Preparation, Preliminary Physicochemical Evaluation and HPLC Analysis of Brahmi ghrita

Introduction

Ghee-based formulations are prescribed for various disorders in Ayurveda. Brahmi ghrita (BG) is a ghee-based poly herbal formulation used in Ayurveda, also called ‘Sneha Kalpana’. BG is a major formulation prescribed for the management of psychological disorders mentioned in classical Ayurvedic texts, with different compositions for treatment of different disorders.1,2 According to Charaka Samhita, BG is composed of Purana Ghrita (old ghee), Brahmi (Bacopa monneri), Kushta (Sassarea lappa), Vacha (Acorus calamus), and Sankhapushpi (Convlulos pluricalis). In addition to Apasmara, additional neurological conditions are treated with BG.¹

Being a classical preparation, mere data is available on the standardization of BG according to modern analytical techniques. The available scientific evidence confirms Bacoside A as the active constituent responsible for the CNS related pharmacological activities of Bacopa.³ Bacosides has been shown to repair injured neurons by boosting kinase activity, neuronal synthesis, synaptic activity and nerve impulse transmission.⁴ Attempts were made to estimate Bacoside A in other Ayurvedic formulations using the HPLC (High-performance liquid chromatography) method, but there is no evidence of estimation of Bacoside A in Brahmi ghrita using the same.^5,6 With this background, the present study was undertaken to quantify the Bacoside A present in the BG, along with assessing its physicochemical characters.

Materials and Methods

Raw material collection and authentication

Fresh Brahmi was collected from the Indian Institute of Horticulture Research (IIHR), Hesaraghatta, Bangalore. Vacha, kushta and shankhapushpi were purchased from M/S Amruthkesari Depot, Bangalore. Identification and authentication of plant materials was done at Regional Ayurveda Research Institute for Metabolic Disorders, Bangalore. Purana Ghrita (5 year old) was collected as a gift sample from Rashtrotthana Goushala, Doddaballpur. It was analyzed and compared with the API standards.

A reference standard Bacoside A was received as a gift sample from Sami Labs Ltd, Bengaluru. The HPLC solvents such as methanol, acetonitrile, and ortho phosphoric acid were procured from Ms. Merck Limited, Mumbai, India. The HPLC-grade water was prepared using a millipore purification system.

Preparation of Ghrita

The BG was prepared as prescribed in Srangadhara Samhita in the ratio of one part of Purana ghrita (old ghee), four parts of Brahmi juice, 1/8th part of paste of the drugs vacha, kushta, shankapushpi, and four parts of water.⁷ Fresh Brahmi was subjected to mechanical pressure to extract the Brahmi swarasa (juice). In a large iron vessel, ghrita was liquified under moderate temperature, later the kalka (paste) of vacha, kushtha, and shankhapushpi (in the ratio of 1/8th of ghee) prepared in a small quantity of Brahmi Swarasa was added; subsequently remaining Brahmi Swarasa and four parts of water were added. The contents were heated for three hours at moderate temperature (70-80ºC) and allowed to cool. This process was repeated for three days. At the end of the third day, the contents were heated till the Sneha siddhi lakshana (end point of ghee preparation).⁸ Then the vessel was removed from the fire and the contents were filtered through a clean, white, thick and dry cloth while its warm and stored in an airtight container till further use.

Organoleptic and Physicochemical Parameters

The prepared ghrita samples were analyzed for organoleptic characteristics such as color, odor and taste. Physicochemical parameters like refractive index, specific gravity, acid value, saponification value, and iodine value were performed as per the Protocol for testing of Ayurveda, Siddha and Unani medicine and Ayurvedic Pharmacopoeia of India.^9,10

Refractive index (RI)

Refractive index was measured using Abbes refractometer. Initially a drop of water was placed on the prism and the drive knob was adjusted in such a way that the boundary line intersects the separatrix exactly at the centre. The reading was noted. RI of melted ghrita samples was determined using one drop of the sample.

Specific gravity

The specific gravity of ghritas was measured using pycnometer attached with thermometer at 40°C. The empty weight of dried pycnometer with thermometer was noted down. Then the pycnometer was filled with sample, the spillage was cleaned with tissue paper and the filled weight was noted down. After the samples, the pycnometer was cleaned with ether and acetone, dried and the weight of the distilled water was noted down. The specific gravity of ghrita samples were estimated in triplicate. The specific gravity of the sample was calculated using the formula:

specific gravity = (weight of sample)/(weight of water) x density of water

where a is volume of 0.1N KOH in ML, and w is the weight of sample in gram.

Iodine value

About 0.5 to 1 g sample was accurately weighed in a dry iodine flask. The sample was dissolved with 10 mL CCl4. To this, 20 mL of iodine mono chloride solution (whijs solution) was added. Stopper was inserted, which was previously moistened with solution of potassium iodide. Flask was kept in a dark place at a temperature of about 17ºC for 30 min. 15 mL of potassium iodide and 100 mL of water were added and the mixture was shaken well. This was titrated with 0.1N sodium thiosulphate, using starch as an indicator. Blank titration was performed omitting the sample. Iodine value was calculated using the formula:

Iodine value = ((b-a)×0.01269×100)/w

where - a is the volume of 0.1 N sodium thiosulfate consumed by sample (mL)

b is the volume consumed during blank titration (mL)

w is the weight of the sample (g)

Saponification value

About 2g of sample was accurately weighed into 250 mL flask. 25 mL of 0.5 M alcoholic potassium hydroxide was added and the contents were refluxed on a water bath for one hour. The contents were cooled and titrated against 0.5 M HCl using phenolphthalein as indicator. A blank titration was performed omitting the sample. The saponification value was calculated using the formula-

Saponification value = ((b-a)×0.02805×1000)/w

Where - a is the volume of 0.1M HCl consumed by sample (mL)

b is the volume consumed during blank titration (mL)

w is the weight of the sample (g)

UV spectroscopy of Brahmi ghrita

Instrumentation

UV absorbance spectroscopy was performed using a Shimadzu Model UV-2501 PC UV-VIS spectro-photometer equipped with a data system using UV probe software version 2.51 (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). The scanning range was set at 200 to 800 nm wavelength.

Sample preparation

Initially, about 0.2 g of BG was weighed and dissolved in 50 mL of petroleum ether. It was extracted four times with 25 mL of 70% methanol. Each fraction was filtered through a 0.45μ filter and subjected to UV scanning and HPLC analysis. A similar procedure was adopted for 0.5, 1, 2, 5 and 8 g of BG samples.

HPLC analysis of Brahmi ghrita (BG)

The HPLC analysis of BG was performed using Bacoside A as a reference standard. The HPLC analysis was performed through modifications of the standard method given in the official monograph of Bacopa monniera in United States Pharmacopoeia (USP).¹¹

Instrumentation

An HPLC instrument (Shimadzu) consisting of an ultraviolet detector equipped with LC solution software with 205 nm was used for the analysis. The chromatographic separations were performed on a ZODIAC C18 (250 × 4.6 mm), 5 μm column, oven temperature maintained at 27°C, with injection volume 50 μL, flow rate of 1.5 mL/min and a total run time of 70 minutes.

Mobile phase composition

Solution A consists of 0.14 g of anhydrous potassium dihydrogen phosphate dissolved in 900 mL water, 0.5 mL of phosphoric acid was added and made-up to 1000 mL with water, filtered and degassed. The solution B comprised of Acetonitrile and 70% v/v methanol in water was used as a diluent.

A gradient elution pattern of solution A: solution B was executed as 70:30, 60:40, 50:50, 40:60, 30:70 with time intervals 0-25 min, 25-35 min, 35-45 min, 45-50 min, and 50-60 min, respectively.

Standard preparation

The standard stock solution was prepared by dissolving 25.5 mg of Bacoside A (24.68%) in 25 mL of 70% v/v methanol (in water), which is equivalent to 251.74 μg/mL. Using the stock solution, serial dilutions corresponding to 5.03, 12.5, 25.17, 50.34, 100.69, 151.04, and 251.74 μg/mL concentrations of Bacoside A were prepared in 70% v/v methanol. A standard calibration curve was plotted using these concentrations.

Sample preparation

After the initial optimization procedure, about 8 g of BG was dissolved in 50 mL of petroleum ether and extracted four times with 25 mL of 70% methanol.The methanol fractions were pooled, evaporated and it was reconstituted with 10 mL of 70% methanol. Subsequently, it was filtered through a 0.45 μ filter and injected to the HPLC system.

Data collection and analysis

Samples were analyzed in triplicates (n=3) for all the physico-chemical parameters. Values were expressed in mean±SEM. The total amount of Bacoside A present in BG was calculated using the standard linearity curve of Bacoside A. The values were expressed as mg/g of BG.

Results

Organoleptic parameters

Purana ghrita (PG) was greyish in color, with a fragrant odor and a characteristic taste. The BG was green in color, with characteristic fragrance and taste.

Physicochemical parameters

Physicochemical parameters such as RI, specific gravity, acid value, iodine value, and saponification value of PG and BG were found to be 1.460 and 1.461; 0.90592 and 0.90486; 6.225 and 6.545; 32.24 and 31.27; 239.2 and 243.6, respectively.

The UV spectra of BG showed three absorption maxima at wavelengths 205 nm, 285 nm and 300 nm. The UV spectrum of BG is depicted in Figure 1.

Optimization of extraction method for HPLC

Methanolic fractions of BG samples weighing 0.5, 1, and 2 g did not show any peaks in the HPLC chromatogram. The peaks were detected only in the first fraction of the 5 g sample, but subsequent fractions did not show any peaks. In 8 g sample, HPLC peaks were noticed up to four fractions, but in the 5th fraction, the desired peaks were not detected. Hence, 8 g of BG sample was used with an extraction cycle of 4x25 mL of 70% methanol.

HPLC chromatogram of Bacoside A

The HPLC chromatogram for standard Bacoside A is depicted in Figure 2 a. The peaks were identified as Bacoside A3 (RT-17.1 min), Bacopaside II (RT-17.71 min), Bacopaside X (RT-19.447 min) and Bacosaponin C (RT- 20.28 min). The RT of the peaks eluted in the chromatogram of BG were found to be matching with the standard chromatogram and were well separated (Figure 2b).

The total area under the curve (AUC) of Bacoside A was calculated by adding the individual areas of Bacoside A3, Bacopaside II, Bacopaside X and Bacosaponin C. By plotting area against Bacoside A concentration, the linearity curve was obtained. The r2 (r-square) value was found to be 0.9997 with slope equation y = 9911.3x-10046 (Table 1, Figure 3). The concentration of Bacoside A was calculated using the equation and the results are given in Table 2.

Discussion

The principal components that compose Brahmi ghrita are Brahmi (Bacopa monneri), Kushta (Sassarea lappa), Vacha (Acorus calamus), Sankhapushpi (Convlulos pluricalis) and Purana ghrita.¹ The classical texts mentioned that the medicinal value of a ghrita increases with time.¹² According to Kaiyadheva nigantu, the ghrita which is older than one year can be considered as Purana ghrita.¹³ In this regard, in the present study, 5-year-old ghrita (ghee) was used for the preparation of BG.

Though Ayurveda has been extensively practiced in India and abroad, the quality of the Ayurvedic products has been a prime concern for the end users. In this context, the standardization of Ayurvedic medicines aids in filling the paucity, and the evaluation of physicochemical parameters help in the identification and authentication of the product and substance. In these lines, in the present study, we made an attempt to standardize the Brahmi ghrita for physicochemical properties and Bacoside A content.

The physicochemical parameters of PG and BG were assessed in terms of specific gravity, refractive index (RI), acid value, iodine value and saponification value.

Refractive Index (RI) is mostly used to determine the identity of a specific material, verify its purity, or calculate its concentration. RI at 40°C can also be correlated with the presence of turbid materials. The RI helps in the identification of adulteration in ghee with vegetable oils.¹⁴ There was no significant difference in the refractive indices of the BG and PG, though there was a slight difference in RI compared to the original ghrita samples. Thus, the addition of plant materials did not significantly influence the RI of the ghrita.

The specific gra vity of a sample can be correlated to the density of arranged molecules, and the total solid content in a sample is indicated by the ratio of mass/volume.¹⁵ The specific gravity of BG was slightly different from the Purana ghrita and may be due to the presence of plant materials.

One of the distinguishing parameters used to define fats and oils is the acid value. It is the amount of potassium hydroxide (KOH) in milligrams required to neutralize 1g of fat's organic acid content and is a measurement of the amount of free fatty acids (FFA) found in oils and fats. Acid values of PG and BG were found to be high. An increase in FFA content in an oil or fat sample signifies the hydrolysis of triglycerides.¹⁶ The higher acid value in PG and BG was due to the time-based and enzyme-induced hydrolysis of triglycerides. According to a report, ghrita made using the conventional method has higher concentrations of Omega-3 fatty acids and docosahexaenoic acid (DHA), and therefore ghritas are considered to be useful in treating various diseases associated with the retina and brain.¹⁷

The iodine value is indicative of the extent of unsaturated fatty material present in the ghrita. The greater the iodine number, the more are the unsaturated bonds present in the fat. The poly unsaturated fatty acids (PFA) possess several health benefits.¹⁸ It was evident from the present study that there was no significant change in the iodine values of PG and BG.

The iodine value is defined as the amount of KOH in milligrams required to neutralize the fatty acids resulting from the complete hydrolysis of 1g of fat or oil. It was evidenced that short chain fatty acids have higher saponification values, while long chain fatty acids have lower ones. The short chain fatty acids are beneficial in maintaining the gut health.¹⁹ The BG showed higher saponification value compared to PG. However, the changes in saponification values were not statistically significant.

Bacoside A is comprised of a mixture of saponins, specifically Bacoside A3, Bacopaside II, Bacosaponin C, and the jujubogenin isomer of Bacosaponin C (Bacoside X.).^5,20 Previously reported analytical methods for Bacoside A suggest that methanol is the ideal solvent for the extraction of Bacoside A.^6,20 Since methanol is miscible with petroleum ether, in the present study, the extraction procedure was established using petroleum ether and methanol (70%). The ghrita was dissolved in petroleum ether, and Bacoside A was partitioned between petroleum ether and methanol (70%). The resultant methanolic portion was analyzed using UV and HPLC. The extraction procedure was optimized using different amounts of ghrita and modifying extraction cycles.

The procedure given in the USP monograph was used for the estimation of Bacoside A.¹¹ During the procedure, the standard chromatogram showed separation of four saponins of Bacoside A, while in the sample chromatogram, there was an interference observed in subsequent injections which may be due to other phytoconstituents present in the formulation and carried over to the next injection. In the present study, the run time was increased so that all the constituents present in the BG were eluted without interfering with Bacoside A.

Conclusion

In the present study, physicochemical parameters (refractive index, specific gravity, acid value, saponification value and iodine value) of prepared Brahmi ghrita were assessed along with the quantification of Bacoside A content by optimizing the extraction procedure. The method of extraction and estimation of Bacoside A in Brahmi ghrita may be helpful in quantifying Bacoside A in ghrita samples and thereby play an important role in pharmacodynamic and pharmacokinetic studies of Brahmi ghrita. Further research is required to determine the other active phytoconstituents present in Brahmi ghrita.

Conflicts of Interest

Authors declares no conflicts of interest