Standardization of an Ayurvedic Formulation- Maharasnadi Kvatha Churna

 

Ashish Sharma¹, Vandna Kalsi^1*, Ashish Suttee¹, Hayat M.Mukhtar², Barinderjit Kaur³

¹Department of Pharmacognosy, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India

²Department of Pharmacognosy, Shaheed Bhagat Singh College of Pharmacy, Patti, Punjab, India

³Department of Pharmacology, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India

 

ABSTRACT:

Standardization of a compound ayurvedic formulation is essential for establishing its authenticity, quality and efficacy. The present study highlights the standardization of Maharasnadi Kvatha Churna (a polyherbal formulation containing 26 plant materials) which can make contributions to the existing literature. Standardization of formulation was done on the basis of its macroscopic and microscopic characters, physico-chemical parameters, phytochemical screening, flouresence studies and thin layer chromatographic analysis. Phytochemical screening has shown the presence of alkaloids, glycosides, saponins, phytosterols, fixed oils, resins, phenols, flavonoids and tannins. Maharasnadi Kvatha Churna has been used for various diseases related to vata conditions and also used for muscular pains. From the investigations of the study, it can be concluded that the pharmacognostical and phytochemical evaluation of Maharasnadi Kvatha Churna can be used as reference standard for the quality control/quality assurance purpose.

 

 

 

INTRODUCTION:

In recent years, there has been a great demand for plant derived products in the developed countries.¹ These products are increasingly being sold as medicinal products, nutraceuticals and cosmaceuticals. There are around 6000 herbal manufacturers in India and more than 4000 units are producing herbal medicines. Due to lack of infrastructures, skilled manpower, reliable methods and stringent regulatory laws, most of these manufacturers produce their product on very tentative basis.² Estimates in India suggest that as many as 3226 out of 4752 communities, around 70% population is dependent on traditional plant-based medicines.

 

According to World Health Organization (WHO), 35% of the out-patients and 22% of the in-patients in rural China are treated with traditional Chinese medicines. About 1400 species of medicinal plants are regularly traded in USA.³ In order to have a good coordination between the quality of raw materials, in process materials and the final products, it has become essential to develop reliable, specific and sensitive quality control methods using a combination of classical and modern instrumental methods of analysis.⁴

 

Maharasnadi Kvatha Churna is a polyherbal preparation which is used in acute vatta (air) conditions, for Kamp-Vata (severe body shivers), paralysis, acute muscular and joint pains due to high vata (which is moving from thighs to lower legs). As a carrier, it increases the efficiency of other natural herbal products. Basically it possesses antiinflammatory, antirheumatic properties and controls degeneration.⁵ Kvatha or Kasaya is the filtered liquid obtained by boiling coarse powder of drug in proportion of 4, 8 or 16 times of water and reduced to one fourth. The fine sieved powder of well dried drug (s) is called churna.⁶

 

Maharasnadi Kvatha Churna contains 26 ingredients that includes 50 parts of rasna and 1 part each of Dhanvayasa, Bala, Eranda mula, Devdaru, Sathi (sati), Vaca, Vasaka (vasa), Nagara (sunthi), Pathya (haritki), Cavya, Musta, Punarnava, Guduchi, Vrddadaru, Satapusha, Asvagandha, Goksura, Prativasa, Krtamala, Satavari, Krsna, Sahakara, Dhanyaka, Kantakari, Brhati.⁶

 

MATERIALS AND METHODS:

Preparation of Maharasnadi Kvatha Churna:

The ingredients of preparation were procured from the Vindhya Herbals, Bhopal and Kvatha Churna was prepared in the Department of Pharmaceutical Sciences, Lovely Professional University, Punjab. Their identities were confirmed by correlating their morphological and microscopical characters with those given in the literature. The drugs were cleaned, dried and coarsely powdered. The drugs were weighed as per formula and mixed well. Kvatha was prepared and the contents were powdered.⁷

 

Macroscopic Evaluation:

The powder was observed for its colour, odour, taste and appearance.^8,9

 

Microscopic Evaluation:

The powder of Maharasnadi Kvatha Churna was cleared with chloral hydrate and stained with respective reagents such as phloroglucinol, hydrochloric acid, iodine solution and ruthenium red. The sample was mounted on glass slide and observed under the microscope (10X and 40X) to observe microscopic characteristics of the powdered formulation.^4,8 The prepared slides were scanned in NIKON eclipse 90 i scanner.

 

Physicochemical Investigations:

In physicochemical investigation, loss on drying, total ash, acid insoluble ash, alcohol and water extractive values, swelling index, foaming index ², moisture content^10,11 were determined as per standard procedures. All the parameters were performed according to the official methods prescribed in Indian Pharmacopoeia and ¹² WHO on quality control methods for medicinal plant materials.²

 

Heavy /toxic metals and microbial load analysis:

The formulation was also tested for the presence of heavy/ toxic metals and for the microbial load as per WHO guidelines.^{10, 13}

 

Preliminary Phytochemical Analysis:

Preliminary phytochemical screening was carried out by using standard procedures.^{14, 15}

 

Fluorescence Analysis:

Fluorescence characters of powdered plant material with different chemical reagents were determined under ordinary day light and ultraviolet light (254 nm).¹⁶

 

Thin Layer Chromatography:

Preparation of test sample:

The powdered formulation was extracted on small scale with ethanol (10 ml) at 90°C, dichloromethane (10ml) at 40°C, ethyl acetate (10ml) at 77°C for 1 hr using reflux condenser.

 

 Development of Plates:

The plates were developed in CAMAG TLC jars presaturated with the solvent system. Anisaldehyde sulphuric acid was used as derivatizing agent followed by drying at 110°C and the plates were observed in day light and under UV light (254 nm).

Mobile Phase

Chloroform: Methanol: Water (60:40:4)^{17, 18}

 

RESULTS AND DISCUSSION:

As a part of standardization procedure the formulation was tested for relevant physical and chemical parameters and also subjected to microbial screening through quality control measures.

 

Macroscopic Evaluation:

The formulation was light brown in colour having a pleasant odour and bitter taste (Table 1).

 

Table 1: Macroscopic evaluation of formulation

 

Microscopic Evaluation:

The powdered formulation has shown the presence of starch grains, fibers, crystal fibers, unicellular and multicellular trichomes on microscopical evaluation shown (Fig. 1-6).

 

Fig.1: Starch grains

Fig.2: Fibers

Fig.3: Crystal fibers

Fig.4: Crystal fibers and trichomes

 

Fig.5: Multicellular trichome

Fig.6: Unicellular trichome

 

Physicochemical Investigations:

Loss on drying gives an idea of moisture present in the drug. Ash value of a drug provides an idea of the earthy matter or the inorganic components and other impurities present along with the drug. Extractive values are useful for the determination of exhausted or adulterated drugs. The water soluble extractive was high in the formulation. Swelling index gives the idea of gums present in the drug and foaming index tells about the saponins. The results of physico-chemical constants of the drug powder are presented in Table 2.

 

Table 2: Physicochemical Parameters: Maharasnadi Kvatha Churna

Parameter	Value %w/w
Loss on drying	12.4%
Total ash value	4.5 mg/g
Acid insoluble ash	1.6 mg/g
Alcohol soluble extractive (hot)	1.45%
Alcohol soluble extractive (cold)	3.25%
Water soluble extractive (hot)	4%
Water soluble extractive (cold)	4.75%
Swelling index	8%
Foaming index	Less than 100
Moisture content	12%

 

Heavy /toxic metals and microbial load analysis:

Heavy metals have been associated with various adverse effects including status epilepticus, fatal infant encephalopathy, hepatotoxicity, congenital paralysis and deafness. Many case studies have reported serious adverse conditions due to heavy metals in ayurvedic and other herbal drugs. The formulation was found free from the arsenic, mercury and lead. Nickel and calcium were present up to the content 2.8mg/l and 337mg/l respectively. Results are tabulated in table 3.

 

Various microorganisms can contaminate herbal drugs and cause serious health hazards. Table 4 represents the count of total bacteria, total yeast and mould. Escherichia coli, Pseudomonas aeruginosa, Salmonella species, Staphylococcus aureus were found to be absent in the formulation. The results revealed the absence of these microorganisms thereby confirming the nontoxic nature of the formulation.

 

Table 3: Heavy/ Toxic metals in the formulation

Metal	Content
Arsenic content	Absent
Mercury	Absent
Lead	Absent
Nickel	2.8mg/l
Calcium	337mg/l

 

Table 4: Microbial load of the formulation

Microbe	Count
Total bacterial count	10.8 x 105 cfu/g
Total yeast and mould	1.6 x 103 cfu/g
Escherichia coli	Absent
Pseudomonas aeruginosa	Absent
Salmonella species	Absent
Staphylococcus aureus	Absent

Preliminary Phytochemical Analysis: 

Preliminary phytochemical analysis has revealed the presence of alkaloids, glycosides, saponins, phytosterols, resins, phenols, flavonoids, diterpenes and tannins. (Table 5)

 

Table 5– Phytochemical investigation of formulation

Phytochemical	Ethyl acetate extract	Ethanolic extract	Dichloromethane extract
1. Alkaloids a) Wagner’s test b) Hager’s test c) Mayer’s test d) Dragendorff’s test	+ + + +	+ + + +	+ + + +
2. Carbohydrates a) Benedict’s test b) Fehling’s test	- -	- -	- -
3. Glycosides a) Modified Borntrager’s test b) Legal’s test	+   +	+   +	+   +
4. Saponin a) Froth test	+	+	+
5. Phytosterols a) Salkowski’s test b) Liebermann Burchard’s test	+ +	+ +	+ +
6. Fixed oils and fats a) Stain test	-	+	-
7. Resins a) Acetone water test	-	+	-
8. Phenols a) Ferric chloride test	+	+	+
9. Flavonoids a) Alkaline reagent test b) Lead acetate test	+ +	+ +	+ +
10. Diterpenes a) Copper acetate test	-	+	-
11. Tannins a) Gelatin test	+	+	+
12. Proteins and amino acids a) Xanthoproteic test b) Ninhydrin test	- -	- -	- -

Fluorescence Analysis:

Fluorescence is an important phenomenon exhibited by various chemical constituents present in the plant material. If the substances themselves are not fluorescent, they may often be converted into fluorescent derivatives by reagents hence some crude drugs are often assessed qualitatively in this way and it is an important parameter of pharmacognostical evaluation. Fluorescence analysis of drug powder treated with various reagents was studied at day light and ultraviolet light (254 nm) and the observations are presented in Table 6.

 

Table 6 – Flourescence analysis of the formulation

Powdered drug	Visible/day light	Ultraviolet light (254 nm)
Powder as such	Dark brown	Brown
Powder + FeCl3	Dark grey	Greyish yellow
Powder + conc. HCl	Orange yellow	Greyish yellow
Powder + 10% HNO3	Orange	Yellow
Powder + 10% K2Cr2O7	Yellow	Green
Powder + 1 M NaOH	Brownish yellow	Green
Powder + AgNO3	Buff brown	Light brown
Powder + conc. HNO3	Orange yellow	Flourescent yellow
Powder + conc. H2SO4	Orange	Flourescent green
Powder + Bromine water	Brown	Light brown
Powder + 5 % H2O2	Brown	Greyish green
Powder + CCl4   Brown	Brown	Dark brown

 

Thin layer chromatography:

Dichloromethane extract of Maharasnadi Kvatha Churna has shown the presence of four compounds (Table 7), whereas Ethanolic extract showed only two components (Table 8) and Ethyl acetate extract showed four components (Table 9). TLC plates are shown in fig. 7.

 

 

 

 

 

 

 

UV (254 nm)           Daylight                   UV (254 nm)           Daylight                  UV (254 nm)          Daylight

(a)                             (a)                         (b)                           (b)                           (c)                          (c)

Fig.7– TLC profile of (a) DCM extract (b) Ethanolic extract (c) Ethyl acetate extract

Table 7– TLC profile of Dichloromethane extract of Maharasnadi Kvatha Churna

Distance travelled by solute (cm)	Rf value	Color of the spot
		UV254 nm	Visually
4.1	0.82	Brown	Black
3.8	0.76	Green	Dark grey
3.5	0.70	Pink	Light grey
3.1	0.62	Orange	Light grey

 

Table 8– TLC profile of Ethanolic extract of Maharasnadi Kvatha Churna

Distance travelled by solute (cm)	Rf value	Color of the spot
		UV254 nm	Visually
3.6	0.82	Orange	Black
3.4	0.76	Fluorescent green	Dark grey

 

Table 9– TLC profile of Ethyl acetate extract of Maharasnadi Kvatha Churna

Distance travelled by solute (cm)	Rf value	Color of the spot
		UV254 nm	Visually
4.3	0.83	Orange	Black
3.7	0.77	Fluorescent green	Dark grey
3.5	0.70	Light orange	Light grey
3.0	0.62	Light orange	Light grey

 

CONCLUSION:

From the above investigation, it can be concluded that the pharmacognostical and phytochemical evaluation of Maharasnadi Kvatha Churna can be used as reference standard for the quality control/quality assurance purpose. It can also serve as an important source of information to ascertain the identity and to determine the quality and purity of the formulation in future studies.

 

ACKNOWLEDGEMENT:

The authors are thankful to the management of Lovely Professional University for providing necessary facilities to carry out this work.

 

CONFLICT OF INTEREST:

The authors declare that they have no competing interests.

 

REFERENCES:

1.        Choudhary N, Sekhon BS. An overview of advances in the standardization of herbal drugs. J Pharm Edu.2011; 2: 55-70.

2.        WHO/PHARM/92.559/rev.1.,Quality Control Methods for Medicinal Plant Materials, Organisation Mondiale De La Sante, Geneva; 1992; pp. 9, 22-34.

3.        Handa SS, Kapoor VK. Textbook of Pharmacognosy. Second ed. Vallabh Prakashan, 1998, New Delhi; .2-5.

4.        Ansari SH. Standardization of crude drugs. Essentials of Pharmacognosy. 14^th ed. Business Horizons; New Delhi, 347.

5.        Pathak R, Joshi R., Sharma R. Ayurveda Sarsangrah. Shri Baidyanath Ayurveda Bhavan Limited, Allahabad; 2005; .603.

6.        Anonymous, Ayurvedic Pharmacopoeia of India, Government of India, Ministry of Health and Family Welfare and Department of Indian System of Medicine and Homoeoathy, 2^nd ed, volume-I, 195-197.

7.        Shah B, Seth AK. Textbook of Pharmacognosy and Phytochemistry, 2^nd ed, CBS Publishers and Distributors Pvt Ltd, New Delhi, 2014,492.

8.        Borhade P, Khandelwal K. Review on standardization of churn. World J Pharm Pharmaceutical Sci. 2012; 1(4):1260-1274.

9.        Gautam A, Sharma P. Identification, evaluation and standardization of herbal drugs: A review. Scholars Research Library. 2010; 22(6): 302-315.

10.     Lohar D R. Protocol for testing of ayurvedic, siddha and unani medicine. Government of India and Department of AYUSH. Ministry of Health and Welfare. Pharmacopoeial Laboratory for Indian Medicine. 2011; 21-22.

11.     Kokare NV, Wadkar KA,. Kondawar MS. Review on standardization of herbal churna. Int J Res Ayurveda Pharm. 2014; 5(3): 397-401.

12.     Indian Pharmacopoeia, 4^th ed., Vol. II, Government of India, Ministry of Health and Welfare, Controller of Publications, New Delhi, 1996; A53-A54.

13.     WHO guidelines for assessing quality of herbal medicines with reference to contaminants and residues, World Health Organization, Geneva, 2007.

14.     Kokate CK. Practical Pharmacognosy, 1^st ed, Vallabh Prakashan, New Delhi, 1986. 15-30.

15.     Brain KR, Turner TD. The Practical Evaluation of Phytopharmaceuticals. Bristol: Wright Scientechnica. 1975; 82.

16.     Satheesh Madhav NV, Upadhyaya K, Bisht A. Phytochemical screening and standardization of polyherbal formulation for dyslipidemia. International Journal of Pharmacy and Pharmaceutical Sciences. 2011; 3: 235-238.

17.     Wagner H, Baldt S, Zgainski EM. Plant Drug Analysis. Berlin: Springer; 1996. 54, 38, 120.

18.     Krebs KG, Heunsen D, Stahl WH. Thin Layer Chromatography: A Laboratory Hand Book. Berlin: Springer; 2005. 60, 67, 854-909.

 

 

 

 

 

Received on 28.09.2017            Modified on 24.10.2017

Accepted on 09.11.2017         © RJPT All right reserved