PHARMACOPOEIAL AND HPTLC FINGERPRINT STUDIES OF DARCHINI Ã‚Â– ANIMMUNITY BOOSTER UNANI SINGLE DRUG

Corona virus disease 2019 (COVID-19) is threatening the worldwide population by causing symptoms like fever, cough, shortness of breathand tirednessetc.A huge population of world is suffering from this disease and no specific vaccine for this pandemic disease has been developed. According to the guidelines of Ministry of AYUSH, this disease can be prevented by taking immunity boosters, as immune system plays a vital role in defence against any disease. In Unani system of medicines, many drugs of plant origin are mentioned in classical literature for strengthening and increasing the immunity of humans. Darchini is one of the potent immune boosters and it is believed that consumption of Darchini decoction with honey strengthens the immune system.The Ministry of AYUSH, Govt. of India in its Advisory has considered Darchini as one of the important ingredient of AYUSH Joshanda/ Kwath/ Kudineer.Since, the drug Darchini is being given to COVID-19 patients as prophylaxis regime, it has become necessary to authenticate and develop its pharmacopoeial standards so that quality raw material can be provided to needy mass. The present study is aimed to develop identity, purity and strength of drug using pharmacognostical, physico-chemical and quality control methods.

Keeping in view of its various medicinal properties, the present study is aimed to develop microscopic, powder microscopy, physico-chemical, quality control standards and HPTLC fingerprints of Darchinifor laying down the pharmacopoeial standards which will be helpful to provide quality raw material to manufacturers of Unani medicines for management of COVID-19 disease.

Authentication:
The sample of drug Darchini (Stem Bark) was procured from local raw drug dealers of Hyderabad and authenticated at National Research Institute of Unani Medicine for Skin Disorders, Hyderabad by Dr. Aslam Siddiqui, Research Officer (Pharmacognosy) 10 . The collected sample was dried under shade and stored at ambient temperature for evaluation of Pharmacognostical, Physico-chemical and Quality controlstandards. Pharmacognostic study: Compound microscope, glass slides, cover slips, watch glass and other common glassware were the basic apparatus and instruments used for the study. Microphotographs were taken using a microscope attached with camera. Dried stem bark were taken for microscopic studies, transverse sections were prepared and stained as per standard procedure and powder microscopy was performed 11, 12 . Physico-chemical study: Physico-chemical study was carried out under following parameters such as foreign matter (%), loss on drying (%), total ash (%) at 450 0 C, acid-insoluble ash (%) at 550 0 C, volatile oil content (%), aqueous, alcohol and hexane extractive matter (%) were carried out as per IPC approved standard methods 13 .

Preparation of Extracts:
Drug sample (2 g) was extracted with 20 ml of respective solvents (chloroform and alcohol) by refluxing on a water bath for 30 min and filtered using Whatman Filterpaper no. 1, concentrate up to 5 ml and thin layer chromatography studies were carried out 14 .
HPTLC studies: HPTLC was performed on 5 cm × 5 cm Precoated Aluminium Sheets of Silica Gel 60 F 254 (Merck). Samples solution of about 10μl were applied as 10 mm width bands using Automatic TLC applicator system of the DESAGA Sarstedt Gruppe (Germany). A Linear ascending development withtoluene: ethyl acetate (9:1 v/v) and toluene: ethyl acetate: methanol (7:2:1 v/v/v) as mobile phase was carried out in a twin trough glass chamber previously saturated with mobile phase vapour for 20 min. at room temperature (25 ± 2°C). The development of solvent distance was 80 mm. After development plates were air-dried. TLC plate was scanned by densitometer of DESAGA Sarstedt Gruppe (Germany) at 366, 254 and 580 nm wavelength and operated by Pro Quant 1.06 version software 15 .

Quality control parameters:
Quality control parameters viz. Microbial contamination, Heavy Metals, Aflatoxins, and Pesticide residues were analyzed as per WHO methods 16, 17 .

Results And Discussion:-Pharmacognostic Standards: Macroscopic features:
The Bark pieces are about 0.5 mm thick brittle; occurs as single or double; closely packed compound guills; upto a metre or more in length and about 1 cm in diameter; outer surface; dull yellowish-brown; marked with pale wavy 247 longitudinal lines with occasional small scars or holes; inner surface darker in colour; striated with longitudinally elongated reticulation; fracture splintery. (Fig.1) Microscopic features: T.S. of bark shows pericyclic sclerenchyma; 3 or 4 rows of diametric cells; multiple vessels; sometimes tangentially elongated; inner and radial walls often being thicker than the outer; some containing starch 27 grains; small groups of pericylic fibres embedded at intervals in the sclerenchyma; phloem of tangential bands of sieve tissue alternating with parenchyma; and containing axially elongated secreting cells containing volatile oil or mucilage; phloem fibres with very thick walls; upto 30µm in diameter; isolated or in short tangential rows; sieve tubes narrow with transverse sieve plates; collapsed in outer periphery; medullary rays of isodiametric cells; cortical parenchyma and medullary rays containing small starch grains below 10µm in diameter; minute acicular crystals of calcium oxalate present. (Fig.2-4) Powder Microscopy: Powder microscopic study shows the drug is light reddish brown in colour fragrant odour, sweet and pungent taste and aromatic smell with sensation of warmth. Epidermal cells, parenchymatous cells and scalariform vessels were also observed( Fig.5-8).

Physico-chemical Standards:
The physico-chemical standards for the dry powder of the Stem bark of Darchini (80mesh) are given in Table 1.
The drug contains negligible amount of silicates; shown by the acid insoluble ash (0.41%), but contains considerable amount of inorganic materials; shown by the higher ash value (4.66%). The extractive values with different solvents viz. n-hexane 2.15%, alcohol9.48% and water10.21% showed that the plant contains mainly the polar compounds soluble in alcohol and water. It also contains considerable amount of volatile oil viz. 1.01%.

Heavy Metal Analysis:
The medicinal plants materials are generally contaminated with arsenic and heavy metals due to environmental pollution. These components even in trace amounts are dangerous and can damage the important human organs such as kidney, liver and heart. The heavy metal contents viz. lead, cadmium, mercury and arsenic were analysed using Atomic Absorption Spectrophotometer and were found within the permissible limits viz. 10, 0.3, 1 and 3 ppm respectively as per WHO guidelines and the results are shown in Table 2. Darchini (Stem Bark) is hence considered non-pollutant drug in the environment.

Microbial Load Analysis:
The microbial load viz. TBC, TFC, E.Coliand other pathogens were analyzed as per the standard methods and the results are shown in Table 3.

Analysis of Aflatoxins:
The aflatoxins can be acute toxic, carcinogenic, mutagenic, teratogenic and immunosuppressive to the human being if these are found in the plant material above prescribed limits 18 . The aflatoxins B 1 , B 2 , G 1 and G 2 were analysed using HPLC and found within permissible limit as shown in Table 4.The toxic effect of the drug sample may be considered as nil and hence is safe to use.

Analysis of Pesticide Residues:
The various pesticidal residues α -HCH, β -HCH, γ -HCH, δ -HCH, op-DDT, pp-DDT, op-DDE, α-Endosulfan, β -Endosulfan, op-DDD and pp-DDD etc. were tested in the drug sample using GC-MS-MS technique and found within permissible limits. The results are shown in Table 5.The drug sample may be considered as pesticide resistant.

Thin layer Chromatography:
The thin layer chromatographic studies of chloroform and ethanol extracts were carried out using solvent systems toluene: ethyl acetate (9:1) and toluene: ethyl acetate: methanol (7:2:1) respectively and the results are tabulated in Table 6.

Conclusion:-
The evaluated standards will be highly useful to authenticate the drug for providing quality raw material to manufacturers of Unani medicine. From the studies of microbial load, heavy metals, aflatoxins and pesticide residues it can be concluded that the drug Darchini (Stem Bark) is free from microbial contamination, nonpollutantin the environment, non-toxic, pesticide resistant, safe to use and can be consideredas important immunity booster for prevention of COVID-19 disease.

Acknowledgement:-
The authors are very grateful to Director General, Central Council for Research in Unani Medicine, New Delhi, Ministry of AYUSH, Govt. of India for the encouragement and providing necessary facilities to carry out the research work.  Loss in wt. on drying at 105 o C (%w/w) 9.85 3.