and II.

 

DIFFERENT SOLVENTS USED:

Carbon Tetra-Chloride, Benzene, Chloroform, Di-Chloro Ethane, Di-Chloro Methane, Ethyl acetate, Acetone, Water and Alcohol.

 

SAMPLE PREPARATION:

The extract sample solutions were used as test solution for HPTLC analysis.

 

SAMPLE APPLICATION:

2µl of test solution was loaded as 6mm band length in the 9 x 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.

 

SPOT DEVELOPMENT:

The samples loaded plate was kept in TLC twin trough developing chamber (after saturated with solvent vapour) with respective mobile phase and the plate was developed in the respective mobile phase up to 90mm.

 

 

PHOTO-DOCUMENTATION:

The developed plate was dried by hot air to evaporate solvents from the plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at White light, UV 254nm and UV366nm.

 

DERIVATIZATION:

The developed plate was sprayed with respective spray reagent and dried at 100° C in Hot air oven.  The plate was photo-documented in White light and UV 366nm mode using Photo-documentation (CAMAG REPROSTAR 3) chamber.

 

SCANNING:

Finally, the plate was fixed in scanner stage and scanning was done at 500nm.  The Peak table, Peak display and Peak densitogram were noted.

 

ANALYSIS DETAILS:

MOBILE PHASE:

Chloroform: Methanol: Water (6.5: 2.5: 0.4)

 

SPRAY REAGENT:

Sprayed Libermann Burchard reagent and dried at 100° C 5min.

 

Detection:

Violet, violet-brown colored zones at Day light mode were present in the tracks, it was observed from the chromatogram after derivatization, which confirmed the Presence of Terpenoids in the given samples.

 

Table – II: EXTRACTS OF CRUDE DRUGS USED FOR HPTLC ANALYSIS:

Sample no

Crude drugs

Solvent extract

1

Ajowan

Water extract

2

Cardamom

Alcoholic extract

3

Clove

Water extract

4

Cumin

Alcoholic extract

5

Mace

Alcoholic extract

6

Nutmeg

Water extract

 

 

TABLE – III: ANALYTICAL EVALUATION REPORTS

HPTLC ANALYSIS OF GIVEN EXTRACT FOR TERPENOID PROFILE

Track

Peak

Rf

Height

Area

Assigned substance

Sample no.1

1

0.02

309.5

3548.8

Unknown

Sample no.1

2

0.16

12.6

197.1

Unknown

Sample no.1

3

0.19

10.0

99.2

Unknown

Sample no.1

4

0.30

18.4

460.3

Unknown

Sample no.1

5

0.32

24.0

351.5

Unknown

Sample no.1

6

0.41

31.1

872.1

Terpenoid 1

Sample no.1

7

0.86

21.2

941.8

Unknown

Sample no.1

8

0.97

10.3

155.1

Unknown

Sample no.2

1

0.06

52.0

905.5

Terpenoid 1

Sample no.2

2

0.12

28.7

502.3

Terpenoid 2

Sample no.2

3

0.14

33.2

780.5

Terpenoid 3

Sample no.2

4

0.33

22.1

549.7

Unknown

Sample no.2

5

0.85

23.1

393.8

Unknown

Sample no.2

6

0.89

30.5

828.6

Unknown

Sample no.2

7

0.94

45.5

2254.3

Unknown

Sample no.2

8

0.97

27.0

398.7

Unknown

Sample no.3

1

0.12

36.6

873.7

Terpenoid 1

Sample no.3

2

z0.14

42.6

1092.1

Terpenoid 2

Sample no.3

3

0.19

18.1

131.5

Unknown

Sample no.3

4

0.32

14.4

226.8

Unknown

Sample no.3

5

0.85

156.5

8372.2

Terpenoid 3

Sample no.4

1

0.02

31.3

230.7

Terpenoid 1

Sample no.4

2

0.05

52.8

806.3

Terpenoid 2

Sample no.4

3

0.11

21.5

590.2

Terpenoid 3

Sample no.4

4

0.14

24.8

684.4

Terpenoid 4

Sample no.4

5

0.29

43.3

1341.9

Terpenoid 5

Sample no.4

6

0.37

19.1

341.7

Unknown

Sample no.4

7

0.40

42.6

1102.2

Unknown

Sample no.4

8

0.45

22.3

589.1

Unknown

Sample no.4

9

0.54

13.4

360.8

Unknown

Sample no.4

10

0.61

17.0

448.7

Terpenoid 6

Sample no.4

11

0.93

76.3

7074.5

Unknown

Sample no.5

1

0.05

79.1

1266.5

Terpenoid 1

Sample no.5

2

0.10

12.5

127.7

Terpenoid 2

Sample no.5

3

0.11

18.2

212.5

Unknown

Sample no.5

4

0.21

11.9

117.3

Unknown

Sample no.5

5

0.25

20.4

194.0

Unknown

Sample no.5

6

0.36

10.5

236.5

Unknown

Sample no.5

7

0.52

18.0

469.1

Unknown

Sample no.5

8

0.73

30.6

825.9

Unknown

Sample no.5

9

0.90

167.0

14542.9

Terpenoid 3

Sample no.6

1

0.07

13.3

87.5

Unknown

Sample no.6

2

0.41

41.8

1282.8

Unknown

Sample no.6

3

0.47

49.9

674.2

Unknown

Sample no.6

4

0.57

82.5

3220.6

Unknown

Sample no.6

5

0.61

99.9

3270.5

Unknown

Sample no.6

6

0.69

153.1

7700.7

Terpenoid 1

Sample no.6

7

0.79

291.6

17946.4

Terpenoid 2

Sample no.6

8

0.87

470.1

26581.5

Unknown

 

 

RESULTS AND DISCUSSION:

The HPTLC study was carried out for terpenoids profile (Table-3) (Fig. I-XII). The studies indicated that water extract of sample no.1 contain 8 components, amongst which the 6th component is terpenoids. The alcoholic extract of sample no. 2 contains 8 components in which 1st, 2nd and 3rd components are terpenoids. The water extract of sample no. 3 contains 5 components in which 1st, 2nd and 5th components are terpenoids. The alcoholic extract of sample no. 4 contains 11 components in which 1st, 2nd. 3rd, 4th, 5th and 10th components are terpenoids. The chloroform extract of sample no. 5 contains 9 components in which 1st, 2nd and 9th components are terpenoids. The alcoholic extract of sample no. 6 contains 9 components in which 6th and 7th components are terpenoids.

 

CHROMATOGRAM

BEFORE DERIVATIZATION

FIGURE – I                              FIGURE – II

FIGURE – III

 

AFTER DERIVATIZATION

FIGURE – IV                                              

FIGURE – V

 

PEAK DENSITOGRAM DISPLAY

FIGURE – VI        

Track 1 (Sample no.1)

 

FIGURE – VII        

Track 2 (Sample no.2)

 

FIGURE – VIII       

Track 3 (Sample no.3)

 

FIGURE – IX

Track 4 (Sample no.4)

 

FIGURE – X

Track 5 (Sample no.6)

 

FIGURE –XI

Track 6 (Sample no.7)

 

FIGURE –XII

 

3-D Display of all Tracks

 

CONCLUSION:

High performance thin layer chromatography plays a vital role to determine the presence of phytoconstituents quantitatively and qualitatively. The crude drugs (Ajowan. Cardamom, Clove, Cumin, Mace, Nutmeg) shows the presence terpenoids, which can be highly efficient in the therapeutic use of these drug formulation.

 

ACKNOWLEDGEMENT:

Our sincere thanks to the Dalmia Centre for Research and Development, Coimbatore, Tamil Nadu, India.

 

REFERENCES:

1.       Subba Rao C, Murthy T.E.G.K, Ravi Kumar A (2007) Studies   on evaluation of Allium sativum Linn in Lasunadivati Ayurvedic formulation, Adv. Pharmacol. Toxicol. Vol. 8 (2), P. No. 45-49.

2.       Kandasamy C.S, Mohammed Basil E, Shimna Thasnim P.S, Siva Kumar R, Gopal V, Venkatnarayanan R (2010) Hepatoprotective Activity of Polyherbal Formulation Containing Some Indigenous Medicinal Plants in Rats, Research J. Pharm. and Tech. 3(3): 828-831.

3.       Saini PK, Singh RM, Mathur SC, Singh GN, Tuteja S, Singh UK (2010) Indian Drugs. Vol 47(5): 42

4.       Heeshma C. Shah, Vinayak Naik, Pratima Tatke, Kamalinder K. Singh. (2009) Indian J. Nat. Prod., Vol 25(2): 10.

5.       Jadhav VM, Mande PP and Kadam VJ (2009) Indian Drugs Vol 46(12): 32.

 

 

Received on 17.07.2011

Accepted on 03.09.2011     

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 3(6): Nov. - Dec. 2011, 261-265