Properties and Uses:

As far as the properties of this formulation is concerned, the action of this drug is Muqawwi-e- Kabid (Liver Strengthening), Muhallil-e-Waram (Anti inflammatory) and Mudirr-e-Baul (Diuretic) while in therapeutic uses, the drug is widely used for the hepatitis and anemia (Anonymous, 1982)

Analytical Profile of Formulation:

Description:

· Qurs-e-Zarishk is a dark brown colored mass with slightly bitter and aromatic taste and having light aromatic odor.

· Qurs-e-Zarishk was powdered, stained and mounted in glycerine, then these prepared slides are thoroughly examined under microscope.

Microscopy:

The different prepared slides were examined under the microscope which showed the presence of the following ingredients on the basis of very contrast characteristic feature of tissues / cells.

a) Sandal Safaid:

Barrel shaped vessels, pitted with transverse to oblique perforations with tail like projections.

b) Gul-e-Surkh:

Rectangular to radially elongated thick walled epidermal cells with yellowish brown contents, few epidermal cells with long single unicellular and non-glandular trichome and triculpate shaped pollen grains.

c) Tukhm-e-Kasni:

Integumentary cells with paraquetry arrangement, rod shaped epicuticular structure and presence of macrosclereids.

d) Maghz-e-Tukhm -e-Khiyarzah:

Cotyledonary parenchymatous cells filled with aleurone grains and oil globules.

e) Maghz-e-Tukhm -e-Khiyar:

Elongated compact parenchymatous cells filled with large aleurone grains, starch grain and fat globules.

f) Asl-us-Soos:

Cork cells with reddish brown amorphous contents.

g) Rewand Chini:

Cortical cells with starch grains, tannins, clusters of calcium oxalate crystals and yellowish contents and uni-or biseriate ray cells.

h) Gul-e- Nilofar:

Unicellular hair attached to upper apidermis, stellate air cavities between spongy parenchyma and pollen grains with thick smooth exine and thin intine.

i) Rewand Chini:

Cortical cells with starch grains, tannins, clusters of calcium oxalate and yellowish contents and uni-or biseriate ray cells.

Result: Drug is in tablet form dark brown in color characteristic odor and aromatic in taste.

Table no. 2: Organoleptic Characters

Appearance	Tablet
Color	Dark brown
Smell	Characteristic Odor
Taste	Aromatic

Chemical Characteristics:

Qualitative Tests:

Inorganic Elements:

Spot tests of ash of the drug indicate copper, Iron, calcium and magnesium while tin, lead, arsenic, antimony and chromium were found to be negative. (-)

Copper (+)

Iron (+)

Calcium (+)

Magnesium (+)

Chloride (+)

Lead (-)

Antimony (-)

Tin (-)

Chromium (-)

Arsenic (-)

Table no. 3: Qualitative analysis of Phyto-constituents

Chemical Constituents	Test / Reagent	Result
Glycoside	Liebermann test	+ive
Sterol	Salkowaski Reaction Liebermann-Burchard’s test	+ive
Phenolics	Phenol	+ive
Tannins	Ferric chloride test	+ive
Resins	Acetic Anhydride test	+ive
Amino acids	Ninhydrin Solution	+ive
Saponin	Sodium bi-carbonate solution	+ive
Protein	Xanthoproteic test Biuret’s test	+ive
Flavonoids	Magnesium Chips test	+ive
Saponins	Foam test	-ive
Alkaloids	Dragondroff’s reagent	+ive

(-ve) Absent and (+ve) Presence of constituent

Quantitative estimations:

Table no. 4: Estimation of Phyto-Constituents

S. No	Phyto-Constituents	Results (%) (Mean Values)
S. No	Phyto-Constituents	Sample I	Sample II	Sample III	Mean + SE
1.	Total phenolics	1.10	1.25	1.18	1.17+ 0.04
2.	Tannins	0.18	0.15	0..20	0.17 + 0.01
3.	Resins	4.90	5.30	5.35	5.18 + 0.16
4.	Alkaloids	0.45	0.40	0.38	0.41 + 0.01

Table no. 5: Extractive Values

S. No	Solvent used	Results (%) (Mean Values)
S. No	Solvent used	Sample I	Sample II	Sample III	Mean + SE
1.	Pet. ether	5.40	5.20	5.00	5.20 + 0.11
2.	Chloroform	2.08	2.50	3.20	2.59 + 0.23
3.	Ethanol	35.50	34.80	34.09	34.79+ 0.40
4.	Distilled Water	5.80	4.98	5.65	5.47 + 0.16

Successive Extractions:

Successive extractive values were obtained in different solvents as per increasing orders of their polarities with the help of Soxhlet apparatus (Jerkins, et al; 2008).

Pharmacopoeial Standards:

Pharmacopoeial parameters e.g. Total Ash, Acid Insoluble Ash, Alcohol and water Soluble extractives, pH values (1 and 10%), Loss in weight on drying at 105⁰C along with the detection of aflatoxin, microbial load, pesticide residue and heavy metals were carried out as per the method described in WHO guidelines (Anonymous, 1998). The study of organic constituents present in the drug was conducted as per the methods mentioned in (Trease and Evans 1983). For the chromatographic estimations, data of Rf values in TLC profile is reported for whereas successive extracts of pet ether, chloroform and ethanol were prepared by using Soxh-let apparatus and were subjected to find out their characteristic pattern of chromatograph in different solvent systems following the method (Stahl,et al, 1969) and (Harborne, et al,1973) on percolated Silica gel 60 F264 TLC plates (Wagner et al; 1984).

Table No. 6: Physico-chemical Parameters of the Compound Formulation Qurs-e-Zarishk (Batch I-III)

Results (%) (Mean Values)
Parameters	Batch I	Batch II	Batch III	Mean + SE
Alcohol Soluble matter (%)	34.91-35.01	35.25-35.53	35.02-35.10	35.11+ 0.11
Hexane Soluble Matters (%)	3.50 -3.80	3.80-3.90	4.00 - 4.20	3.83 + 0.19
Water Soluble matter (%)	38.32-38.40	38.52-38.56	38.65-38.68	38.30 + 0.17
Bulk Density (gm/ml)	1.290	1.286	1.291	1.256 + 0.02
Total ash (%)	6.95-7.10	6.39-6.60	6.58-7.00	6.74 + 0.16
Acid Insoluble ash (%)	3.20-3.40	3.25-3.41	3.15-3.28	3.32 + 0.06
Loss in wt. on drying at 105⁰C	4.80-5.30	4.10-5.00	5.00-5.70	4.80 + 0.17
pH of 1% aqueous solution	6.38	6.37	6.38	6.34 + 0.01
pH of 10% aqueous solution	4.70	4.55	4.38	4.54 + 0.09

Values are expressed as mean % + SE except Bulk density and pH values

The drug samples were subjected for the evaluation of physicochemical parameters and analyzed as per standard methods (Anonymous, 1987)

Quality control parameters:

Microbial load, Heavy metals, Aflatoxins and Pesticidal residue:

In order to ensure the quality of drug, the modern techniques and standard methods were adopted. The parameters such as microbial load and heavy metals were carried out as per WHO guidelines (Anonymous, 1998). Aflatoxins and pesticide residues carried out by standard methods (Anonymous, 2000). The heavy metals viz. lead was present within the permissible limit and cadmium mercury and arsenic were found to be negative while aflatoxins B1, B2, G1, G2 were not detected and pesticide residues DDT and Endosulfan were found below the limit of quantification. On the other hand parameters in the estimation of microbial load viz. Escherichia coli and Total Fungal count (TFC) were not detected while Total Bacterial load and Salmonella spp were found to be within the permissible limit as stated by WHO.

Table no. 7: Quantitative estimations Heavy Metals

S. No	Name of Element analysed	WHO and FDA limits of detection	Results
1.	Mercury	1 ppm	Below Detection Value
2.	Lead	10 ppm	Below Detection Value
3.	Cadmium	0.3 ppm	Below Detection Value
4.	Arsenic	3 ppm	Below Detection Value

Table no. 8: Analysis of Microbial Load

S. No.	Parameter analysed	Result	WHO limit
1.	Total Bacterial Count	3 x 10³ CFU/gm	10⁵ CFU/gm
2.	Total Fungal Count	Nil	10³ CFU/gm
3	Salmonella spp	Absent	Nil
4.	Escherichia coli	Absent	Nil
5.	Staphylococcus aureus	Absent	Nil
6.	Enterobacteriaceae	Absent	10³ CFU/gm

Table no. 9: Estimations of Aflatoxin

S. No	Aflatoxin	Result	WHO limit (ppm)
1.	B1	Not detected	0.5
2.	B2	Not detected	0.1
3.	G1	Not detected	0.5
4.	G2	Not detected	0.1

Table no. 10: Analysis of Pesticide Residues

S. No	Pesticide Residues	Result	Limit
1	Alachor	Not Detected	LOQ: 0.01
2	Aldrin	Not Detected	LOQ: 0.01
3	Azinphos –methyl	Not Detected	LOQ: 0.01
4	Chlordane (cisand trans)	Not Detected	LOQ: 0.01
5	Chlorfenvinphos	Not Detected	LOQ: 0.01
6	Chlorpyrifos	Not Detected	0.071
7	Chlorpyrifos-methyl	Not Detected	LOQ: 0.01
8	Cypermethrin	Not Detected	LOQ: 0.01
9	DDT	Not Detected	LOQ: 0.01
10	Deltamethrin	Not Detected	LOQ: 0.01
11	Diazinon	Not Detected	LOQ: 0.01
12	Dichlorvos	Not Detected	LOQ: 0.01
13	Dimethoate	Not Detected	LOQ: 0.01
14	Dieldrin	Not Detected	LOQ: 0.01
15	Endosulphan	Not Detected	LOQ: 0.01
16	Endrin	Not Detected	LOQ: 0.01
17	Ethion	Not Detected	LOQ: 0.01
18	Fenitrothion	Not Detected	LOQ: 0.01
19	Fenvalerate	Not Detected	LOQ: 0.01
20	Heptachlor	Not Detected	LOQ: 0.01
21	Hexacholobenzene	Not Detected	LOQ: 0.01
22	Lindane (gamma HCH)	Not Detected	LOQ: 0.01
23	Malathion	Not Detected	LOQ: 0.01
24	Parathion	Not Detected	LOQ: 0.01
25	Parathion-methyl	Not Detected	LOQ: 0.01
26	Permethrin	Not Detected	LOQ: 0.01
27	Phosalone	Not Detected	LOQ: 0.01
28	Primiphos methyl	Not Detected	LOQ: 0.01

LOQ (Limit of quantification)

Chromatographic estimation

Thin Layer Chromatography:

Successive extracts of petroleum ether, Chloroform and ethanol so obtained of the drug were spotted on a pre-coated silica gel 60 F254 TLC (Merck Germany) as an absorbent and developed the plate using Pet. ether: Diethyl ether (9: 1), Chloroform: Methanol (19: 1) and Toluene: Ethyl acetate: Formic acid (8: 1: 1) respectively as a mobile phase. After developing, plate was dried and studied under UV 254nm, 366nm as well as spraying with 2% ethanolic sulphuric acid, Exposed to I₂ Vapors and 5% Vaniline Sulphuric acid with the respective extracts followed by heating at 105⁰C for 10 minutes in an oven (Anonymous, 1992) and reported the Rf values of spots observed as given in the following table no. 11.

Table no. 11. TLC profile of different extracts of “Qurs-e-Zarishk”

Extracts/ solvent systems	Rf Values of spots observed under UV		Rf Values
Extracts/ solvent systems	254nm	366nm	Spray/Treatment
Pet. ether extract Pet. ether: Diethyl ether (9:1)	0.18 (violet) 0.30 (Pink) 0.40 (violet)	0.30 (purple) 0.40(light violet)	with 2% Ethanolic Sulphuric acid 0.40 (black) 0.69(brown) 0.85(Brownish black)
Chloroform Extract Chloroform: Methanol (19: 1)	0.35 (violet)	0.41, 0.50 (blue)	Exposed to I2 Vapors 0.30 (Yellow) 0.88 (Yellow) 0.92 (Brownish blue)
Ethanol extract Toluene: Ethyl acetate: Formic acid (8: 1: 1)	0.08 (violet) 0.25(purple)	0.15 (purple) 0.30 (violet)	with 5% Vanillin Sulphuric acid 0.18 (brown) 0.36 (grayish black) 0.48 (purple) 0.77 (black) 0.92(violet)

RESULTS AND DISCUSSION:

The chemical compounds may be responsible for the therapeutic efficacy of that drug. Phytochemical screening of the chemical constituents (Overtone, et al., 1963) present in the drug revealed that it contains glycosides, steroids, phenolic, tannins, resins, proteins, flavonoids and amino acids while the saponins was found to be negative. These phyto-constituents so obtained during the analysis has been shown in Table no. 3. In quantitative estimation for physico-chemical parameters three experiments were conducted for each sample and their % mean values of total phenolics, tannins, resins and alkaloids are mentioned in Table no. 4. The solubility and extractive values sometimes are used as an index of purity for formulations. Therefore, for establishing the standards of any drug, these extractive values and solubility play an important role These physico- are considered as tools of checking the quality, identity, purity and strength of Unani drugs as the adulterated or exhausted drug material will give different values rather than the extractive percentage of the genuine one (Jerkins et al.,1967). Quantitative standards revealed that the loss of weight on drying obtained in the drug was 3.5-4.5% shows the amount of moisture content present in the drug. Estimation of ash values are an important criterion for judging the impurities along with identity and quality of the drugs because adulteration of herbal drugs with unwanted materials like earthy matters resulting in higher ash percentage. Estimation of ash values are an important criterion for judging the impurities along with identity and quality of the drugs because adulteration of herbal drugs with unwanted materials like earthy matters resulting in higher ash percentage. The values of the total ash and acid insoluble ash were 6.39-7.10% and 3.15-3.40% respectively which indicate that the amount of mineral matter present in the drug. Water soluble extractives 38.68%, indicates the presence of inorganic content while alcoholic soluble extractives 35.53% indicates the extraction of polar constituents. The pH values of all three samples were found to be acidic. The study was extended for the successive extraction in different solvents as per increasing orders of their polarities are given in table no. 5 and chromatographic profile of these extracts can be seen in table no. 11. In quality control parameters the bacterial load and total fungal count of microbial studies, aflatoxin, heavy metals and pesticide residues were either within the permissible limits or found to be absent as shown in Table no. 7, 8, 9 and 10 respectively. At the same time bacteria Enterobacteriaceae, Salmonella spp, Escherichia coli, Staphylococcus aureus, were found to be absent which shows that the drug is not the carrier of these organisms. TLC profile of successive extracts petroleum ether, chloroform and ethanol can be seen in table no. 11.

CONCLUSION:

The results of all the biological and physico-chemical studies presented here will be useful not only for monitoring the purity and quality of the drug but also check adulteration in raw material as well. Besides, the data regarding the macro and microscopic studies of ingredients particularly plant based drugs further helps in authentication of the product.

The evaluated data such as powder microscopy, physicochemical, TLC and analysis of quality control parameters indicate that the genuine raw drugs were added in the formulation and there is no variation in the batch to batch consistency of the drug. The quality control study revealed the absence of microbial load, heavy metals, aflatoxins and pesticide residue which shows the non-toxic effect of the drug. The evaluated standards will be much useful for laying down the pharmacopoeial standards of Qurs-e-Zarishk and also the providing the quality medicine to the needy mass. From the present studt it can be concluded that the formulation Qurs-e-Zarishk is quite safe, effective and affordable drug.

ACKNOWLEDGEMENT:

The authors are grateful to the Director General, Central Council for Research in Unani Medicine for providing necessary facilities.

REFERENCES:

1. Mukherjee, PK: Exploring Botanicals in Indian System of Medicine Regulatory Perspective, Clin. Res. Reg affairs 20(3): 249-264 (2003)

2. Shailajan S, Yeragi M, Purohit A. Optimized separation and quantification of eugenol from traditional Unani medicine Jawarish‑e‑Bisbasa using HPTLC. Int J Pharm Sci Rev Res 2011;9:146‑51.

3. Anonymous, (1950, 1962, 1976), The Wealth of India Raw Materials, Publication and Information Directorate, CSIR, New Delhi

4. Anonymous, (1982). National Formulary of Unani Medicine, Ministry of Health, Govt. of India, New Delhi, Part I, pp. 46

5. Anonymous, (1987), Pharmacopoeal Standards for Ayurvedic Formulations, (CCRAS), Ministry of Health, Govt. of India, New Delhi. Pp 599-603.

6. Arthur, I. V., (1971), Practical Organic Chemistry Including Qualitative Organic Analysis, 3^rd Edn., English, Language Ball Society and Longman Limited London, pp 152-154.

7. Anonymous, 1998, Quality Control methods for medicinal plant materials, World Health organisation, Geneva, Pp. 25-28.

8. Harborne, J.B., 1973. Phytochemical Methods, Chapman and Hall, London, P. 70.

9. Stahl., 1969, Thin layer Chromatography: A laboratory hand book, Springer Verlag, Berlin, pp. 52-86, 127-128, 900

10. Trease, G. E. and Evans, W. C., 1983: Pharmacognosy, 10^th Edition, BailliereTindel, London.

11. Wagner, H. Bladt, S., 1984, Plant Drug Analysis,: A Thin Layer Chromatography Atlas (2^nd Edition) Springer-Verlag, Germany.

12. Overtone, K. H., 1963, Isolation, purification and preliminary observation in elucidation of structures by physical and chemical methods, Bentley Interscience, Pub., New York Pp. 34.

13. Anonymous, 2000 Association of Official Analytical Chemists (AOAC), 17^th edition

14. Jerkins, G. L., Knevel, A. M. and Digangi, F. E. 2008. Qualitative Pharmaceuitical Chemistry, 6^thedn. CDS Publishers, New Delhi PP.225

15. Anonymous, 1987, Physicochemical Standards of Unani Formulations part II, CCRUM, M/O Health and F. W, New Delhi, pp 300-317.

16. Anonymous, 1992, Standardization of Single Drugs of Unani Medicine, part II, CCRUM, M/O Health and F. W, New Delhi, pp 201

17. Bayaz-e-kabir, Hakim Kabiruddin, part II, Sheikh-e-Jamia, Jamia Tibbia, Delhi, pp. 106

18. Wagner, H. Bladt, S., 1984, Plant Drug Analysis,: A Thin Layer Chromatography Atlas (2^nd Edition) Springer-Verlag, Germany.

Received on 22.09.2021 Modified on 03.10.2021

Res. J. Pharmacognosy and Phytochem. 2022; 14(1):5-10.

DOI: 10.52711/0975-4385.2022.00002

S.No.	Ingredients	Botanical / English Name	Part used	Qty
1.	Zarishk	Berberis aristata DC	Extract	40 g
2.	Rewand Chini	Rheum emodi Wall.	Root	10 g
3.	Gul-e-Surkh	Rosa damascena Mill	Petals	10 g
4.	Maghz-e-Tukhm-e-Khayarain	i) Cucumis sativus Linn. ii) C.melo Linn	Kernel	10 g
5.	Sandal Safaid	Santatum album Linn.	Wood	10 g
6.	Tukhm-e-Kasni	Cichorium intybus Linn	Seed	10 g
7.	Luk Maghsool	Lac	-	5 g
8.	Asal-us-Soos	Glycyrrhiza glabra Linn	Root	5 g
9.	Gul-e-Nilofar	Nymphaea alba Linn	Flower	5 g
10.	Tabasheer	Bambusa bambos Druce	Concretion	5 g