Effect of infusing ghee with Ayurvedic herb mix (Murchana) on its oxidative stability and its comparative analysis with medicated ghee (Siddha Ghrita)

Abstract

Background

The preparation of medicated ghee, known as siddha ghrita in Ayurveda, faces significant challenges due to lipid susceptibility to oxidation, which reduces shelf life and potentially leads to the loss of bioactive compounds. A popular lipid-stabilization process, murchana, has been recommended in Ayurveda as a pre-treatment for ghee with an Ayurvedic herb mix, which may improve its stability.

Objective

This study investigates the effect of murchana process on the oxidative stability of Centella asiatica (CA) ghee.

Materials and Methods

Plain ghee (PG), murchit ghee (MG), CA with PG (PGCA), and CA with MG (MGCA) were assessed for their oxidative stability via peroxide value (PV) and Rancimat induction time. Physicochemical properties (granulation, saponification value, color, free fatty acids), antioxidant capacity (DPPH-RSA), and Total Polyphenolic Content (TPC) were evaluated over 160 days at 45 °C, 85% RH.

Results

MGCA showed higher oxidative stability (PV: 6.49 vs. safe limit of 10 m.eq. O₂/kg), longer induction time (MGCA: 38.8 h vs. PGCA: 24.32 h as measured by Rancimat), higher TPC (273.27 μg GAE/g), and antioxidant activity (DPPH-RSA: 5.37%) compared to PGCA. No detectable cholesterol oxidation products (COPs) were observed under the TLC conditions employed.

Conclusion

The results indicated that the murchana process delayed rancidity and enhanced the oxidative stability of the medicated ghee. However, the underlying phytochemical changes were not directly investigated in this study. Further studies are warranted to elucidate the contribution of these changes to the oxidative stability and therapeutic potential of Ayurvedic medicated ghee preparations.

1. Introduction

Medicated lipid-based formulations hold a prominent place in Ayurvedic therapeutics, serving vital roles in both internal and external clinical applications [[1], [2]]. These formulations are prepared by processing herbs or botanicals in a lipid-based medium to facilitate the extraction and bioavailability of their phytoconstituents. The herbal formulations have been suggested to deliver phytoconstituents to the peripheral circulation via the lymphatic system, thereby prolonging their half-lives. Phytoconstituents from aqueous-based formulations, on the other hand, enter the bloodstream directly through capillaries. The portal vein then carries them to the liver, thereby reducing the half-lives of the active ingredients [3].

The process for preparing lipid-based formulations in Ayurveda is called sneha kalpana, as are the products produced by this process. The base lipid used in sneha kalpana can be ghee, oil, or other fatty substances. When herbs are processed with ghee according to the principles of sneha kalpana, the resulting medicated ghee is called siddha ghrita (or medicated ghee). The Ayurveda Formulary of India lists 44 ghee-based sneha kalpanas and 63 oil-based sneha kalpana. The general process of sneha kalpana involves heating ghee or oil with decoctions (kashaya) and finely ground, smooth pastes (kalka) of select herbs specific to the formulation at a temperature near the boiling point of water, thereby facilitating the transfer of bioactive molecules into the lipid phase. The herbal decoction and paste bring water and insoluble solids to the mixture. The heating is continued until all the water has evaporated and the insoluble solids have settled out. The ghee or oil is then filtered, leaving it free of insolubles and with moisture content of <0.5%. A generic ratio of the various components used in many sneha kalpana or lipid-based medicated formulations involves 1 part herb paste (kalka), 4 parts lipid base, and 16 parts herbal decoction (kasaya) [[4], [5]].

Bhaishajya Ratnavali, a 16th-century text, suggests a two-stage infusing process for sneha kalpana. The first stage involves infusing ghee with a specific set of murchana herbs. This step yields an intermediate lipid formulation, referred to as murchit ghee (MG). MG was then used as the base for the second stage, in which the herbal decoction, and paste were added to MG, followed by heating to remove water and separate the insoluble solids. The MG is then filtered out to yield the final medicated ghee. The heating of lipids in the presence of water during the infusion step may lead to lipid deterioration and the breakdown of triglycerides into diglycerides, monoglycerides, and free fatty acids, as well as their hydrolytic oxidation [6]. Such changes can affect sensory quality, acceptability, and shelf life of lipid-based products. More importantly, the oxidized fatty acids are harmful to health [7]. Therefore, it is essential to investigate the sneha kalpana process to assess the oxidative stability of medicated ghee and the significance of the murchana step.

The assessment of lipid quality and stability often relies on physicochemical parameters such as acid value, saponification value, peroxide value (PV), and free fatty acid content. However, these methods are not direct measures of lipid oxidative stability. The saponification value is an indicator of the length of the fatty acid carbon chains. It is commonly used to characterize the average molecular weight of fatty acids in an oil and is not a direct test of stability [[7], [8]]. The free fatty acid content indicates the readily oxidizable species and, again, is not a true indicator of stability [9]. The PV measures only the transient species generated by lipid oxidation at any given time [10]. While traditional methods provide valuable, immediate data, they offer limited insight into the long-term oxidative stability of lipids. Recognizing this shortcoming, the current study evaluated the stability of medicated ghee using Rancimat method. The Rancimat assay offers a complementary and valuable approach to assessing lipid stability through accelerated aging. The test measures induction time, determining the precise inflection point at which rapid oxidative degradation begins, providing an important indicator of the oxidative stability of lipid-based systems. This method provides a dynamic assessment of lipid stability by simulating accelerated thermal-oxidative stress through exposing a lipid sample to controlled heat and a continuous air stream, thereby inducing auto-oxidation [11]. Complementing induction time data with cholesterol oxidation products (COPs) and traditional measures such as PV can give new insights into product stability. Further, a single-stage process is used as a control in which the herbal decoction and paste were directly added to the lipid of choice, followed by heating to remove water and separate insoluble components. Insights gained from this study will not only help improve the quality of Ayurveda sneha kalpana products but also benefit the manufacture of medicated oils that are gaining popularity for non-therapeutic applications.

2. Materials and methods

Dry whole herbs for murchana process, including Emblica officinalis (Amla), Terminalia chebula (Haritaki), Terminalia belerica (Bibhitaki), Curcuma longa (turmeric), Cyperus rotundus (Musta), were obtained from the pharmacy of Institute of Ayurveda and Integrative Medicine (I-AIM), Bengaluru, Karnataka, India, NABH-accredited healthcare facility. Fresh Citrus medica (Matulunga) fruits and fresh Centella asiatica (Mandukparni) leaves were procured from the Herbal Garden and Nursery located at the I-AIM hospital, Bengaluru, Karnataka, India. The synthetic antioxidant Butylated Hydroxytoluene (BHT; Sigma-Aldrich) was obtained from SRV Enterprises, Bengaluru. All chemicals used in the study were of analytical grade.

2.1. Preparation of ghee (Plain Ghee, PG)

Ghee was prepared from milk of the Deoni cow breed, which was procured from the dairy farm of the Southern Regional Station, National Dairy Research Institute (Bengaluru, Karnataka, India). Milk was pasteurized and inoculated with a 2% starter culture of Streptococcus lactis sbsp. Diacetylactis, followed by incubation at 37 °C for 20–22 h.

The starter culture of Streptococcus lactis ssp. diacetylactis was obtained from the National Consortium of Dairy Cultures (NCDC) at the National Dairy Research Institute, Karnal, Haryana, India. The set curd was churned to extract butter. The butter was clarified at 120 °C to produce ghee. Clarification was continued until the moisture content of the ghee reduced to less than 0.5%. Fresh ghee was filtered and stored in air-tight glass bottles for analysis. PG containing 0.02% BHT was used as a positive control to compare the oxidative stability of herbs with that of a synthetic antioxidant. The 0.02% concentration of BHT was selected as the positive control since this level was in accordance with the maximum regulatory limit for synthetic antioxidants in clarified butterfat under FSSAI guidelines [[12], [13]].

2.2. Preparation of murchit ghee (MG)

Murchana herbs were powdered in a grinder and sieved through an 80-mesh sieve to obtain fine powder. The paste was made by mixing Citrus medica juice and water. The quantities of herbs used are given in Table 1. This paste was added to melted ghee and water (in a 1:4 ratio) in a stainless-steel vessel. The vessel was heated on a low flame with intermittent stirring. The mixture's temperature was monitored, and heating ceased at 120 °C upon ‘wick’ formation. The murchit ghee was filtered and stored in a glass bottle [14].

Table 1.

Proportions of ingredients used in Murchit Ghee (MG).

S.No.	Ingredients	Scientific name	Plant part used	Amount
1	Haritaki	Terminalia chebula	Fruit (dried pericarp)	48 g
2	Dhatri/Amla	Emblica officinalis	Fruit (dried pericarp)	48 g
3	Bibhitaki	Terminalia belerica	Fruit (dried pericarp)	48 g
4	Musta	Cyperus rotundus	Rhizome	48 g
5	Haridra	Curcuma longa	Rhizome	48 g
6	Matulunga	Citrus medica	Fresh fruit juice	48 g
7	Ghee			768 g
8	Water			3.072 L

2.3. Processing of Centella asiatica (CA) ghrita

Fresh Centella asiatica leaves were thoroughly washed and homogenized to obtain the fresh leaf juice (Brahmi swarasa). The juice thus obtained was filtered through a muslin cloth and added to plain ghee (PG) and murchit ghee (MG) to make two medicated ghee; (1) plain ghee with CA (PGCA) and (2) murchit ghee with CA (MGCA).

For medicated ghee processing, one part ghee was heated mildly and mixed with two parts CA juice in a stainless-steel vessel. The ratio of Brahmi svarasa (1.536 L) and ghee (768 g) used in the preparation was based on the classical formulation of Brahmi ghrita described in Ashtanga Hridayam. The mixtures were heated under mild heating conditions (mridu agni) with continuous stirring until attainment of paka siddhi lakshana, indicated by subsidence of froth and formation of a dry-rolled wick (varti). The medicated ghee was then filtered while warm and stored in airtight glass bottles for further analysis. Fig. 1 illustrates the three-step preparation process of the medicated ghrita formulations.

Fig. 1.

Steps for making plain ghee, murchit ghee, and Centella asiatica ghrita.

Schematic representation of the preparation of medicated ghee -Centella asiatica Ghrita (CA Ghrita), illustrating the sequential steps of ghee preparation, Murchana (pre-processing), and medicated ghrita preparation. Kalka Dravya: Mixture of the herbs; Kalka: Thick paste of the herbs made with Matulunga juice (Citrus medica) and water; Ghrita paka: Processing/heating of the herbal mixture (kalka) with ghee and water.

2.4. Physico-chemical analysis of ghee

2.4.1. Moisture content

The moisture content of freshly prepared ghee samples was determined using a moisture analyzer (RADWAG, model MA 50. R, Poland, EU) equipped with infrared heating. One gram of the sample was placed in a tared aluminium plate within the moisture meter. The drying temperature was set to 120 °C, and the process was set to auto-switch-off mode. The analysis was conducted in triplicate, and the mean with standard deviation was recorded [15].

2.4.2. Colour assessment

The colour of ghee samples was assessed using a digital colorimeter (Make: Konica Minolta). Ghee samples were heated at 25 °C for 3 h in a water bath before the analysis. Approximately 15 g of ghee was placed in a dark-coloured container for colour analysis. The colorimeter was set to take three readings for each test, and the average l, a, b values were recorded. Mean values of the three determinations were directly obtained from the output device of the digital colorimeter.

2.4.3. Granulation

Grain formation during ghee granulation was evaluated using modifications from the method by Arumughan and Narayanan (1979). Ghee samples were melted at 40 °C in a water bath. Ghee (30 mL) was placed in a tightly capped 50 mL centrifuge tube and incubated at 29 °C for 24 h for complete granulation. Subsequently, the samples were centrifuged at 250 × g for 10 min to separate grains. The quantity of solid granulated ghee settled at the bottom of the tube was recorded and expressed as a percentage of the total fat [16].

2.4.4. Specific gravity

The specific gravity of ghee samples was assessed using the Pycnometer method as described by the Food Safety and Standards Authority of India [17].

2.4.5. Saponification value

The saponification value of the ghee samples was assessed according to the method outlined in the Manual of Analysis of Fats and Oils, published by the Food Safety and Standards Authority of India [17].

2.4.6. Free fatty acid (FFA) value

10 g of ghee sample was taken in a 250 ml conical flask. 50 mL of ethanol was neutralized and added to the ghee. The contents were heated to ensure proper mixing and then titrated with 0.1 N NaOH while vigorously shaking. The endpoint was reached when a single drop produced a faint but distinct color change that lasted at least 15 s. The acidity of ghee was calculated as the percentage of free fatty acids in the sample (oleic acid), where (1 ml of N NaOH = 1 ml of N oleic acid = 0.282 g of oleic acid) [18].

2.5. Storage stability

Ghee samples were stored in airtight glass containers in a stability chamber at 45 °C and 85% relative humidity (RH) for 160 days. The samples were withdrawn at 10-day intervals up to 60 days, then at 100 days (160 days) for analysis of oxidative stability and rancidity.

2.5.1. Peroxide value (PV)

The PV of ghee samples was determined using the method described in IS: 3508 (1966) with some modifications. The ghee sample was accurately weighed to 5 g into a 250 ml glass-stoppered Erlenmeyer flask. 20 mL of a solvent mixture of glacial acetic acid and chloroform (3:2) was added to the flask and swirled to dissolve the fat. Freshly prepared saturated potassium iodide (0.5 mL) was added to the flask and left undisturbed for 1 min. The Erlenmeyer flask was capped with a glass stopper and heated in a boiling water bath for 30 s. Then, the flask was placed under a stream of cold water for about 1 min to condense the vapours produced during boiling. Distilled water (25 mL) was added to the cooled solvent mixture, followed by 0.5 mL of 1% freshly prepared starch indicator, which act as a color indicator. The mixture was titrated against 0.01 N sodium thiosulfate, and the disappearance of blue colour was recorded as the end of the titration [18].

2.5.2. Induction period by rancimat

The induction period of the ghee samples was measured through the Rancimat apparatus (Metrohm Rancimat Model 743, Switzerland) at ITC, Bengaluru, Karnataka, India. The operating parameters for analyzing the induction period were set to 120 °C and an airflow rate of 20 L/h. A molten ghee sample (3 g) was placed in a vessel and heated in the heating block of the Rancimat apparatus. The capped vessel was connected to the measuring vessel via connecting tubes. Deionized water (60 ml) was added to each measuring vessel with enclosed conductors. The test was done at 120 °C until the endpoint was reached [13].

2.6. Cholesterol oxidation products (COPs)

COPs in ghee were extracted by the cold saponification method [19]. Ghee (2g) was weighed and mixed with 50 mL of 0.5 N ethanolic potassium hydroxide solution in a conical flask. The mixture was left in the dark for 24 h to ensure complete saponification. The unsaponifiable matter (USM) was extracted by mixing 25 mL portions of diethyl ether three times, and the pooled ether extracts were washed three times with 25 mL portions of 0.5 N aqueous potassium hydroxide, then with distilled water until free of alkali. The collected extracts were filtered, dried over anhydrous sodium sulfate, and the ether was removed under a gentle stream of nitrogen. The resulting dried USM was further analyzed after mixing with 5 mL of chloroform.

gheeFor thin-layer chromatography (TLC) analysis, a 2 μL aliquot of unsaponifiable matter (USM) extracted from the ghee samples was applied to a pre-coated silica gel TLC plate (F₂₅₄) using a capillary tube. The plate was developed in a saturated chamber using a mobile phase of heptane and ethyl acetate (1:1, v/v). Following development, the plate was air-dried and visually developed by uniformly spraying it with a derivatization reagent containing 10% copper sulfate mixed in 8% orthophosphoric acid. The chromatogram was then heated on a laboratory hot plate at 180 °C for 5 min to char and visualize the spots. Qualitative evaluation of the TLC plates was performed using an HPTLC densitometer (CAMAG TLC Scanner 4) controlled by dedicated evaluation software (visionCATS). Scanning was performed in absorbance mode under visible-light (R white) illumination for the charred spots. Identification of cholesterol oxidation products (COPs) was achieved by comparing the migration distances and calculated retention factors (Rf values) of the sample zones with those of standard COPs resolved concurrently on the same plate. Cholesterol oxidation products were assessed only at the predefined study time points; therefore, potential changes in COPs beyond the evaluated storage period were not investigated in this study.

2.7. Radical-scavenging activity (RSA)

The radical scavenging activity (RSA) of medicated ghee samples was assessed in fresh (day 0) ghee and in ghee stored for 60 days at 45 °C, then for up to 160 days. Ghee (0.2 g) was placed in a glass test tube and diluted to a final volume of 5 mL with ethyl acetate for the highly coloured medicated ghee samples. An aliquot (60 μl) of the diluted ghee in ethyl acetate was mixed with 500 μl of 0.1 mM 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) solution (2 mg in 50 ml ethyl acetate), and the volume was adjusted to 1 ml with ethyl acetate. After incubation in the dark for 30 min, absorbance was measured at 520 nm in a spectrophotometer. The reference sample was 500 μl ethyl acetate in 500 μl DPPH solution. The RSA of ghee samples was expressed as the percentage inhibition and compared with a Trolox standard to obtain Trolox Equivalent Antioxidant Capacity (TEAC) [20].

RSA was calculated as follows:

$$\text{RSA}(\%)=\frac{\left(\text{Absorbance}\text{of}\text{control}\right)-\left(\text{bsorbance}\text{of}\text{test}\text{sample}\right)}{\left(\text{Absorbance}\text{of}\text{control}\right)}\times 100$$

2.8. Total phenolic content (TPC)

The TPC of ghee was estimated using the Folin-Ciocalteu method. The phenolic compounds were extracted with a methanol/water solution (80/20, v/v). A centrifuge tube was filled with a 2 mL aliquot of ethanolic extract, 1 mL of Folin-Ciocalteu reagent, 5 mL of distilled water, and 5 mL of a 10% sodium carbonate solution. The mixture was left in the dark for 30 min. A spectrophotometer was used to measure the absorbance of each solution at 760 nm. Gallic acid standard solutions with known concentrations were used to create a calibration curve. The results were expressed as millimolar Gallic Acid Equivalent per gram (mM GAE/g) [21].

2.9. Statistical analysis

The data obtained from each analysis were statistically assessed using Statistical Software for the Social Sciences (SPSS) (IBM Corporation, Armonk, NY). One-way analysis of variance (ANOVA) was applied with a significance level (p < 0.05), and the samples were compared with Tukey's multiple comparison test. The results of the various analyses were presented as mean ± standard deviation.

3. Results and discussion

Sneha siddhi of ghee and CA was used as a model system in this study to understand the effects of the two processes on lipid oxidation – the single-stage and two-stage processes. The CA was directly and infused into ghee (PGCA) in the single-stage direct process. In the two-stage process, the ghee was first treated with the ‘murchana’ herbs to make MG, which was then used as the base lipid for infusing CA. The oxidative stability of the two medicated ghee samples (MGCA and PGCA) was compared with that of plain ghee (PG) and the intermediate MG to understand the contribution of the ‘murchana’ step to the overall stability of ghee. In addition to oxidative stability, the four ghee samples — PG, MG, MGCA, and PGCA — were also compared for their physicochemical properties, antioxidant capacity, and Total polyphenolic content (TPC).

3.1. Physicochemical properties

Table 2 presents the physicochemical characteristics of the ghee samples. The moisture content and free fatty acid levels of all formulations remained within the regulatory limits of 0.5% and 3%, respectively [17]. Processing and herbal treatment significantly influenced the granulation characteristics of ghee, with the extent of reduction corresponding to the degree of thermal processing and treatment with herbs. PG, which underwent a single heating cycle, exhibited a granulation value of 88.00 ± 0.82%, whereas MG, subjected to an additional heating cycle during murchana, showed a slight reduction to 83.33 ± 0.47%. A more pronounced decrease was observed in the medicated formulations, with PGCA and MGCA exhibiting granulation values of 52.17 ± 0.62% and 40.17 ± 1.03%, respectively (p < 0.05). These changes may be attributed to changes in the lipid matrix resulting from repeated heating and to the transfer of herbal constituents into the ghee phase during processing. Incorporation of phytoconstituents from the herbs may have contributed to changes in consistency and melting behaviour of the lipid system.

Table 2.

Physico-chemical properties of medicated ghee samples.

S.No.	Samples	Granulation (% solids)	Specific gravity (at 30 °C)	Saponification value	Moisture (%)	Free fatty acid value (%oleic acid)	Color values
							L*	a*	b*
1	Plain ghee, Control (PG)	88.00 ± 0.82a	0.9123 ± 0.00a	234.17 ± 1.11a	0.17 ± 0.00	0.25 ± 0.001	60.96	−6.32	21.64
2	Plain ghee with Centella asiatica (PGCA)	52.17 ± 0.62c	0.9085 ± 0.00b	225.35 ± 0.60c	0.18 ± 0.01	0.24 ± 0.001	18.88	7.29	−4.65
3	Murchit ghee (MG)	83.33 ± 0.47d	0.9096 ± 0.00b	209.52 ± 0.79b	0.26 ± 0.01	0.23 ± 0.001	53.03	−10.45	43.61
4	Murchit ghee with Centella asiatica (MGCA)	40.17 ± 1.03e	0.9031 ± 0.00c	224.35 ± 0.52d	0.29 ± 0.01	0.23 ± 0.001	32.94	0.61	1.75

*Means with different superscripts in each column differ significantly (p < 0.05). The statistical difference was analyzed through one-way ANOVA with Tukey's HSD in SPSS, IBM software.

** Results are expressed as mean ± standard deviation of three measurements.

The observed reduction in granulation is consistent with previous reports on medicated lipid formulations and murchit ghee preparations [19]. Although reduced granulation is often associated with increased unsaturation and lower oxidative stability, the processed samples (MG, PGCA, and MGCA) demonstrated greater oxidative stability than PG, suggesting that the transferred phytochemicals may have exerted protective antioxidant effects within the lipid matrix.

The specific gravity of the processed ghee samples was marginally lower than that of PG. Similarly, the saponification value decreased from 234.17 ± 1.11 in PG to 209.52 ± 0.79 in MG, 225.35 ± 0.60 in PGCA, and 224.35 ± 0.52 in MGCA. Such variations may reflect changes in the fatty acid profile and lipid composition induced by thermal processing and the incorporation of herbs. Previous studies on murchit ghee have also reported variable changes in saponification values depending on the formulation and processing conditions [[14], [22]].

Noticeable colour changes were observed following processing. MG exhibited a bright golden-yellow hue due to transfer of curcuminoids from turmeric during murchana, reflected by a marked increase in the b* value from 21.64 in PG to 43.61 in MG. The negative a* value (−10.45) further indicated the development of a deeper yellow colour tone. These changes were consistent with the incorporation of pigment-rich phytoconstituents into the lipid base during herbal processing. In contrast, PGCA and MGCA developed a greenish tinge, likely due to migration of chlorophyll and other plant pigments from Centella asiatica into the lipid matrix during medicated ghee preparation. This was reflected by significantly reduced L* and b* values along with increased a* values.

3.2. Oxidative stability of medicated ghee samples

Acid value measures the free fatty acids in the lipid. A higher acid value in a lipid implies that the lipid sample has a higher amount of free fatty acids, which would suggest the lipid is more prone to oxidation. In this study, the acid value test did not differentiate among the four ghee samples, as their acid values were not significantly different. However, the PV test, which measures the amount of hydroperoxides at a given time, the primary oxidation products of lipids, showed differences between the samples. The PV of ghee increased marginally during processing, with PG at 0.40 m.eq., while MG, MGCA, and PGCA had PVs of 0.83, 1.36, and 1.43 m.eq., respectively. O₂/Kg ghee. Further, all four ghee samples were incubated at 45 °C for 160 days, and PV was assessed on days 20, 30, 40, 50, 60, and 160. The results are given in Table 3. The PV of PG increased from 0.40 m.eq. O₂/Kg ghee to 1.58 m.eq. O₂/Kg on day 60 and then came down to 1.07 m.eq. O₂/Kg on day 160. The PV of PGCA increased from 1.43 m.eq. O₂/Kg ghee on day 0 to 9.50 m.eq. O₂/Kg ghee on day 60, which was nearly seven times greater than its initial value. The PV increased to 10.98 on day 160, slightly higher than the safe limit of 10 m.eq. O₂/Kg. In contrast, the PV of MGCA increased from 1.36 mEq. O₂/Kg to 5.0 m.eq. O₂/Kg on day 60, then to 6.49 mEq. O₂/Kg on day 160. Thus, even after 160 days, the PV of MGCA remained well below the safe limit of 10 m.eq. O₂/Kg prescribed in FSSAI (2018) standards [23]. In terms of the fold increase of PV, MGCA increased only 3.5-fold compared to PGCA, which increased 7-fold without stabilizing (murchana) process (Table 3). The results show that the two-stage process, which uses MG as the base, has a lower PV even at 160 days under accelerated storage conditions of 45 °C and 85% of relative humidity.

Table 3.

Peroxide Value (PV) (m.eq. O₂/Kg ghee) of ghee samples during storage at 45 °C with 85% RH.

Samples	0 d	10 d	20 d	30 d	40 d	50 d	60 d	160 d
Plain ghee, Control (PG)	0.40	0.87	1.00	1.50	1.50	1.54	1.58	1.07
Plain ghee with Centella asiatica (PGCA)	1.43	1.67	2.00	2.00	4.00	4.00	9.50	10.98
Murchit ghee (MG)	0.83	1.00	1.50	1.50	1.61	1.67	2.00	1.64
Murchit ghee with Centella asiatica (MGCA)	1.36	1.50	2.00	2.22	2.50	4.62	5.00	6.49

Some studies have used acid value and PV as indicators of lipid quality and oxidative stability [24]. However, free fatty acids and hydroperoxides are transient species that are consumed in subsequent oxidation reactions, generating a variety of secondary oxidation products [25]. Consequently, acid value and PV alone may not accurately reflect cumulative oxidative deterioration or the remaining shelf life of lipid systems. Furthermore, the relationship between PV and the sensory quality of oils, fats, or lipid-based foods has been reported to be inconsistent in certain studies [26].

Therefore, in the present study, the effect of the sneha kalpana process on oxidative stability was evaluated using the Rancimat method (Fig. 2b), along with acid value and PV analyses. The Rancimat test determines oxidative stability by monitoring volatile secondary oxidation products generated under accelerated oxidation conditions [27]. The rate of fatty acid oxidation is represented by the induction period, the time required to reach the initial rate of lipid oxidation under accelerated conditions. A higher induction period is associated with higher oxidative stability [28]. PG exhibited an induction period of 11.99 h at 120 °C. Following the murchana process, MG showed a marked increase in induction period to 42.8 h, indicating significantly improved oxidative stability. In comparison, the synthetic antioxidant Butylated Hydroxytoluene (BHT) ghee exhibited an induction period of 16.25 h, which was higher than that of PG but considerably lower than that of MG. These findings suggest that the murchana process enhanced oxidative stability and may contribute to improved shelf life of the ghee.

Fig. 2.

Oxidative stability, induction time and assessment of COPs in ghee samples

a. A fold increase in peroxide value of ghee samples during storage at 45 °C. b. Induction period of ghee samples analyzed at 120 °C using Rancimat. c. TLC chromatograms of unsaponifiable matter isolated from herbal ghee samples for COPs (i) 0-day storage (ii) 60-day storage [A = 5α-Cholestane 3,5,6, triol, B = 5-Cholesten-3β-ol-7-one, C = 5α, 6α-epoxide, D = 25-Hydroxycholesterol, and E = Cholesterol. Samples 1 to 4 (1 = PG, 2 = PGCA, 3 = MG, 4 = MGCA).

The medicated ghee formulations also demonstrated improved oxidative stability compared to PG. PGCA exhibited an induction period of 24.32 h, whereas MGCA showed a higher induction period of 38.8 h. The superior stability of MGCA compared with PGCA suggests that using murchit ghee as the lipid base may enhance the oxidative stability of Sneha Kalpana formulations, possibly due to the transfer and retention of antioxidant phytoconstituents during processing.

3.3. Antioxidant Potential and Total Polyphenolic Content (TPC)

The enhanced oxidative stability may be attributed to the antioxidative potential of the phenolic and flavonoid components of Centella asiatica, as hypothesized in the literature [29]. To validate this hypothesis, the DPPH radical scavenging assay (RSA), Trolox equivalent antioxidant capacity (TEAC), and TPC were measured in the four ghee samples. The results are given in Table 4. The TPC of the three processed ghee samples (MG, PGCA, and MGCA) was 5- to 7-fold higher than that of the PG. While PG exhibited a total phenolic content (TPC) of 40.33 GAE μg/g ghee, the values increased to 208 in MG, 228.20 in PGCA, and 273.27 GAE μg/g ghee in MGCA. The marked increase in TPC following murchana and the preparation of medicated ghee indicated effective transfer and retention of phenolic phytoconstituents from the herbs into the lipid matrix during processing. The transfer of polyphenols to the ghee is reflected in the higher antioxidant potential for MG, PGCA, and MGCA. This was assessed using the DPPH-RSA. The test involved measuring the reduction in DPPH radical absorbance in the presence of antioxidants, indicating the free-radical-scavenging potential and antioxidative ability of the tested sample. The samples' TEAC was also calculated by treating the DPPH radical with a Trolox standard antioxidant, using the calibration curve y = 215.7x + 1.572 (R² = 0.9952).

Table 4.

DPPH Radical Scavenging Activity (RSA) and Trolox Equivalent Antioxidant Capacity (TEAC) of ghee samples.

S.No.	Sample	DPPH (RSA) (%RSA/0.2 g)		Trolox Equivalent (μM TEAC/g ghee)		TPC (GAE μg/g ghee)
		d0	d60	d0	d60	d0	d60
1	Plain ghee, Control (PG)	3.91 ± 1.04a	2.37 ± 0.65a	127.05 ± 19.77a	91.43 ± 15.05b	40.33 ± 2.36a	38.29 ± 0.59a
2	Plain ghee with Centella asiatica (PGCA)	4.03 ± 1.71a	5.66 ± 0.52a	129.91 ± 32.33a	167.57 ± 11.96b	228.20 ± 2.64b	67.97 ± 0.56d
3	Murchit ghee (MG)	12.85 ± 1.13b	12.47 ± 0.43b	334.35 ± 21.44c	325.49 ± 9.97c	208.36 ± 1.79c	215.23 ± 1.55c
4	Murchit ghee with Centella asiatica (MGCA)	5.37 ± 0.56a	4.81 ± 0.76a	160.85 ± 10.59	147.83 ± 17.72	273.27 ± 0.99c	220.09 ± 0.63c

*Means with different superscripts in each column and row differ significantly (p < 0.05).

MG exhibited significantly higher free radical scavenging activity, with a DPPH radical scavenging activity (RSA) of 12.47 ± 0.43%, compared to PG, which showed only 3.91 ± 0.10% RSA. These findings are consistent with previous reports demonstrating the enhanced antioxidant potential of murchit ghee formulations [30]. Phytochemical analysis indicates that the murchana herbs, including Curcuma longa, Cyperus rotundus, Terminalia chebula, Terminalia belerica, Emblica officinalis, and Citrus medica, contain potent antioxidants such as flavonoids, coumarins, tannins, and phenolic acids (e.g., gallic acid, ellagic acid, chebulinic acid, and curcumin), all of which exhibit free radical scavenging properties [[29], [31], [32], [33], [34]]. The presence of these compounds likely contributes to the observed antioxidant potential and stabilizing effect of the murchana process.

The MGCA had a DPPH-RSA of 5.37%, which is higher than that of PG. Thus, despite the thermal exposure in the presence of water, the antioxidant capacity of MGCA was higher than that of PG. Centella asiatica ghee (PGCA) had a DPPH-RSA (4.03%) similar to the PG (3.91%), suggesting a comparatively weaker antioxidant effect of Centella asiatica compared to MGCA. It is noteworthy that the heating time for MGCA was twice that of PGCA, resulting in substantially longer heating duration and greater aqueous-phase evaporation than with PGCA, yet the formulation retained enhanced antioxidant potential.

DPPH and TPC were also evaluated after 60 days of storage. Both parameters remain relatively constant over the 60-day period in all formulations, except for the TPC value for PGCA. The TPC for PGCA decreased significantly from 228 to 67 GAE μg/g ghee over the 60-day period. Although the exact mechanism underlying this reduction could not be conclusively established, it is possible that the polyphenols were depleted in sequestering the free radicals generated during storage. This may also explain the comparatively higher peroxide value observed for PGCA at day 60 than in other samples. In contrast, MG and MGCA demonstrated substantial retention of phenolic content and antioxidant activity, as indicated by higher TPC and DPPH-RSA values, which may have contributed to their improved oxidative stability at day 60. These findings are consistent with the extended shelf-life attributes traditionally associated with medicated ghee formulations, as documented in classical Ayurvedic texts (Sarangdhara Samhita), suggesting that the antioxidative potential of herbs used in the murchana process may help preserve the lipid matrix.

A few studies have suggested that heating ghee can generate cholesterol oxidative products (COPs) from the cholesterol present in ghee. COPs are considered a potential concern for human health due to their numerous harmful effects, including mutagenic, cytotoxic, and atherogenic properties [[35], [36], [37]]. Therefore, a preliminary analysis of COPs was performed for all four samples using thin-layer chromatography. Fig. 2c shows the COPs bands obtained from the unsaponifiable matter of medicated ghee. While no COPs were expected in PG, the high thermal exposure in the presence of water during the processing of the MG, PGCA, and MGCA also did not result in COPs generation. Even after 60 days of accelerated storage, none of the processed ghee samples showed COPs.

4. Study limitations

The current research has some limitations. The proposed association between granulation and the presence of unsaturated fatty acids could not be directly confirmed in the study, as fatty acid profiling was not performed. Similarly, phytochemical analysis and standardization were not performed, thereby limiting direct evidence of specific bioactive ingredients transferred to lipid base during herbal processing, despite increases in overall phenolic content and antioxidant activity in the medicated ghee formulations were observed. Evaluation of secondary oxidation products and advanced compositional studies using GC-MS were also outside the purview of the investigation. Furthermore, the biochemical changes that occurred during extended storage were not examined, and COPs were evaluated only at specific time intervals. Future research should therefore, include detailed chemical characterization and extended storage assessments to further understand the mechanisms underlying the improved oxidative stability of the medicated lipid-based formulations.

5. Conclusion

The processing of medicated ghee involves exposure to high temperatures for extended periods in the presence of a large amount of water. Although these conditions are ideal for the oxidation of fatty acids and cholesterol, the stability data of the medicated lipids studied in this work showed that, despite this exposure, oxidation remained limited. The murchana step appeared to be critical for imparting this stability, as MG and MGCA had much longer induction times than their respective controls, PG and PGCA. This oxidative stability could be attributed to the high level of polyphenols transferred from the herbs to the ghee during the infusion step, thereby increasing the radical-scavenging activity of the polyphenol-rich ghee samples.

The insights gained from this work can lead to the development of newer medicated lipid formulations with longer shelf lives, beyond those described in Ayurvedic sneha kalpana formulations. The knowledge can also be helpful to the growing number of herbs-infused oils being developed and marketed for culinary, aromatherapy and skincare applications.

The insights also suggest the potential to develop natural antioxidants from the herbs used in this study, as the 4-fold increase in induction time of MG over PG (42.8 h vs 11.9 h) was significantly higher than that obtained by BHT, which was only a 1.4-fold increase (16.25 h for BHT vs 42.8 h for MG). A deconstruction of the murchana recipe could identify the ingredient that imparts stability to the lipid, which may, in turn, facilitate the development of natural, safer, and more effective antioxidants for edible oil stabilization.

Author contribution

DK- Writing original draft, investigation, writing review and editing, methodology, and formal analysis.

MNH-writing review and editing, methodology, and formal analysis.

GS- Conceptualization, Supervision, writing-review and editing, methodology, formal analysis, and funding acquisition.

Declaration of generative AI in scientific writing

In this study, a few AI tools were used to enhance the readability, fluency, and clarity of the manuscript.

Funding sources

This work was supported by the Rural India Supporting Trust (RIST).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.