Role of Hridayarnava Rasa on inflammatory responses in rabbits with high fat diet induced atherosclerosis

Subramani Chitra; Rathinam Arunadevi; Gaidhani Sudesh; Raju Ilavarasan; Veeraswamy Sharmila Devi; Erram Narasimha Thri Vikram; Gautam K Manish

doi:10.4103/AYUHOM.AYUHOM_46_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 8 | Issue : 2 | Page : 73-81

Role of Hridayarnava Rasa on inflammatory responses in rabbits with high fat diet induced atherosclerosis

Subramani Chitra¹, Rathinam Arunadevi¹, Gaidhani Sudesh², Raju Ilavarasan¹, Veeraswamy Sharmila Devi¹, Erram Narasimha Thri Vikram¹, Gautam K Manish²
¹ Captain Srinivasa Murthy Central Ayurveda Research Institute, Central Council for Research in Ayurvedic Sciences, M/o AYUSH, Government of India, Anna Hospital Campus, Chennai, Tamil Nadu, India
² Central Council for Research in Ayurvedic Sciences, M/o AYUSH, Government of India, New Delhi, India

Date of Submission	29-Sep-2021
Date of Decision	03-Feb-2022
Date of Acceptance	11-Feb-2022
Date of Web Publication	29-Jun-2022

Correspondence Address:
Subramani Chitra
Captain Srinivasa Murthy Central Ayurveda Research Institute, Central Council for Research in Ayurvedic Sciences, M/o AYUSH, Government of India, Anna Hospital Campus, Arumbakkam, Chennai - 600 106, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/AYUHOM.AYUHOM_46_21

Abstract

Background: Atherosclerotic plaque formation is a chain of events that begins with fatty streak accumulation followed by monocytes infiltration and lipid core formation. Monocytes/macrophages play an important role in the initiation and progression of atherosclerosis. The role of inflammation and atherosclerosis provides a mechanistic framework for understanding the clinical benefits of lipid-lowering therapies in high-fat diet (HFD) induced atherosclerosis rabbits. Identifying triggers for inflammation and uncovering the details of inflammatory pathways may ultimately present new therapeutic targets. H. Rasa maintains the heart by providing cardioprotective activity along with changes in certain inflammatory markers in atherosclerosis. Objectives: The primary objective of the study is to evaluate the role of oxidized low-density lipoprotein cholesterol (LDLc) in the inflammatory response and how this inflammation triggers the level of white blood cells. The secondary objective is how this Hridayarnava Rasa, an Ayurvedic formulation inhibits the oxidation of LDLc and protects cells from inflammation in HFD-induced atherosclerosis model rabbits. Materials and Methods: Newzealand white rabbits of 24 were randomly divided into 6 groups of 4 animals each. Group I rabbits fed with standard pellet diet; group II rabbits fed with HFD; group III, IV, and V were fed with HFD and different doses of H. Rasa and group VI rabbits were fed with HFD plus Atorvastatin. Results: Total leucocytes, lymphocytes, monocytes, LDLc: high-density lipoprotein cholesterol (HDLc) ratio and total cholesterol (TC): HDLc ratio were increased in group II, III, IV, and VI of 30, 60, and 90 days when compared to group I. The levels of total leukocytes, lymphocytes, monocytes, LDLc: HDLc ratio, and TC: HDLc ratio were significantly reduced in group IV and V of 30, 60, and 90 days when compared to group II. In the present study, treatment with H. Rasa (group V) (i.e., 41.07 mg/kg. b. wt/p. o) was shown to be most effective over 90 days. Conclusion: These results suggest that HFD accelerates the development of atherosclerosis by increasing the inflammatory markers such as oxidized LDL (oxLDLc) and leukocyte counts in a time-dependent manner and H. Rasa protects the aorta by preventing the oxidative damage of LDLc which inturn maintains the inflammatory markers and provided the anti-inflammatory responses and protects the aorta from atherosclerotic plaque formation in a dose-and time-dependent manner. Limitation of the Study: This study focused on the characteristics of the rabbit lipoprotein pathway and pathophysiology of atherosclerotic lesions via inflammatory markers. This paper primarily determines how H. Rasa protects the aorta from the formation of atherosclerotic plaques caused by oxidative low-density lipoprotein. Further studies will need to focus specifically on the inflammatory pathways and the role of H. Rasa.

Keywords: Atherosclerosis, Hridayarnava Rasa, lymphocytes, rabbits, total leucocytes

How to cite this article:
Chitra S, Arunadevi R, Sudesh G, Ilavarasan R, Devi VS, Thri Vikram EN, Manish GK. Role of Hridayarnava Rasa on inflammatory responses in rabbits with high fat diet induced atherosclerosis. AYUHOM 2021;8:73-81

How to cite this URL:
Chitra S, Arunadevi R, Sudesh G, Ilavarasan R, Devi VS, Thri Vikram EN, Manish GK. Role of Hridayarnava Rasa on inflammatory responses in rabbits with high fat diet induced atherosclerosis. AYUHOM [serial online] 2021 [cited 2022 Aug 17];8:73-81. Available from: http://www.ayuhom.com/text.asp?2021/8/2/73/348861

Introduction

Inflammation plays a key role in the initiation and progression of atherosclerosis and caused by several mediators, including high concentration of circulating low-density lipoprotein cholesterol (LDLc). The arterial wall affected by atherosclerosis is augmented with lymphocytes and dendritic cells (DC). A range of pro-inflammatory immune cells, like macrophages, DCs, T-cells, natural killer cells, and neutrophils are present in atherosclerotic plaques.^[1] Macrophages play a central role in the early stages of atherogenesis by engulfing oxidized LDL (oxLDL) in the intima which leads to the transformation to foam cells. The dying macrophage in atherogenic plaque forms a necrotic core which further aggravates the lesion.^[2] Atherosclerosis is a chronic inflammatory disease caused by LDL. Leukocytes and macrophages can prevent atherosclerosis by scavenging and eliminating excess lipoproteins from the arterial wall.^[3] Leukocytosis and neutrophilia are independent risk factors for the development of coronary heart disease.^[4] Neutrophils, mast cells, and platelets promote atherosclerosis by intensifying inflammation. Mast cells have a role in allergy and anaphylaxis and also promote atherosclerosis by releasing the contents of their protease-cytokine-autacoid-rich granules.^[5] Platelets are the megakaryocyte-derived thrombocytes which play an important role in atherosclerosis, they adhere to the endothelium and help monocytes to enter into the lesions^[6] and form thrombus that causes ischemia of downstream tissue when plaque ruptures.^[7] If the inflammatory process reaches optimum, repair process starts, and the plaque can become stable. However, failure to resolve the inflammatory response; result in the formation of the most dangerous unstable plaque that can cause thrombosis. This study aims to evaluate the oxidized LDLc in the inflammatory response and how this inflammation triggers the level of white blood cells (WBC) and also how H. Rasa inhibits the oxidation of LDLc and protects cells from inflammation in high-fat diet (HFD)-induced atherosclerosis model rabbits.

New vessel formation inside the arterial wall and atherosclerotic plaque play a critical role in pathogenesis of heart attacks and strokes. The two known mechanisms resulting in the formation of neovascularisation within the plaque are local ischemia and inflammation. The imbalance between monocytes and macrophages toward the pro-inflammatory phenotype and a lack of normal inflammation is also present in atherosclerosis. Circulating oxLDLc has increased atherogenicity when compared to native LDLc. Chemical modification of LDLc plays a prominent role in atherosclerosis development.^[8] The atheroprotective potential of high-density lipoprotein cholesterol (HDLc) is mediated through reverse cholesterol transport, anti-inflammatory, antioxidant, and vascular protective properties.^[9] H. Rasa, an Ayurvedic drug composed of Triphala choorna (Terminalia chebula Retz., Terminalia bellerica [Gaertn] Roxb, Embelica officinallis Gaertn); Tamra bhasma (Calx of purified copper); Shodhita parada (Mercury); and Gandhaka (Sulphur) were processed in Kakamachi (Solanum nigrum Linn.). H. Rasa cures coronary heart diseases^[10] and have antihyperlipidemic, antiobesity, and antioxidant activity.^[11],[12] There were no such studies available on anti-inflammatory action of H. Rasa on atherosclerosis. Therefore the objective of the present study is to evaluate the role of monocytes, macrophages, and LDLc in atherosclerosis development and in relation to plaque formation and the potential role of H. Rasa in alleviating these changes in atherosclerosis-induced rabbits. Mouse and rabbit models are commonly used to study atherosclerosis. Compared to the mouse model, the rabbit is the most widely accepted laboratory animal model nowadays. In Ayurvedic classical text indicated that H. Rasa is used to treat hyperlipidemic (Medovridhi) condition as well as it shows cardioprotective.^[13] In general, it is used to treat cardiovascular diseases by the Ayurvedic Physicians. Atherosclerosis is one of the major cardiovascular diseases, hence, the present study is attempted to find out the role of H. Rasa on HFD induced atherosclerosis rabbits. Furthermore, literature proved that H. Rasa posses hypolipidemic activity, as hyperlipidemia is a major risk factor in the development of atherosclerosis. H. Rasa is thereby useful in managing Hridroga in specific, Atherosclerosis. Therefore, this formulation can be used in place of statins of conventional medicine.^[14] Kakamach, Haritaki, Amalaki and Tamra bhasma are proved as cardioprotective. The anupana varies according to different authors. H. Rasa is safe for therapeutic use at its normal dose. The main objective of the present study is to evaluate the role of H. Rasa in inflammatory response markers and protection of cells from inflammation in HFD-induced atherosclerosis.

Materials and Methods

Animals

The male Newzealand white rabbits were purchased from Biogen Laboratory Animal Facility, Bangalore, Karnataka and acclimatized to the laboratory conditions for 7 days before the experiment. The average body weight of rabbits chosen for the study was ranging from 1.9 to 2.2 kg. The variation in body weight of animals on randomization was not exceeded ±20% of the mean body weight. Each rabbit was housed in an individual cage and rabbit pellet feed and reverse osmosis water was fed ad libitum. Temperature and relative humidity were maintained at 23°C ± 5°C and 45%–60%, respectively, and illumination was controlled by approximately 12/12 h light/dark cycle. This study was approved by Institutional Animal Ethics Committee (IAEC/CSMRADDI/17/2017).

Atherogenic diet

Atherogenic diet was procured from National Institute of Nutrition, Indian Council for Medical Research, Hyderabad. A slight modification in the procedure was used to induce atherosclerosis by feeding HFD consisted of 1% cholesterol, 5% egg yolk powder, 5% lard and 89% normal diet, and it was prepared freshly for every day.^[15]

Procurement and storage of drugs

H. Rasa, an Ayurvedic drug procured from Arya vaidya sala, Kottakkal, Kerala, India (Batch No. 191248; (MFG. LIC. Number: 1/25D/76; Date of Manufacture: 03/2018 and Date of Expiry: 02/2023). The drugs were kept under the temperature of 25°C ± 3°C and humidity of 52% ± 10% RH until experiment gets completed. The composition [Table 1] of each tablet of H. Rasa contains 83 mg of dehydrated ground mass and composed of the following:

Table 1: Composition of Hridayarnava Rasa

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Experimental protocol -1

A total of 24 rabbits were randomly divided into 6 groups of 4 rabbits in each group is given in [Table 2] and [Table 3].

Table 2: Description of groups

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Table 3: Description of groups with drug doses

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Experimental protocol -2

A dose volume (10 mL/kg b. wt/p. o) of vehicle (0.5% solution of sodium carboxyl methylcellulose) was administered by oral route from day 1 to day 90 to all animals in control group. The volume of vehicle administration was calculated based on the most recent body weights of animals. The drug was prepared in 0.5% solution of sodium carboxyl methylcellulose. Drug dose was calculated based on the human dose (mg/kg) x conversion factor (3.08).

Biochemical analysis

At the end of 30, 60, and 90 days of treated groups (group II to VI) and control (group I) rabbits, blood were collected from saphenous vein in the back leg of rabbit under thiopental sodium anesthesia. Blood was collected in K₂ EDTA tubes for hematological analysis (Automated digital cell counter) and used heparin tubes for blood biochemistry. Biochemical parameters were estimated using clinical chemistry fully auto analyzer (EM-200; Transasia) using ERBA kits.

Statistical analysis

Statistical analysis was carried out using graph pad prism software, version 8.4. All the values were expressed as mean ± standard deviation (n = 4). One-way analysis of variance with multiple comparisons using Turkey's test and P < 0.05 was considered as statistically significant.

Results

Disorders of lipid metabolism have strong relationship in the development and progression of coronary heart diseases. [Figure 1] shows the status of total leukocytes (WBC) of control and treated groups of different doses and at different time intervals of treatment. WBC were increased in group II (P < 0.01), III, IV, and VI (P < 0.05) of 30 days; group II (P < 0.001), III, IV (P < 0.01), VI (P < 0.05) of 60 days; group II (P < 0.001), III (P < 0.0001), IV and VI (P < 0.05) of 90 days when compared to group I. The levels were significantly reduced in group IV (P < 0.05), V (P < 0.01) of 30 days; group IV (P < 0.01), V (P < 0.0001), group VI (P < 0.001) of 60 days; group III (P < 0.01), IV (P < 0.001), V and VI (P < 0.0001) of 90 days when compared to group II. The levels were significantly decreased in group V (P < 0.01) of 60 days; group IV (P < 0.05) and V (P < 0.001) of 90 days when compared to group III.

Figure 1: Status of total leukocyte counts in experimental groups. (Gp I-Control; Gp II-Disease Control; Gp III-Dose 1; Gp IV- Dose 2; Gp V-Dose 3; Gp VI-Standard drug). (Value are expressed as mean ± SD of 4 rabbits; *P < 0.05). $Statistical analysis of one-way ANOVA was used to compare result with group I. #Statistical analysis of one-way ANOVA was used to compare result with group II. @Statistical analysis of one way ANOVA was used to compare result with group IV and V with group III

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[Figure 2] depicts the status of lymphocytes of control and treatment groups of different doses and at different time intervals of treatment. Lymphocytes were increased in group II, III, IV, and VI (P < 0.05) of 30 days; group VI (P < 0.05) of 60 and 90 days when compared to group I. The levels were significantly reduced in group V (P < 0.05) of 60 and 90 days when compared to group II. [Figure 3] depicts the status of monocytes of control and treatment groups of different doses and at different time intervals of treatment. The increased monocytes levels were observed in group II (P < 0.05) of 30, 60, and 90 days when compared to group I. There was a significant reduction in group V (P < 0.05) of 90 days were noticed when compared to group II.

Figure 2: Status of lymphocyte in experimental groups. (Gp I-Control; Gp II-Disease Control; Gp III-Dose 1; Gp IV- Dose 2; Gp V-Dose 3; Gp VI-Standard drug). (Value is expressed as mean ± SD of 4 rabbits; *P < 0.05). $Statistical analysis of one way ANOVA was used to compare result with group I. #Statistical analysis of one way ANOVA was used to compare result with group II

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Figure 3: Status of monocyte counts in experimental groups. (Gp I-Control; Gp II-Disease Control; Gp III-Dose 1; Gp IV- Dose 2; Gp V-Dose 3; Gp VI-Standard drug). (Value is expressed as mean ± SD of 4 rabbits; *P < 0.05). $Statistical analysis of one way ANOVA was used to compare result with group I. #Statistical analysis of one way ANOVA was used to compare result with group II

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[Figure 4] depicts the status of platelets of control and treated groups of different doses and at different time intervals of treatment. The decreased platelet level was observed in group II of (P < 0.05) 30 days; group II and VI of (P < 0.05) 60 and 90 days when compared to group I. The levels were significantly increased in group V (P < 0.05) of 30, 60, and 90 days compared to group II. [Table 4] shows the ratio of LDLc: HDLc in plasma of control and drug-treated groups of different doses and at different time intervals of treatment. The ratio of LDLc: HDLc was significantly increased in group II to IV and VI (P < 0.0001) of 30 and 60 days; group II, III, and VI (P < 0.0001) of 90 days of treatment when compared to respective control groups. The ratio was significantly decreased in group IV, V (P < 0.0001), IV (P < 0.001) of 30 days; group III to V (P < 0.0001), VI (P < 0.001) of 60 days; group III to VI (P < 0.0001) of 90 days treatment when compared to group II. The ratio was significantly reduced in group IV (P < 0.05) and V (P < 0.01) of 30, 60, and 90 days of drug treatment when compared to dose-1. The ratio of LDLc: HDLc in group II (P < 0.0001) was significantly increased and group IV (P < 0.01) was significantly reduced when compared to respective 30 days of treatment.

Figure 4: Status blood platelets in experimental groups. (Gp I-Control; Gp II-Disease Control; Gp III-Dose 1; Gp IV- Dose 2; Gp V-Dose 3; Gp VI-Standard drug). (Value is expressed as mean ± SD of 4 rabbits; *P < 0.05). $Statistical analysis of one way ANOVA was used to compare result with group I. #Statistical analysis of one way ANOVA was used to compare result with group II

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Table 4: Status of low-density lipoprotein cholesterol: high-density lipoprotein ratio in plasma of experimental groups

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[Table 5] shows the ratio of total cholesterol (TC): HDLc in plasma of control and drug-treated groups of different doses and at different time intervals of treatment. The ratio of TC: HDLc was significantly increased in group II, III, VI (P < 0.0001), IV (P < 0.01) and V (P < 0.05) of 30 days; group II, III, VI (P < 0.0001), IV and V (P < 0.01) of 60 days; group II to IV, VI (P < 0.0001) and V (P < 0.01) of 90 days treatment when compared to respective control groups. There was a significantly decrease in group III (P < 0.05), IV to VI (P < 0.0001) of 30 days; group III to VI (P < 0.0001) of 60 and 90 days of treatment when compared to group II of respective days. The ratio was reduced significantly in group IV (P < 0.05), V (P < 0.01) of 30 and 60 days and group V (P < 0.01) of 90 days when compared to dose-1. A significant increase was observed in group II (P < 0.01) of 60 and 90 (P < 0.0001) days when compared to 30 days of HFD induced rabbits. Plasma and tissue concentrations of mercury, copper, and sulfur were analyzed by ICP-OES after 90 days of treatment. No detectable limit was observed in both plasma and tissues of major organs such as liver, heart, kidney, spleen, and aorta (data not shown).

Table 5: Status of total cholesterol: high density lipoprotein cholesterol (total cholesterol: high-density lipoprotein) ratio in plasma of experimental groups

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Discussion

Oxidation of these atherogenic lipoproteins such as VLDLc and LDLc may generate lipid-derived inflammatory mediators; fatty acid peroxidation induces atherogenic inflammatory responses in arterial wall by monocytes.^[16] Total WBC count was increased in the HFD fed group of the present study; this might be due to WBC participate in the chronic inflammatory process and affect the development of cardiovascular diseases through multiple mechanisms that mediate inflammation, cause proteolytic oxidative damage to the endothelial cells which promote infarct expansion in the arterial wall.^[3] WBC counts >8100 cells/mm3 were associated with increased risk of coronary heart disease.^[17] Monocytic activation leads to the generation of macrophage foam cells and the initiation of fatty streaks.^[18] The present study demonstrated that the progression of atherosclerosis in the coronary arteries of hypercholesterolemic rabbits, this might be due to smooth muscle cell proliferation and transformation in foam cells which was evident in histopathological studies on abdominal aorta of hypercholesterolemic rabbits (data not shown). The result of our present study was very well correlated with others' findings.

Oxidised LDL plays a significant role in the initiation and progression of the cardiovascular dysfunction associated with atherosclerosis. An atherogenic index in plasma (AIP) is supposed to be a vital and stand-alone index for the estimation of cardiac risk. AIP is the best determinant for fractionated etherification rate of HDLc and more useful than regular lipid parameters. AIP estimates the values of “Zone of atherogenic risk.” Furthermore, it is a predictive marker for plasma atherogenicity. Furthermore, this AIP is associated with lipoproteins such as HDLc, LDLc, and VLDLc. However, AIP is a sensitive marker for identifying cardiovascular diseases. The major aherogenic risk parameters include (i) TC: HDLc ratio, (ii) LDLc: HDLc ratio, (iii) TC-HDLc: HDLc, if these parameters appear normal, the AIP may be the alternative diagnostic risk parameters. Our present study the AIP index was significantly higher in HFD induced rabbits. The range of AIP between-0.3 and 0.1; 0.1-0.24; >0.24 is considered to be a low risk; medium risk, and high risk, respectively, of getting CVD. Hence, AIP is the strongest risk assessment factor for atherosclerosis. Considering the above ranges, dose-3 of H. Rasa treatment showed near normal value like control group. AIP index of atorvastatin-treated group clearly showed the value nearer to dose-2 of H. Rasa-treated rabbits. It is obviously evident that a rabbit treated with high dose of H. Rasa provides beneficial effects.

The TC: HDLc ratio is known to be an athrogenic or Castelli index-I; this is a significant marker of cardiovascular risk. TC: HDLc ratio is >4 in human considered to be a high risk of cardiovascular diseases, particularly atherosclerosis. This ratio is more powerful coronary risk predictor than independently used TC, HDLc, and LDLc. TC: HDLc ratio is very high from 30 days to 90 days of HFD fed rabbits. The ratio was higher from 9 to 17 times more when compared to human range. On treatment with H. Rasa the ratio was reduced drastically from 3 to 5 times in dose-1 to dose-3 at different intervals of time. It is obvious that the major vascular risk factor is reduced on H. Rasa treatment. The LDLc: HDLc ratio is also known to be a Castelli index-II; is an important marker of cardiovascular risk. In human the normal range of LDLc: HDLc ratio is <3.0. If it exceeds, >3.0 is considered to be a high risk of atherosclerosis. Our present study showed that the ratio of LDLc: HDLc the ratio ranges from 4 to 7 times more from 30 to 90 days in HFD induced rabbits. This indicates that 30 days of HFD fed rabbit induces atherosclerosis in rabbits. On treatment with H. Rasa gradually decrease this ratio to near normalcy from 60 days of second dose treatment and 30 days of third dose of treatment. This might be due to the presence of Tamira bhasma, a rich source of copper, and it is a central component in H. Rasa possesses hypolipidemic activity, antioxidant, cardio-protective, and free radical scavenging activities.^[19] Solanum nigrum Linn., one of the components of H. Rasa plays a vital role in anti-hyperlipidemic activity.^[12] The primary objective of the study is to evaluate the role of oxidized LDLc in the inflammatory response and how this inflammation triggers the level of WBC. The secondary objective is how this Hridayarnava Rasa, an Ayurvedic formulation inhibits the oxidation of LDLc and protects cells from inflammation in HFD-induced atherosclerosis model rabbits.

Conclusion

These results suggest that HFD accelerates the development of atherosclerosis by increasing the inflammatory markers such as oxLDLc and leukocyte counts in a time-dependent manner and H. Rasa protects the aorta by preventing the oxidative damage of LDLc which inturn maintains the inflammatory markers and provided the anti-inflammatory responses and protects the aorta from atherosclerotic plaque formation in a dose-and time-dependent manner.

Acknowledgment

Authors gratefully acknowledge the Director General, Central Council for Research in Ayurvedic Sciences, M/o AYUSH, Government of India, New Delhi, for the financial support through Intra Mural Research Scheme.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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