Standardization and Quality Control Analysis of Marichadi tailam:
An Ayurvedic Formulation
Manas Ranjan Sahoo*, Ramesh Raghava Varier, Anithakumar Rajendran, Ramesh K.
AVN Ayurveda Formulation Pvt Ltd, Quality Control and Research and Development Department,
Madurai, Tamil Nadu, India, 625004.
*Corresponding Author E-mail: manasranjansahoo09@yahoo.com
ABSTRACT:
The Ayurvedic medicines are having great therapeutic application due to their natural holistic way of treatment of the disease. To ensure the effectiveness of the medicine standardization of the drug essential. Ayurvedic tailam is an important group of formulations used for treatment of various types of diseases. The principle of using tailam is better absorption of active ingredients by skin when applied through a lipophilic vehicle like vegetable oils. Marichadi Tailam is a herbal oil used for treatment of skin diseases such as eczema and dermatitis. In the present study, an attempt was made to develop and validate a HPTLC method for quantitative determination piperine in the Marichadi coconut oil and its standardization. The HTPLC separation was performed on precoated silica gel 60 F254 aluminum plate (10 × 10cm, 250μm thickness) using toluene:ethyl acetate:diethyl amine (7:2:1) as a mobile phase. The detection and quantification was performed at 340nm. The formulation contains 2.35% of piperine. Linearity studies indicated piperine in the linear ranges, while recovery studies revealed 99.32% (w/w) of piperine. The developed HPTLC method resolved and quantified piperine effectively, so it could be used as a simple reliable method for QC of polyherbal formulations.
As Ayurvedic medicines are now a days being used across the world for therapeutic and preventive action against various diseases and wellness attributes, the quality control of herbal medicines and ingredients is required to ensure the consistency in therapeutic activity and efficacy13-16. The ayurvedic Tailam are one of various formulation where the herbal drugs are processed with the vegetable oil or ghee in order to transfer the desired compounds of the herbs to oil medium17. Marichadi tailam is one of the Ayurvedic tailam preparation prescribed for in treatment of various skin diseases such as eczema, dermatitis etc. The ingredients used in the preparation of MT are Piper nigrum (fruit) and milk processed with Coconut oil. As the quality control is an essential part in the standardization of Ayurvedic formulation and herbal medicine to ensure efficacy and proper quality. Thus in the present study an attempt has been made to standardize MT using HPTLC method on basis of phytochemical marker piperine, HPTLC fingerprinting and physicochemical parameters.
MATERIALS AND METHODS:
All the solvents and chemicals used in the formulation were of analytical grade. The Marichadi Coconut oil was procured from manufacturing department of the organization. TLC plates coated with silica gel 60F254 for HPTLC were purchased from Merck, Germany.
Physico-chemical Evaluation of Marichadi tailam:
Various physical properties which are specific for tailam preparation such as iodine value, saponification value, acid value, water soluble extractive value, alcohol soluble extractive value, specific gravity and refractive index. All these parameters were determined as per Indian pharmacopoeia guidelines [Ref]
Density measurement:
The density of the tailam was measured by an RD bottle with a capacity of 25mL.
Refractive index measurement:
The refractive index was measured by using an Abbe Refractometer (Advance research instruments company-Delhi)
Iodine value (IV) measurement:
Iodine value (IV) is the measure of the degree of unsaturation in a fat or vegetable oil.
Iodine value was calculated by iodimetric titration method. An accurately weighed 0.2gm of substance to be tested is dissolved in 10mL of carbon tetrachloride in iodine flask followed by addition of 20mL of iodine monochloride solution. Then insert the stopper of iodine flask and keep aside in dark at temperature between 15 to 25 for 30 minutes. Then add 15mL of potassium iodide solution and titrate with 0.1M sodium thiosulphate solution using starch as indicator. Note the number of ml required (a) and repeat the experiment with blank without the substance and note the no of ml required. The IV is calculated using the formula
Iodine value (IV) = 1.269 (b-a)/w
Where b= weight of the substance in gram.
Saponification value:
The saponification value is the number of number of milligrams of potassium hydroxide necessary to nutralise the free fatty acids and to saponify the esters present in 1 g of substances. For determination of SV take 2g of accurately weighed substance in conical flask and reflux under condenser with 25mL of 0.5M ethanolic potassium hydroxide. Let the reflux continue for 30 minutes. Immediately titrate with 0.5M hydrochloric acid (a) using phenolphthalin indicator. Then carry out the blank titration using without the test substance. Calculate the saponification value from the formula
Saponification value (SV) = 28.05 x (b-a)/w
Where: w= weight of substance in gram.
Peroxide value:
The peroxide value is the number of milliequivalents of active oxygen that expresses the amount of peroxide in 1000gram of the substance. To determine the peroxide value accurately weighed 5gm of the substances dissolved in 30ml mixture of glacial acetic acid and chloroform in ration of 3:2 (v/v) and 0.5mL of saturated potassium iodide solution is added. Then allow the mixture to stand for 1 minutes with occasional shaking. Add 30ml of water and titrate with 0.01M sodium thiosulphate until yellow color disappear and add 0.5mL of starch solution and titrate until blue color disappear (a mL). carryout the blank titration without the test substance (bmL). The volume of 0.01M sodium thiosulphate should not exceeds 0.1mL. The peroxide value is calculated using the formula
Peroxide value (PV) = 10 ( a - b)/w
Where, w= weight of substance in gram.
Acid value:
The acid value is the number which indicates the milligrams of amount of potassium hydroxide required to neutralize the free acid present in the 1gram of substances. Acid value was evaluated by potassium hydroxide titration method. 10gram of accurately weighed test substance is dissolved in 50mL of mixture of equal volume of 95% ethanol and ether previously neutralised with 0.1M potassium hydroxide to phenolphthalein solution. To the above solution add 1mL of phenolphthalein indicator and titrate with 0.1M potassium hydroxide until the solution remains faintly pink after shaking for 30 seconds.
Calculate the acid value from formula
Acid value (AV) = n x 5.61/ w
n= The number of ml of 0.1 M potassium hydroxide required
w= The weight in gram of the substance
Determination of extractive values of MT:
The extractive value of MT was determined using methanol and water to get medium-polar extractive value and high polar extractive values. for determination of extractive value 25ml of MT was taken and sonicated with 100ml of alcohol and alcohol soluble layer was separated and dried over water bath and the methanol soluble extractive value was calculated in percentage. The water soluble extractive value was calculated similar to that of alcohol soluble extractive value.
Preparation of test and standard samples:
25mL of Marichadi tailam was sonicated with 10mL of methanol and methanol soluble fraction was separated and dissolved in 0.25ml of methanol and used in HPTLC analysis. The standard solution of piperine was prepared by dissolving accurately weighed 10mg of piperine in 10 mL of methanol, which gives a 1mg/mL of concentration.
HPTLC analysis of Piper nigrum extract with MT:
For comparison methanolic extract and hexane extract of Piper nigrum fruits were prepared under reflux. The extract was dried and dissolved in methanol and hexane at concentration 10mg per ml for comparative TLC analysis. For comparative analysis of Marichadi tailam the tailam was subjected to saponification and the unsaponified fraction of MT was compared with Piper nigrum extract. The saponification was done to remove the fatty acid of the tailam.
HPTLC fingerprint of Marichadi tailam:
A Camag HPTLC system consist of Linomat V applicator with 100mL Hamilton syringe, and CAMAG Scanner III equipped with winCATS software version 1.4.3 was used for scanning of the plates. Densitometric scanning was done in absorbance-reflectance mode at 340 nm. Precoated TLC plate with silica gel 60F254 of 0.2 mm thickness (Merck-Germany) was used for HPTLC fingerprint. The plate was run in a Camag twin trough glass chamber using an optimized mobile phase consist of toluene: ethyl acetate:diethyl-amine (7.5:2:0.5; v:v:v:v). The mobile phase for TLC was optimized after using various solvents of different polarities consist of hexane, ethyl acetate, chloroform, ethanol and methanol of different ratios with saturation time for chamber of 10 minutes. The plate was visualized in Camag-UV cabinet under 254 nm and 366 nm. After the development the plate was derivatized with drgendorff’s reagent for detection of alkaloids.
Method Validation:
The HPTLC method was validated as per ICH guidelines analytical procedures. The method was validated by for parameters linearity, precision, accuracy, LOD and LOQ. The linear relation between peak area and concentration of piperine was evaluated by regression coefficient over the concentration of range of 2000 ng to 10, 000ng/band of piperine. Amount of piperine in the product was calculated using calibration curve of standard piperine. The precision was evaluated by in terms of intra-day precision and inter-day precision by analyzing 2mg to 10g of piperine on the same day and alternate day and the result was expressed as %CV. The recovery study was done by addition at three levels 80%, 100% and 120% of piperine to known amount of piperine standard solution. As per the ICH guideline, the LOD and LOQ were computed from the standard deviation of the response, peak area and slope of the calibration curve of markers using the formulas LOD =3.3×σ/S and LOQ =10×σ/S, where σ is the standard deviation of response, peak area, S is the slope of the calibration curve.
Table 1. Physicochemical properties of Marichadi tailam at room temperature 30℃
|
S. No |
Properties |
Marichadi Coconut Oil |
Coconut Oil |
|
1 |
Density (weight/mL) |
0.921 |
0.9229 |
|
2 |
Refractive index |
1.457 |
1.4485 |
|
3 |
Saponification Value (mg) |
286 |
165 |
|
4 |
Iodine Value (g) |
17.34 |
6.32 |
|
5 |
Peroxide value (meq/kg) |
6.2 |
3.8 |
|
6 |
Acid value |
2.89 |
2.00 |
|
7 |
Alcohol soluble extractive value (%w/w) |
4.12 |
3.90 |
|
8 |
Water soluble extractive value(%w/w) |
2.55 |
0.84 |
RESULTS AND DISCUSSION:
Physicochemical analysis of the tailam:
The quality of the Marichadi tailam was evaluated for physicochemical parameters such as density, refractive index, saponification value, iodine value, peroxide value and acid value.
HPTLC fingerprint:
Well resolved HPTLC fingerprint was developed for marichadi tailam formulation (figure 1). The unsaponified fraction of marichadi tailam, hexane extract and methanolic extract of piper nigrum extract showed spots ar Rf of 0.29, 0.41, 0.48, 0.56, 0.78, and 0.84 in 254nm, under 366nm they showed common fluorescent bluish, yellow and green bands at 0.29, 0.31, 0.36, 0.42 and 0.62 and after derivatization orange colored spots were visible at Rf of 0.24, 0.30, 0.42 and 0.49 which are common in the hexane and methanolic extract of Piper nigrum as well. The bands from the plant was more visible in methanolic fraction of the Marichadi tailam due to enrichment of tailam sample while the same bands were not visible in the direct tailam sample. As none of these common bands were not visible in coconut oil and its methanolic fraction of coconut oil, it infers that these compounds were extracted from the herb Piper nigrum processed in preparation of Marichadi tailam.
Figure 1. HPTLC Fingerprint of Marichadi tailam and Piper nigrum extracts
Track-1: Coconut oil, Track-2: Coconut oil methanolic fraction, Track-3: Marichadi oil, Track-4: Methanolic fraction of marichadi oil, Track-5: Piper nigrum methanol extract, Track-6: Piper nigrum hexane extract.
Table 2. Rf values of samples and Marichadi tailam at 254nm and 366 nm and under 520 nm post derivatization
|
Track |
Rf value (254 nm) |
Rf value (366 nm) |
Rf value (520 nm) Dragendorf reagent |
Rf value (520nm) Anisaldehyde H2SO4 |
|
1 (CO) |
0.80, 0.93 |
0.63, 0.70, 0.81, 0.88 |
0.83, 0.93 |
0.63, 0.70, 0.81, 0.88, |
|
2 (COMF) |
0.80, 0.93 |
0.63, 0.72, 0.77, 0.87 |
0.46 |
0.63, 0.72, 0.77, 0.87 |
|
3 (MCO) |
0.07, 0.10, 0.22, 0.31, 0.37, 0.46, 0.74, 0.90 |
0.19, 0.29, 0.35, 0.59, 0.77 |
0.23, 0.27, 0.38, 0.76, 0.79 |
0.19, 0.29, 0.35, 0.59, 0.77 |
|
4 (MCOMF) |
0.05, 0.12, 0.17, 0.23, 0.29, 0.41, 0.48, 0.56, 0.61, 0.72, 0.78, 0.84, 0.90 |
0.20, 0.28, 0.31, 0.36, 0.46, 0.62, 0.71, 0.75, 0.87, 0.95 |
0.24, 0.3, 0.41, 0.48, 0.55, 0.65, 0.73 |
0.20, 0.28, 0.31, 0.36, 0.46, 0.62, 0.71, 0.75, 0.87. |
|
5 (PNPF) |
0.08, 0.28, 0.34, 0.41, 0.49, 0.55, 0.66, 0.78, 0.86 |
0.06, 0.15, 0.19, 0.31, 0.40, 0.45, 0.56, 0.62, 0.68, 0.77 |
0.09, 0.25, 0.28, 0.3, 0.42, 0.49 |
0.06, 0.15, 0.19, 0.31, 0.40, 0.45, 0.56, 0.62, 0.68, 0.77 |
|
6 (PNMF) |
0.09, 0.16, 0.24, 0.32, 0.40, 0.47, 0.53, 0.66, 0.76, 0.92 |
0.06, 0.19, 0.29, 0.36, 0.43, 0.60 |
0.03, 0.11, 0.17, 0.19, 0.24, 0.3, 0.41, 0.48 |
0.06, 0.19, 0.29, 0.36, 0.43, 0.60 |
CO-Coconut Oil, COMF: Coconut oil methanolic fraction, MT: Marichadi tailam; MCOMF: Marichadi tailam methanolic fraction; PNPE: Piper nigrum hexane extract; PNEE: Piper nigrum methanol extract
Method Validation:
The method was validated in accordance with ICH guide lines with determination of validation parameters. Standard solution of piperine was applied over silica gel TLC plates at volume of 2mg to 10mg. The plate was developed and analyzed to generate a calibration plot (Table 1 and Figure 1) for quantification of piperine in Marichadi tailam. The spots were well separated in the chromatogram of the formulation samples, extract of Piper nigrum and the spot of standard piperine was located at Rf value of 0.33. The linearity studies were carried out and there exists linearity in the concentration range of 2 to 10mg/ml. The limit of detection (LOD) and limit of quantification was found to be 0.6mg and 2mg/ml (table 2) respectively. Amount of piperine in 100 mg of oil was found to be 10.13mg. The recoveries for piperine was found to be 98.10 to 101.30. The method developed was found to be accurate, precise and a suitable and cost effective method for estimation of piperine in Ayurvedic Formulation Marichadi tailam.
Figure 2.- Standard curve of piperine
Table 3. HPTLC Scanning of the plate at 340nm
|
S. No |
Applied volume of piperine solution |
Rf Value |
Area |
|
1 |
2.0 |
0.33 |
22720.22 |
|
2 |
4.0 |
0.32 |
27324.00 |
|
3 |
6.0 |
0.32 |
29889.53 |
|
4 |
8.0 |
0.33 |
32042.37 |
|
5 |
10.0 |
0.33 |
33641.91 |
|
(A) |
(B) |
|
High‑performance thin layer chromatography chromatogram of standard with Marichadi tailam (A) 254 nm and (B)-366nm |
|
|
Chromatogram of piperine |
Chromatogram of piperine in sample |
|
Absorption spectrum of piperine |
Absorption spectrum of piperine in marichadi tailam sample |
Figure 3. High‑performance thin layer chromatography chromatogram of standard with Marichadi tailam for quantification of piperine
Table 4. Linear regression parameters for piperine
|
Parameters |
piperine |
|
Rf |
0.32 |
|
Concentration range(mg/band) |
2 mg to 10mg |
|
Regression Equation |
y = 1328.083x + 21155.129 |
|
Correlation coefficient (R2) |
0.9780 |
|
LOD |
0.66 mg |
|
LOQ |
2 mg |
|
Precision |
0.03 |
|
Accuracy (% recovery) |
102.72% |
|
Amount found in 100mg |
10.13 µg |
CONCLUSION:
The developed and validated HPTLC method for estimation of the piperine as a marker component may be useful for the quality control and standardization of traditional ayurvedic medicines and herbal drugs. It will also help physicians and pharmaceutical researchers to understand the role of piperine present in Marichadi tailam in therapeutic application and to further understanding its mechanism of actions.
ACKNOWLEDGEMENT:
we are deeply thankful to Dr. Ramesh Raghava Varier, the Managing Director of AVN Ayurveda Formulation Pvt Ltd for his immense support, inspiration and motivation to carry out research work on Ayurveda and to provide all the necessary facilities.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
REFERENCES:
1. Roy U, David B, Stefan G, Sabine G. Botanical ingredient identification and quality assessment: strengths and limitations of analytical techniques. Phytochemistry Reviews volume (Phytochem Rev.). 2020; 19: 1157-77.
2. Jon CT, Ted JK. Herbal medicine research and global health: an ethical analysis. Bull World Health Organ. 2008; 86(8): 594-9.
3. Sen S, Chakraborty R. Revival, modernization and integration of Indian traditional herbal medicine in clinical practice: Importance, challenges and future. J Tradit Complement Med. 2017; 7(2):234–244.
4. Li Y, Shen Y, Yao C-liang, Guo D-an, Quality assessment of herbal medicines based on chemical fingerprints combined with chemometrics approach: A review. Journal of Pharmaceutical and Biomedical Analysis. 20205; 185:113215.
5. WHO. Guidelines for assessing quality of herbal medicines with reference to contaminants and residues. Geneva: World Health Organization; 2007.
6. Jaiswal S, Chavhan SA, Shinde SA, Wawge NK. New Tools for Herbal Drug Standardization. Asian J. Res. Pharm. Sci. 2018; 8(3): 161-169.
7. Patel U, Girme A, Patel K, Ghule C, Lal H, Gandhi T.A validated HPTLC method for quantification of cordifolioside A, 20‑β‑hydroxyecdysone and columbin with HPTLC–ESI–MS/MS characterization in stems of Tinospora cordifolia. JPC-Journal of Planar Chromatography-Modern TLC. 2021; 34:217-28.
8. Bhakti JL, Vijay MM, Urmi DK, Rina HG. Marker based standardization of polyherbal formulation (SJT-DI-02) by high performance thin layer chromatography method. J Pharm Bioallied Sci. 2014; 6(3): 213-9.
9. Jain V, Jain T, Saraf S. HPTLC method for routine quality control of Ayurvedic formulation Drakshadi gutika. Asian Journal of Pharmaceutical Analysis. J. Pharm. Ana. 2013; 3(4):111-4.
10. Arivukkarasu A, Rajasekaran A. Detection and Quantification of Anti-oxidant markers like Rutin, Catechin, Quercetin, Gallic acid, Ellagic acid, Ferulic acid, Vitexin and Mangiferin in Herbal raw materials available in market belongs to Rutaceae family by HPTLC Technique. Asian Journal of Pharmaceutical Analysis. 2021; 11(2):151-5.
11. Ghumare P, Dolas RT, Aher V. Development of HPTLC Method for the Estimation of Andrgrapholide from Arjuna Tablet. Asian Journal of Pharmaceutical Analysis. 2021; 11(2):156-8.
12. Rawat S, Gupta A. Development of Novel HPTLC Method for Estimation of Qurcetine in Ocimum sanctum. Asian J. Pharm. Tech. 1(4): Oct. - Dec. 2011; Page 149-151.
13. Kaviarasan L, Sarojini S, Yoganandam PG, Gopal V. Preparation and Standardization of Deepavali Avaleha - A Traditional Polyherbal Ayurvedic Formulation. Asian J. Pharm. Tech. 2016; 6(4): 231-237.
14. Sharma R, Kumari N, Ashawat MS, Verma CPS. Standardization and Phytochemical Screening analysis for Herbal Extracts: Zingiber officinalis, Rosc., Curcuma longa Linn., Cinnamonum zeylanicum Nees., Piper longum, Linn., Boerhaavia diffussa Linn. Asian J. Pharm. Tech. 2020; 10(3):127-133.
15. Yeotkar US, Nimbhorkar M, Deshmukh TA, Patil VR. Quantitative Estimation of Piperine in Ayurvedic Formulations by HPTLC. Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 898-902.
16. Rohan RV, Talokar S, Dhole AR, Mohite SK, Magdum CS. Comparative Standardization Study of Two Marketed Shatavari Churna Formulation. Asian J. Pharm. Ana. 2016; 6(1):1-6.
17. Singh N, Chaudhary A.A comparative review study of Sneha Kalpana (Paka) vis-a-vis liposome. Ayu. 2011; 32(1):103-8.
Received on 27.10.2022 Modified on 13.01.2023
Accepted on 25.02.2023 ©AJRC All right reserved
Asian J. Research Chem. 2023; 16(2):133-138.
DOI: 10.52711/0974-4150.2023.00022