Standardization and HPTLC Fingerprinting of Unani compound formulation Habb-e-Muqil Jadeed

Negi R. K.¹*, Rasheed Naikodi M. A.², Sajwan S.¹, Asim Ali Khan³, Khan A. S.¹, Meena R. P.³

¹Drug Standardization Research Institute, CCRUM, Ministry of AYUSH., Govt. of India, PLIM Campus,

Kamla Nehru Nagar, Ghaziabad, U.P., India.

²National Research Institute of Unani Medicine for Skin Disorders, A.G. Colony Road, Hyderabad,

Under CCRUM, Ministry of AYUSH., Govt. of India.

³Central Council for Research in Unani Medicine, 61-65, Institutional Area, Opp: D Block Janakpuri,

New Delhi, India.

*Corresponding Author E-mail: ashraydsri@gmail.com

ABSTRACT:

Standardization and evaluation of Unani formulations is very much essential to ascertain the quality of medicine. Unani medicines have played a significant role in maintaining the good health among the masses around the globe. Unani medicines are accepted as important therapeutic agents for treatment of various kinds of diseases, because of its harmonious nature with the human body. Habb-e-Muqil Jadeed an Unani Compound formulation is frequently prescribed for the treatment of Kabz (Constipation) in Unani system of medicine. In many instances, it has been noticed that due to adulterated raw herbs it is very difficult to find the genuine product in market place. The present communication deals with the proper authentification, identification, determination of physico-chemical constants, chromatographic profile, aflatoxin contamination, pesticide residue and heavy metal analysis of the formulation. The present standardization and HPTLC fingerprinting developing sets the standard for quality assurance of the formulation in the global marketing.

KEYWORDS: Kabz (Constipation), Aflatoxin contamination, pesticide residue, chromatographic profile.

INTRODUCTION:

Habb are small, rounded and uniformly shaped medicinal preparations used in Unani System of Medicine¹. Habb-e-Muqil Jadeed was frequently prescribed by unani physician in the treatment of Kabz (Constipation)². The composition of Habb-e-Muqil Jadeed was mentioned in NFUM consisting of ingredients as given in Table 1.

MATERIAL AND METHODS:

The raw drug ingredients of the Habb-e-Muqil Jadeed (figure 1) were procured form the Pharmacy of NRIUMSD, Hyderabad. All the ingredients (figure 2) are identified by expert Botanist using pharmacopoeial standards³.

The physico-chemical studies of the Habb-e-Muqil Jadeed were carried out according to UPI, for HPTLC Profile “Desaga sarstedt Gruppe” system with automatic sample applicator was used and photographs were taken with the help of Desaga photo-documentation system^4,5.

Microbial contamination, aflatoxin contamination, Heavy metal estimation, pesticide residue were studied as per UPI.

Fig. 1. Habb-e-Muqil Jadeed

Table 1. Formulation composition ^2,6,7,8

S. No	Name	Botanical/sci. Name	Part used	Qty.
1	Muqil Azraq UPI-I^6	Commiphora mukul (Hook ex Stocks) Engl.	Oleo-gum resin	250gm
2	Rasuat Zard UPI^11	Berberis aristata Dc.	Extract	280gm
3	Satt-e-Podina API-VI^7	Mentha arvensis Linn.	Purified extract	5gm
4	Ararot^8,9	Maranta arundinaceae Linn.	Starch	Q.S.
5	Roughan-e-Kunjad UPI-II^11	Oil of Sesamum indicum Linn.	Oil	Q.S.
6	Aab-e-Adrak UPI-I(iv),^10	Zingiber officinale Rosc.	water	Q.S.

UPI= The Unani Pharmacopoeia of India

API= The Ayurvedic Pharmacopoeia of India

Rasaut

Ararot

Muqil Ajraq

Sat Pudina

Fig.2: Photographs of Ingredients of Habb-e-Muqil

Method of preparation:

It is prepared according to the composition of the formulation composition given above is as follows.

All the ingredients were taken of pharmacopoeial quality and cleaned by removing the foreign materials. Muqil Azraq and Rasuat Zard are purified and powdered. Satt-e-Podina was added to the powered mixture. The mixture so obtained was rubbed (charb) in Raughan-e-Kunjad. Ararot Paste was made using Aab-e-Adrak. All ingredients were mixed and passed through mesh no. 40 to make the granules. Granules are subjected to make the Habb (Tablets).

Thin layer chromatography and HPTLC:

Two gram of drug sample was extracted with 20ml of Petroleum ether (40-60°C) by refluxing on a water bath for 30 min. The extract so obtained was filtered and concentrated up to 5ml to carry out the thin layer chromatography. Concentrated Petroleum ether extract was applied on TLC plate.

RESULT AND DISCUSSION:

Analytical Profile:

Organoleptic characters:

Habb-e-Muqil Jadeed is dark brown in colour, solid, mint like odour and slightly bitter in taste. The surface of the tablet is smooth as shown in the figure1. Organoleptic evaluations are carried out based on the method described by Siddique et al¹².

Physico-chemical analysis:

The Physico-chemical parameters of the formulations Habb-e-Muqil Jadeed were studied such as total ash, acid insoluble ash, solubility in water and alcohol, loss in weight on drying at 105°C, and the results are tabulated in table 2. Safety evaluation parameters such as microbial load, aflatoxins, heavy metals and pesticide residues were studied as per method described in WHO guidelines¹³.

Safety evaluation:

Safety evaluation such as heavy metal analysis, microbial load estimation, aflatoxin contamination, and pesticide residue analysis were analyzed and the results are tabulated in the tables 3-6.

Table 2. Physico chemical parameters of Unani Compound formulation Habb-e-Muqil Jadeed          

S. No	Parameter	Sample-I	Sample-II	Sample-III
1.	Colour	Dark Brown	Dark Brown	Dark Brown
2.	Odour	mint like	mint like	mint like
3.	Taste	slightly bitter	slightly bitter	slightly bitter
4.	Total ash (%w/w)	25.6728	26.1026	26.3256
5.	Acid insoluble ash (%w/w)	22.4625	22.3562	22.4126
6.	Alcohol soluble matter (%w/w)	17.2632	17.1706	17.4281
7.	Water soluble matter (%w/w)	34.2736	34.3688	34.2876
8.	Loss in wt. on drying at 105 ^0C (%w/w)	5.0016	5.0156	5.0562
9.	pH of 1% aqueous solution	5.70	5.74	5.72
10.	pH of 10% aqueous solution	5.65	5.69	5.67

Table-3. Heavy metal analysis (Method of testing as per WHO¹³ and AOAC¹⁴)

S.No.	Parameter analyzed	Results			WHO  permissible limit
		Batch 1	Batch 2	Batch 3
1	Lead (Pb)	ND	ND	ND	10 ppm
2	Cadmium (Cd)	ND	ND	ND	0.3 ppm
3	Arsenic (As)	ND	ND	ND	3.0 ppm
4	Mercury (Hg)	ND	ND	ND	1.0 ppm

Table-4. Microbial contamination

S.No.	Parameter analyzed	Results	WHO permissible limit
1	Total Bacterial load	25x10^2	not more than 10^5/g
2	Salmonella spp.	Nil	Nil
3	Escherichia coli	Nil	Nil
4	Total Fungal count	10x10	not more than 10^3/g

Table-5. Aflatoxin contamination

S.No.	Parameter analyzed	Results	WHO permissible limit
1	B1	Nil	not more than 0.50 ppm
2	B2	Nil	not more than 0.10 ppm
3	G1	Nil	not more than 0.50 ppm
4	G2	Nil	not more than 0.10 ppm

Table-6. Pesticide residue: (Method of Testing AOAC 2007.01 by GC MSMS / LC MSMS)

S. No	Test parameters	Results (mg/kg)
1	Aldrin	BLQ (LOQ-0.01)
2	Chlordane (cis & trans)	BLQ (LOQ-0.01)
3	Alachlor	BLQ (LOQ-0.01)
4	Azinphos-methyl	BLQ (LOQ-0.01)
5	Chlorfenviniphos	BLQ (LOQ-0.01)
6	Endosulphan (all isomers)	BLQ (LOQ-0.01)
7	Endrin	BLQ (LOQ-0.01)
8	Chlorpyrifos	BLQ (LOQ-0.01)
9	Chlorpyrifos-methyl	BLQ (LOQ-0.01)
10	Cypermethrin	BLQ (LOQ-0.01)
11	DDT	BLQ (LOQ-0.01)
12	Deltamethrin	BLQ (LOQ-0.01)
13	Diazinon	BLQ (LOQ-0.01)
14	Dichlorvos	BLQ (LOQ-0.01)
15	Ethion	BLQ (LOQ-0.01)
16	Fenitrothion	BLQ (LOQ-0.01)
17	Fenvalerate	BLQ (LOQ-0.01)
18	Heptachlor	BLQ (LOQ-0.01)
19	Hexachlorobenzene	BLQ (LOQ-0.01)
20	Lindane(gamma-HCH)	BLQ (LOQ-0.01)
21	Malathion	BLQ (LOQ-0.01)
22	Parathion	BLQ (LOQ-0.01)
23	Parathion methyl	BLQ (LOQ-0.01)
24	Permethrin	BLQ (LOQ-0.01)
25	Phosalone	BLQ (LOQ-0.01)
26	Pirimiphos methyl	BLQ (LOQ-0.01)

*BLQ- Below limit of Quantification / LOQ-limit of quantification

At UV 366nm	At UV 254nm
Expose to Iodine vapours	After derivatization with anisaldehyde sulphuric acid

Fig-3: HPTLC of Petroleum ether extract of Habb-e-Muqil Jadeed

Thin layer chromatography:

The TLC studies of Petroleum ether extract are tabulated in table 7. Petroleum ether (40-60°C) extract was spotted on silica gel “G” plate and developed with Toluene: Ethyl acetate (9:1) as mobile phase shows eight spots under UV 366nm at R_f values 0.03 (Fluorescent Blue), 0.06 (Fluorescent Blue), 0.31 (Fluorescent Blue), 0.39 (Fluorescent Blue), 0.49 (Fluorescent Blue), 0.60 (Fluorescent Blue), 0.69 (Blue), and 0.82  (blue); and under UV 254nm shows seven spots at R_f values 0.11, 0.17, 0.21, 0.29, 0.46, 0.60, 0.90 (All black); and under Iodine vapours shows seven spots at R_f values 0.11, 0.29, 0.33, 0.49, 0.60, 0.64, 0.90 (All brown); and under visible region after derivatizing with anisaldehyde sulphuric acid shows ten spots at R_f values 0.06 (Grey), 0.11 (brown), 0.29 (reddish pink), 0.33 (Grey), 0.49 (blue), 0.60 (Purple), 0.63 (Purple), 0.67 (Dark Purple), 0.90 (Brown) and 0.99 (Purple) as shown in the figure 3.

Table-7.  Rf Values of petroleum ether (40-60°C) extract.

Solvent System	Rf Values
	Uv-366 nm 8 Spots	UV-254 nm 7 Spots	Under Iodine Vapour 7 Spots	under visible region after derivatizing with anisaldehyde sulphuric acid 10 Spots
Toluene: Ethyl acetate (9:1)	0.03 (Fluorescent Blue)	0.11 (Black)	0.11 (Brown)	0.06 (Grey)
	0.06 (Fluorescent Blue)	0.17 (Black)	0.29 (Brown)	0.11 (brown)
	0.31 (Fluorescent Blue)	0.21 (Black)	0.33 (Brown)	0.29 (reddish pink)
	0.39 (Fluorescent Blue)	0.29 (Black)	0.49 (Brown)	0.33 (Grey)
	0.49 (Fluorescent Blue)	0.46 (Black)	0.60 (Brown)	0.49 (blue)
	0.60 (Fluorescent Blue)	0.60 (Black)	0.64 (Brown)	0.60 (Purple)
	0.69 (Blue)	0.90 (Black)	0.90 (Brown)	0.63 (Purple)
	0.82 (Blue)			0.67 (Dark Purple)
				0.90 (Brown)
				0.99 (Purple)

Table-8. Peak list of Petroleum ether extract of Habb-e-Muqil Jadeed at UV 366nm (Fig-4)

Peak no	Y-Pos	Area	Area %	Height	Rf value
1	11.3	476.44	16.39	192.94	0.03
2	15.3	364.37	12.54	123.73	0.09
3	18.5	179.06	6.16	53.61	0.13
4	23.9	463.41	15.94	173.88	0.20
5	28.1	217.77	7.49	88.76	0.26
6	36.0	9.22	0.32	5.19	0.36
7	45.0	509.79	17.54	107.26	0.49
8	52.0	519.30	17.87	163.88	0.58
9	57.8	5.94	0.20	5.27	0.66
10	60.7	61.73	2.12	21.60	0.70
11	76.1	9.48	0.33	4.85	0.91
12	79.1	38.88	1.34	11.14	0.95
13	82.4	51.18	1.76	22.46	0.99

at UV 366nm	at UV 254nm
upon exposure to Iodine vapours at 580 nm	after derivatization with anisaldehyde sulphuric acid at 580 nm

Fig. 4. Densitogram of Petroleum ether extract of Habb-e-Muqil Jadeed

HPTLC analysis:

Petroleum ether (40-60°C) extract was spotted on silica gel “G” plate and developed with toluene: ethyl acetate (9: 1). The TLC plate under various detection systems such as UV 366nm, UV 254nm, upon exposure to iodine vapours and after derivatization with anisaldehyde sulphuric acid and heating at 105°C was subjected to scanning using the HPTLC densitometer. The corresponding densitograms were obtained in which peaks for the components are appeared as shown in the figure 4. The corresponding peak areas obtained in various detection systems were recorded and depicted in the tables 8-11.

Table-9. Peak list of Petroleum ether extract of Habb-e-Muqil Jadeed at UV 254 nm

Peak no	Y-Pos	Area	Area %	Height	Rf value
1	11.5	1334.12	7.46	432.94	0.03
2	15.4	923.35	5.17	390.65	0.09
3	18.1	1676.90	9.38	557.58	0.12
4	21.8	2016.44	11.28	706.49	0.17
5	26.0	3287.47	18.39	884.54	0.23
6	31.5	1423.97	7.97	338.97	0.30
7	36.9	779.78	4.36	134.16	0.38
8	45.5	1596.66	8.93	391.90	0.49
9	51.6	1520.32	8.50	436.35	0.58
10	60.2	842.18	4.71	129.38	0.69
11	73.3	872.99	4.88	148.06	0.87
12	80.7	1602.54	8.96	360.48	0.97

Table-10. Peak list of Petroleum ether extract of Habb-e-Muqil Jadeed upon exposure to Iodine vapours at 580 nm

Peak no	Y-Pos	Area	Area %	Height	Rf value
1	11.3	115.94	2.85	61.14	0.03
2	17.0	721.13	17.72	161.27	0.11
3	22.4	31.45	0.77	22.05	0.18
4	27.3	38.14	0.94	21.15	0.25
5	31.0	198.14	4.87	67.85	0.30
6	33.7	114.48	2.81	49.16	0.33
7	44.0	523.24	12.86	155.29	0.47
8	51.7	231.88	5.70	66.31	0.58
9	56.0	462.30	11.36	165.13	0.64
10	76.3	1128.60	27.73	162.80	0.91
11	81.3	504.28	12.39	146.60	0.98

Table-11. Peak list of Petroleum ether extract of Habb-e-Muqil Jadeed after derivatization with anisaldehyde sulphuric acid at 580nm

Peak no	Y-Pos	Area	Area %	Height	Rf value
1	14.5	5526.42	7.81	1263.67	0.07
2	18.0	10439.06	14.75	1594.28	0.12
3	29.0	2045.95	2.89	698.94	0.26
4	32.6	4729.20	6.68	1155.83	0.31
5	35.9	3897.33	5.51	1005.83	0.35
6	46.3	12738.99	18.00	1631.12	0.49
7	51.7	3050.83	4.31	955.49	0.56
8	58.8	6845.14	9.67	1497.83	0.66
9	66.7	2182.80	3.08	481.58	0.76
10	78.6	12042.20	17.02	1261.55	0.92
11	84.0	7266.16	10.27	1464.47	0.99

CONCLUSION:

The present study had developed a standard of pharmacopoeial quality for the unani compound formulation Habb-e-Muqil Jadeed with respect to physico-chemical analysis, HPTLC analysis and safety evaluation such as heavy metal analysis, aflatoxin contamination, pesticide residue analysis and microbial load estimation. All the safety parameters were found within permissible limits as per WHO guidelines. Thus the present study serves as a reference standard for the study formation in future and also helps to check the quality control studies.

ACKNOWLEDGEMENT:

The authors are highly thankful to Director-General CCRUM, New Delhi for continuous encouragement and providing necessary research facilities to the researchers of the Council.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 25.08.2020                    Modified on 09.10.2020

Accepted on 16.11.2020                   ©AJRC All right reserved

Asian J. Research Chem. 2021; 14(1):56-60.

DOI: 10.5958/0974-4150.2021.00009.2