1. INTRODUCTION

Since origin of human’s life, plants continue to play a curative and therapeutic role in preserving human health against disease and decay.The plants and animals including humans live in harmony and both complement with each other for their healthy existence (Meena et al., 2013). India having a rich heritage of traditional medicine constituting with its different components like Ayurveda, Siddha and Unani (ASU) medicine system. Plant origin materialare major part of these traditional medicines. The development of these traditional medicines systems with the perspectives of safety, efficacy and quality will helps not only to preserve the traditional heritage but also to rationalize the use of natural products in healthcare (Mukherjee et al., 2003, 2006). Ayurveda is a living example for using plants, minerals and animal origin products for maintaining the health and curing the diseases (WHO, 1992).

The ayurvedic system of medicine has described various herbal formulations in the treatment of diseases, which plays an important role in modern health care and curing various ailments or diseases (Patwardhan et al., 2003). The uses of herbal medicines are increasing as dietary supplements to fight or prevent common diseases (Pandey et al., 2008). Therefore, in recent years, great demand for plant derived products in developed countries for primary health care because plant based medicines, health products, pharmaceuticals, food supplement, cosmetics etc. are thought to be nontoxic, have less side effects, it is easily available and affordable (Eisenberg et al., 1998; Ernstet al., 2000). Herbal formulations show the number of problems when quality aspect is considered, because nature of the herbal ingredients and different secondary metabolites present in the plant. Mainly, variation in the chemical profile of the herbal due to intrinsic and extrinsic factors like growing, harvesting, storage and drying processes (Gilani et al., 2005; WHO 2002; Zidorn et al., 2005). Standardization is an essential factor for polyherbal formulation in order to assess the quality of drugs based on the concentration of their active principle. Plant material when used in bulk quantity may vary in its chemical content. Therefore, in its therapeutic effect according to different batches of collection e.g. collection in different season and/or collection from sites with different environmental surrounding or geographical location (Aswatha et al., 2009).

The mixture of powdered form of herbal materials used in Ayurvedic formulations is known as churna. Avipattikara churna was prepared as per Ayurvedic Formulary of India, Part-I. Agnimandya (digestive impairment), malabandha (constipation), amlapitta (dyspepsia), arsa (haemorrhoids), mutrabandha (retention of urine), prameha (urinary disorders) and piles are important therapeutic uses of avipattikarachurna (AFI 2003;Shastri18^th edition).

The present study aimed to develop standardization parameters and HPTLC finger print profile of avipattikara churna based on organoleptic evaluation, physical characteristics, physicochemical studies like pH, total ash, acid insoluble ash, loss on drying at 105°C, extractive valuesetc. and microbial load were carried out as per the API, IP, WHO and AOAC guidelines to substantiate the adulteration for quality control of herbal drugs(Meena et al., 2011; IP 2018; WHO 1998; AOAC 1995).

2. MATERIALS AND METHODS:

(i) Procurement of raw drugs:

Avipattikara churna contains 14 ingredients Tabulated in Table 1, all these 14 ingredients were collected from local market. Samples were identified and authenticated by Botany/ Pharmacognosy Section.

(ii) Preparation of Avipattikara Churna:

Avipattikara churna was prepared as per Ayurvedic Formulary of India Part-I, 7:2 (Bhaisajyaratnavali, Amlapittadhikara: 24-27). All ingredients were dried below 60°C, powered individually in a pulveriser and pass through 85#sieve and stored in air tight containers. Each ingredient was weighed separately required weight and mixed together to obtain a homogeneous blend(AFI 2003)details given in Table 1.

(iii) TLC/HPTLC fingerprinting profile (Wagner et al., 1996; Sethi 1996):

4g of the sample was soaked with 40 ml ethanol overnight, boiled for 10 minutes, concentrated and made up to 10 ml volumetric flask. The test samples solution and selected important marker compounds were applied on E.Merck aluminium plate precoated with Silica gel 60F₂₅₄ of 0.2 mm thickness using CAMAG ATS IV sample applicator. The plate was developed in the solvent system and dried. The plate was observed through CAMAG TLC Visualizer under UV at 254 nm and 366 nm and photos were documented after that plate was derivatized and photo was documented under white light. Before derivatization the plate was scanned using CAMAG TLC Scanner with WINCATS software at a wavelength of UV 254 and 366 nm using deuterium and mercury lamps respectively. The R_fvalues and fingerprint data were recorded.

(iv) Microbial Contamination (WHO 1998, API 2008;Agarwal et al., 2014):

a) Fungal Load:

Petriplates of Rose Bengal Agar medium. 1 ml of sample was transferred to the individual plates for determination of Total Fungal Count (TFC) followed by incubation at 25°C for five-six days. Total number of colonies on plate were counted with the help of colony counter and colony forming unit (CFU) was counted using formula.

CFU = Number of colonies x Dilution factor

----------------------------------------------

Sample volume (ml)

b) Bacterial load:

Petriplates of soyabean casein digest agar medium. 1 ml of sample was transferred to the individual plates for determination of Total Bacterial Count (TBC) followed by incubation at 37°C for 24-48 hrs. Total number of colonies on plate were counted with the help of colony counter and colony forming unit (CFU) was counted using formula.

CFU = Number of colonies x Dilution factor

---------------------------------------------

Sample volume (ml)

For Escherichia coli, homogenized pretreated sample was transferred to Eosine methylene blue agar incubated at 35-37°C for 18-24 hrs.

Salmonella Spp. homogenized pretreated sample was transferred to brilliant green agar incubated at 35-37°C for 24-48 hrs.

Pseudomona Spp. homogenized pretreated sample was transferred to centrimide agar incubated at 35-37°C for 24-48 hrs.

Staphylococci Spp. homogenized pretreated sample was transferred to Mannitol salt agar incubated at 35-37°C for 24-48 hrs.

3. RESULT AND DISCUSSION

1) Organoleptic evaluation:

Organoleptic evaluation refers to evaluate that the formulation by color, odour, taste and texture. The organoleptic characters of the avipattikara churna were evaluated (API 2008; Tekeshwar et al., 2011) and tabulated in Table 2.

Table 2.Organolepaticcharacter of Avipattikara churna:

Appearance	Color	Odour	Taste
Fine Powder	Light brown	Characteristic	Sweet pungent

2) Determination of fineness of particle Avipattikara churna:

Avipattikara churna sample all the particles of which pass through a sieve with a nominal mesh aperture of 180µm and not more than 40 % by weight through a sieve with a nominal mesh aperture of 125µm.

Percentage of sample passing through each sieve =

(Weight of the sample taken – Weight of the sample remaining)

-------------------------------------------------------------- X100

Weight of the sample taken

It is observed that, the avipattikara churna sample is Fine powder.

3) Determination of physical characteristics of Avipattikara churna:

Physical characteristics like bulk density, tap density, Hausner’s ratio and Carr's index were determined for avipattikara churna and results given in Table 3 (Neha et al., 2011;Srivastava et al., 2010; Martin 2001; Subrahmanayam 1998).

Table 3.Physical characteristics of Avipattikara churna.

S. No.	Parameters	Results
1.	Bulk density (gm/cm³)	0.3448
2.	Tap density (gm/cm³)	0.5263
3.	Hausner’s ratio	1.5263
4.	Carr’s Index (%)	34.48

Table 1. Ingredients of Avipattikarachurna

S. No.	Sanskrit Name	Botanical Name	Common name	Part used	Chemical Constituent	Quantity used
1.	Sunthi	Zingiberofficinale	Dry ginger	Rhizome	6-Shogaol	1 part
2.	Marica	Piper nigrum	Black pepper	Fruit	Piperine	1 part
3.	Pippali	Piper longum	Long pepper	Fruit	Piperlongumine	1 part
4.	Haritaki	Terminaliachebula	Harad	Pericarp	Chebulic acid	1 part
5.	Bibhitaka	Terminaliabellirica	Behada	Pericarp	Gallic acid	1 part
6.	Amalaki	Phyllanthusembilica	Indian gooseberry	Pericarp	Syringic acid	1 part
7.	Musta	Cyperusrotundus	Nut grass	Rhizome	Cyperene-1	1 part
8.	Vida (Vida lavana)	-	Salt (NaCl, Sulphate & Iron)	-	-	1 part
9.	Vidanga	Embeliaribes	False black pepper	Fruit	Embelin	1 part
10.	Ela (Suksmaila)	Elettariacardamomum	Cardamom	Seed	1,8-cineole	1 part
11.	Patra (Tejapatra)	Cinnamomumtamala	Indian bay leaf	Leaf	Linalool	1 part
12.	Lavanga	Syzygiumaromaticum	Cloves	Flower buds	Eugenol	11 part
13.	Trivrt	Operculinaturpethum (Merremiaturpethum)	Pitohri (Indian Jalap)	Root	Scopoletin	44 part
14.	Sarkara	-	-	-	-	66 part

4) Evaluation of physicochemical parameters: (Meena et al., 2011; IP 2018; WHO 1998; AOAC 1995).

The formulation was standardized based on the physico -chemical parameters like pH value, loss on drying at 105 ºC, total ash, acid-insoluble ash and alcohol & water soluble extractive values were carried out as per the API, IP, WHO and AOAC guidelines for avipattikara churna results tabulated in Table 4.10% w/v aqueous solution of sample was prepared and used for determining the pH value by pH meter.

Table 4. Physicochemical parameters of avipattikara churna.

S. No.	Parameters	Results
a)	pH (10 % w/v aqueous solution)	4.95
b)	Loss on drying at 105°C (% w/w)	3.92
c)	Ash value (% w/w)	4.39
d)	Acid-insoluble ash (% w/w)	0.15
e)	Water-soluble extractive (% w/w)	61.59
f)	Alcohol soluble extractive (% w/w)	20.95

Deterioration time of the plant material depends upon the amount of water present in plant material. If the water content is high, the plant can be easily deteriorated due to fungal attack.The loss on drying at 105°C of avipattikara churna was found to be 3.92(% w/w).Total ash value of plant material indicated the amount of minerals and earthy materials attached to the plant material and amount of acid-insoluble ash value is siliceous matter present in the sample. The amount of total ash andacid-insoluble ash in the avipattikara churna was 4.39 and 0.15(% w/w) respectively.

The water-soluble extractive value indicated the presence of sugar, acids and inorganic compounds and alcohol soluble extractive values indicated the presence of polar constituents like phenols, alkaloids, steroids, glycosides, flavonoid and secondary metabolites present in the plant sample. The water soluble extractive value in the churna sample was found to be 61.59(%w/w) and alcohol soluble extractive value was 20.95 (%w/w).

5) TLC/HPTLC fingerprinting profile:

The HPTLC method can be used for phytochemical profiling of plants and quantification of chemical constituents present in plants. The test samples solution and selected important marker compounds were applied 10 µl on Tracks 1 & 2,8 µl on Tracks 3, 4 & 5 and 20 µl on Track 6 & 7for Piper longumine, Piperine, Syringic acid, avipattikara churna, Gallic acid, Ellagic acid and 6-shogaol respectively on TLC plate using CAMAG ATS IV sample applicator. The plate was developed in the solvent system of Toluene: Ethylacetate: Formic acid (5: 5: 0.5) and dried. The plate was observed through TLC Visualizer under UV at 254 nm and 366 nm and photos were documented. Finally the plate was dipped in Vanillin Sulphuric acid reagent and heated in hot air oven at 105°C until the colour of the spots were appeared and photo was documented under white light given in Figure 1. HPTLC profile of Ethanol extracts of Avipattikara churna and Marker compounds (Track-1Piperlongumine, Track -2Piperine, Track -3Syringic acid, Track-4 Avipattikara churna, Track-5 Gallic acid, Track-6 Ellagic acid andTrack-7 6- Shogaol ) at UV 254 nm, UV 366 nm and after derivatization. Before derivatization the plate was scanned using TLC Scanner with WINCATS software at a wavelength of UV 254 and 366 nm using deuterium and mercury lamps respectively. The R_fvalues and fingerprint data were recorded.The marker compounds used in this study are Piper longumine, Piperine, Syringic acid, avipattikara churna, Gallic acid, Ellagic acid and 6-shogaol present in formulation and ingredients of avipattikara churna. The chromatograms of the individual components of avipattikara churna and standards marker compounds given in Figure 2 to 4.

1 2 3 4 5 6 7	1 2 3 4 5 6 7	1 2 3 4 5 6 7
UV at 254 nm	UV at 366 nm	Derivatised with Vanillin H₂SO₄

Figure 1. HPTLC profile of Ethanol extract of Avipattikara churna and Marker compounds at UV 254 nm, UV 366 nm and after derivatization.

Track-1 Piper longumine (10µl), Track -2 Piperine (10µl), Track -3 Syringic acid (8µl), Track-4 Avipattikara churna (8µl), Track-5 Gallic acid (8µl), Track-6 Ellagic acid (20µl) and Track-7 6Shogaol (20µl). Solvent system : Toluene: Ethylacetate: Formic acid (5: 5: 0.5)


Track-1	Track-2

Track-3	Track-4

Track-5	Track-6

Track-7

Figure 2. R_fvalues and HPTLC Fingerprint profile of test sample and Marker compounds at UV 254 nm

(Track-1 Piperlongumine, Track -2 Piperine, Track -3 Syringic acid, Track-4 Avipattikara churna, Track-5 Gallic acid, Track-6 Ellagic acid and Track-7 6-Shogaol )


Track-1	Track-2

Track-3	Track-4

Track-5	Track-6

Track-7

Figure 3. R_fvalues and HPTLC Fingerprint profile of test sample and Marker compounds at UV 366 nm

(Track-1 Piperlongumine, Track -2 Piperine, Track -3 Syringic acid, Track-4 Avipattikara churna, Track-5 Gallic acid, Track-6 Ellagic acid and Track-7 6-Shogaol )

at UV 254 nm	at UV 366 nm

Figure 4. 3D view of all tracks of Densitometric chromatogram of test sample and Marker compounds at UV 254 and 366 nm.

(Track-1 Piper longumine, Track -2 Piperine, Track -3 Syringic acid, Track-4 Avipattikara churna, Track-5 Gallic acid, Track-6 Ellagic acid and Track-7 6- Shogaol )

6) Microbial Contamination (WHO 1998, API 2008;Agarwal et al., 2014):

The results of the microbial load of avipattikara churna are presented in Tables 5.Study revealed that total bacterial count and total fungal count were found within the permissible limit.

Table 5. Microbial load of Avipattikara churna.

S. No.	Parameter	Results	API Limits	WHO Limits
1.	Total Bacterial Count (cfu/gm)	1.9x10⁴	10⁵	10⁵
2.	Total Fungal Count (cfu/gm)	<10³	10³	10³
3.	Enterobacteriaceae (cfu/gm)	Absent	Absent	10³
4.	Salmonella spp. (per gm)	Absent	Absent	Nil
5.	Staphylococcus spp. (per gm)	Absent	Absent	Nil
6.	Pseudomonas aeroginosa (per gm)	Absent	Absent	-
7.	Escherichia coli (per gm)	Absent	Absent	-

4. CONCLUSION:

Ayurvedic medicine avipattikara churna has been standardized by intervention of modern scientific quality control parameters in the traditional Ayurvedic preparation described in classical texts. The avipattikara churna were standardised as per WHO, IP, AOAC guidelines and Ayurvedic Pharmacopoeia of India etc. The sample was studied along with organoleptic, physical characteristics, physio-chemical studies, HPTLC finger print profile and microbial load. Study revealed that total bacterial count and total fungal count were found within the permissible limit.

5. ACKNOWLEDGEMENT:

The authors are very grateful to Director General, DDG and Programme Officers (Chemistry), CCRAS, New Delhi for providing encouragement and facilities for carrying out this work.

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Received on 31.01.2020 Modified on 22.02.2020

Research J. Pharm. and Tech 2020; 13(3):1361-1367.

DOI: 10.5958/0974-360X.2020.00251.6