An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia...

[Full title: An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) for Hypercholesterolemia in Albino Rats]

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[Find the meaning and references behind the names: Vikas, Real, Vinod, Kumar, Fast, Heart, Rao, Prashant, April, Good]

ORIGINAL ARTICLE October 2024 Journal of Ayurveda and Integrated Medical Sciences | October 2024 | Vol. 9 | Issue 10 26 An experimental evaluation of bark of Chirbilva ( Holoptelea integrifolia Planch.) for Hypercholesterolemia in Albino Rats Vinod Katoch 1 , Prashant Kumar Jha 2 , Vikas Kumar 3 1 Associate Professor, Department of Dravyaguna, Abhilashi Ayurvedic Medical College and Research Institute Mandi, Himachal Pradesh, India. 2 Professor, Quality Control Laboratory, A.L.N. Rao Memorial Ayurvedic Medical College, Koppa, Karnataka, India. 3 Associate Professor, Department of Samhita Siddhant, Abhilashi Ayurvedic Medical College and Research Institute Mandi, Himachal Pradesh, India. I NTRODUCTION Global Health observatory data W.H.O risk of heart disease and stoke due to raised cholesterol levels. Globally ischemic heart disease is due to high cholesterol [1] Overall, raised cholesterol is estimated to cause 2.6 million deaths (4.5% of total) and 29.7 million disability adjusted life years (DALYS), or 2.0% of total DALYS. According to Journal of Clinical Lipidology April 2020 Rates of atherosclerotic cardiovascular disease is increasing in India as compared to western countries also found in younger age with hypercholesterolemia Address for correspondence: Dr. Vinod Katoch Associate Professor, Department of Dravyaguna, Abhilashi Ayurvedic Medical College and Research Institute Mandi, Himachal Pradesh, India. E-mail: drvinodkatoch@gmail.com Submission Date: 11/09/2024 Accepted Date: 24/10/2024 Access this article online Quick Response Code Website: www.jaims.in DOI: 10.21760/jaims.9.10.4 CVD is an important cause of mortality and morbidity in India too. According to data of Registrar General of India, age-adjusted CVD is more in southern and eastern India. Studies reported the various factors. In real sense fast moving life-style and getting away from nature have invited number of physiological problems in general human being. Industrialization, heavy work load bringing the stress, changed dietary habits, lack of exercise, good consumption of beverages, fast food and lower intake of fruit and vegetables etc. have added fuel with physiological problems especially when high total serum cholesterol related problems are considered. World Health Organization has set the goal to reduce the global death rate by 2% a year up to 2015 [2] The goal is attributed with lowering of high total serum cholesterol as this is directly related with cardiovascular diseases. Various combination of statins are suggested as safe and potent medication for the problem throughout globe, but adverse effects are also reported with use as Australian Adverse Drug Reactions Advisory Committee had received 22 reports of paraesthesia associated with simvastatin [3] Symptoms most frequently involved the face, scalp, tongue, and limbs and ranged from hypoaesthetic to hyperaesthetic sensations, although 4 cases of more A B S T R A C T Hypercholesterolemia is an emerging problem affecting population now a days and responsible for CVD, HTN, Diabetes. Not following Dincharya, Ritucharya , smoking, consuming alcohol, lack of exercise, stress, intake of fast food are one of common cause for hypercholesterolemia. Hypercholesterolemia can be compared in Ayurveda as Medodusti which is due to intake of excessive Sleshmavradhaka Ahara (heavy food) leads to Agnimandhya (diminished digestive fire) and in turn Medodusti that is abnormal accumulation of Meda (Cholesterol). Chirbilva in Sushruta Samhita mentioned as Kaphamedovishoshaka . Hence to trace out its hypercholesteremic effect present study is taken. Key words: Hypercholesterolemia, Medodusti, Chirbilva, Holoptelea integrifolia

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[Find the meaning and references behind the names: Long, Pranav, Ama, Sneha, Medo, Sama]

Vinod Katoch et al. An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) ISSN: 2456-3110 ORIGINAL ARTICLE October 2024 Journal of Ayurveda and Integrated Medical Sciences | October 2024 | Vol. 9 | Issue 10 27 serious neurological damage had also been reported. To avoid such side-effects, the whole world is looking towards alternative medicine and Ayurveda comes ahead amongst different traditional system of medicines with long lists of medicine for the purpose. The problem In Ayurvedic classical texts, a condition described by name of Medo Dhatu Dusti very much resembles hypercholesterolemia and this is resulted from imbalance of energy consumption and intake. Number of tissues in any human body is rich in Sneha (oil) and they are Medodhatu , Vasa and Majjadhatu . They have Snehatva as common feature but they differ in their site and function [4] Meda’s is present mainly in Udara , but some are also present in Mamsa and Asthi . Medas present in small Asthi is called as Sarakta Medas and those present in large Asthi , is termed as Majja . Pure Meda present in Mamsa is known as Vasa. [5] It is Medodhatu which is having significant role in developing many metabolic disorders like Medoroga etc. There are two types of Medodhatu by name of Poshaka and Poshya . Poshaka Medodhatu is mobile in nature which is circulated in the whole body along with the Rasarakta Dhatu . Its purpose is to provide nutrition to Poshya Medodhatu and recent researches have proved that cholesterol is circulated with blood, means Gatiyukta . Poshya Medo Dhatu is having immobile nature, which is stored in Medodharakala . The site of Medodharakala is Udara , Anuasthi, Sphika , Stana and Gala . Such conditions are result of derangement of metabolism of Medas . Excessive intake of Slesma Vardhaka Ahara produces Agnimandya . This Agnimandya brings Ama Dosa resulting in Ama Annarasa which is circulated in body. This vitiates Dosa, Dhatu, Srotasa, Bhutagni and Dhatwagni . Excessive vitiation occurs in Medodhatwagni which brings more production of Sama Medasa . This Sama Medasa circulates in body which brings Posaka and Posaka Dhatus in Sama condition. Due to this Medo Dhatu Dusti occurs. Medodushti is abnormal accumulation of Meda Dhatu in body. It includes several numbers of other Medovikaras , which are collectively known as Medoroga . Various formulations and plants are suggested for Medoroga viz., Musta, Haridra, Daruharidra, Vacha, Agnimantha, Agaru, Ashoka, Triphala, Trikatu, Chirabilva, Shimsapa etc. Reference regarding Chiravilva is found in Sutrasthana 38/8-9 of Sushruta Samhita as Kaphamedovishoshana . Less references are found regarding hypocholesterolemic effect of Chirbilva ( Holoptlea integrifolia ). So, present work is taken for study. M ATERIALS AND M ETHODS Pharmacological Study [6-10] Animals Procurement and Standard Setup for Experiment 1 Male Wister Strain Albino rats weighing 150-250 g were used for the study. 2 The animals were obtained from the animal house attached to the A.L.N Rao Memorial Ayurvedic Medical College, with Reg. No. -191/ CPCSEA, IAEC Approval no: A.E.D.G.03/11. 3 They were maintained on “Amrut” brand animal pellet feed of Pranav Agro Industries and plain tap water given ad libitum. 4 Animals were exposed to natural day and night cycles with ideal laboratory conditions in terms of ambient temperature (22 ± 3ºC) and humidity (50- 60%). 5 The experiments were carried out after obtaining permission from “Institutional Animal Ethics Committee” Pilot Study Maximum dose of cholesterol powder in rat is around 25 g/kg/day. Average weight of rat is around 200 gm. Hence, dose of cholesterol comes as:25×200/1000 = 5 mg A pilot study was conducted to fix the dose of the rat. For that 5 mg of cholesterol powder was mixed with 5 ml of vanaspati ghee by making it in lukewarm state, the 5 ml of saturated solution was administered with the help of gastric tube, but on the next day the rat was not survived because of excess dose of vanaspati ghee. Therefore, dose of ghee was reduced to 2 ml and was mixed with same 5 mg of cholesterol powder in

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[Find the meaning and references behind the names: Low, Pala]

Vinod Katoch et al. An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) ISSN: 2456-3110 ORIGINAL ARTICLE October 2024 Journal of Ayurveda and Integrated Medical Sciences | October 2024 | Vol. 9 | Issue 10 28 lukewarm state, it was observed that this particular dose was suitable for rat and easily digestible. Autopsy Finding On autopsy, for 5 mg cholesterol in 5 ml vanaspati ghee, it was found that ghee regurgitated back and entered the airways of rat and to lungs, due to these, rat by choking. Dose Fixation and Schedule As, per FDA approved The Jounal of Korean Oriental Medicine formula for conversion of Human dose to rat dose is: Rat dose (mg/kg) = HED (mg/kg) × Conversion factor Whereas, Conversion factor for rat is 6.17 (As per reference for specified weight of rats) Conversion of the dose obtained above to dose in mg/kg/day by multiplying with suitable conversion factor based on the average weight of the animal. Dose of Kashaya for human being = 2 pala = 2×48 =96 ml/60 kg body weight So, HED = 1.6 ml/kg body weight This can be calculated as per formula that is: Animal dose = 1.6 × 6.17 = 9.872/kg body weight As, the average weight of rat taken is in grams so, 9.872 × 210/ 1000 = 2.07 ml/210 gm body weight of rat 2 ml of freshly prepared Kashaya was given for experiment. Dose fixation of standard drug: Human dose of Atorvastatin is 10-80 mg once in a day. As per earlier studies both 20 mg and 40 mg human dose was proved effective in reduction of serum lipids in rats and there was no significant difference between them. Hence the dose selected was 20 mg, conversion of human dose to rat dose based on formula. Rat dose = HED × Conversion factor for rat that is 6.17 As, dose of Atorvostatin for human is 20 mg. Average weight of an individual is 60 kg So, according to formula, 20/ 60 = 1/3 mg is per kg bodyweight for human Therefore, for rat 1/3 x 6.17 = 2.05 mg is per kg bodyweight For 210 gm rat, 2.05 mg x 0.210 = 0.43 mg 20 mg of standard was dissolved in 25 ml of distilled water. 0.5 ml of Atorvostatin was administered in morning hours to the standard group. Preparation of Kashaya Fehling method was applied for removal of bark, then it was cut into small pieces and was allowed to dry in shade. When bark of both the drugs got dried then they are pounded well and make to coarse powder form by passing through the sieve no. 2000/355, According to W.H.O. guidelines. Then powders of both the bark were stored in air tight container in dark. Test Drug: To prepare freshly Twaka Kashaya of drug Chirbilva ( Holoptelea integrifolia Planch . ) as mentioned in classics ( Sarangadhara ) i.e., 1 Pala (48 gms) of powdered drug was taken with 16 parts of water. It was reduced to 1/8 th part. The mentioned methods were adopted to prepare Kashaya of bark of drugs Chirbilva ( Holoptelea integrifolia Planch . ) Roxb.). Fresh Kashaya was prepared under Mandagni (low flame) as per told in Sharangdhara every day to give to rats. The dose given was 2 ml as obtained by dose conversion formula. The drug was administered in morning hours to both trial groups. Experimental study design and protocol Sample: 18 Albino rats of either sex will be randomly selected. Inclusion: Criteria Healthy, active rats each weighing in between 150-200 g

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Vinod Katoch et al. An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) ISSN: 2456-3110 ORIGINAL ARTICLE October 2024 Journal of Ayurveda and Integrated Medical Sciences | October 2024 | Vol. 9 | Issue 10 29 Exclusion Criteria: Pregnant, diseased rats weighing below 150 g or above 200 g and rats under trial for other experiments will be excluded. Grouping Groups No. of Albino rats Drugs Forms Dose / 200 g Body Weight Purpose Control Group 6 Distilled water 2 ml To serve as prophylac tic control Standar d Group 6 Atorvastati n 20 mg Liquid 0.4 ml As positive Control Trial Drug I 6 Stem bark of Chirabilva Kashaya 2 ml To bring about Medohara effect Initial body weights of all the animals were taken and the animals were fed on the hyperlipidemic diet except control (Group-1) where they were kept on pellet feed and water. Rats of groups 2 and 3 were given with hypercholesterolemic diet. In Group-2 standard drug Atorvastatin 20 mg diluted in 25 ml of water and 0.5 ml of saturated solution was administered (in morning hrs) whereas Groups-3 received Chirabilva Twak Kshaya respectively for 30 days. The hypercholesterolemic diet included hydrogenated vegetable oil (Vanaspati Ghee and Cholesterol extra pure powder) made into suspension form. The suspension was administered in the dose of 0.5 ml/210 g bodyweight of rats daily for 30 days (at evening hours) to all the rats. On the 30 th day final weight of all the rats will be taken and on the 31 st day after overnight fasting, rats were anaesthetized by giving mild dose of Ketamine subcutaneously. Then, the blood was collected by Retro-orbital puncture and was sent to laboratory for biocicalestimations [8-10] Duration: 31 days Statistical Test: Anova followed by Post hoc. Scheffe test was done. Observation 1) Subjective Criteria: Weight (B.T. & A.T.) are taken 2) Objective Criteria: HDL, LDL, VLDL, Total Cholestrol, Triglycerides Collection of Blood According to guidelines for Collection of Blood from Laboratory Animals following procedure is opted for the collection of blood: Under general anesthesia the rats were grasped so that its back rested on the palm with its head toward thumb. The thumb was placed just lateral to the animal's trachea so that the jugular vein on the same side as the eye from which occluded blood was collected. The fur on the animals head was drawn into the palm, this caused the animal’s eye to proptose (bulge) slightly. A 50 μL sterile micro hematocrit tube was directed into the medial canthus (junction of eyelids closest to the animal's nose) of the eye rotating slightly as the tube was directed to a point directly behind the globe. Sufficient pressure was applied to cut through the fibrous layer that surrounded the sinus. Blood flowed through the tube once the sinus had been penetrated. After blood collection, the tube was removed and the eyelids were closed and a dry cotton pad was applied over the eye with gentle pressure to prevent retro-orbital hemorrhage. Blood was not collected from the same eye more than 2 times. An antibiotic ophthalmic ointment was applied following bleeding. Mode of Drug Administration: Oral route was selected for administration of drug to respective group of animals by using syringe with an attached gastric tube. Criteria for assessment of results: Blood cholesterol levels were checked before and after treatment. Blood was drawn from the rats by retroorbital method. These values were subjected to statistical analysis to evaluate the hypercholesterolemic activity.

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Vinod Katoch et al. An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) ISSN: 2456-3110 ORIGINAL ARTICLE October 2024 Journal of Ayurveda and Integrated Medical Sciences | October 2024 | Vol. 9 | Issue 10 30 R ESULTS Animal Experiment Animals (Rats) in Cage Feeding to Rat Collection of Blood by Retro-orbital Puncture Bodyweight Comparison: Group Bodyweight (Before Treatment) Mean + SD Bodyweight (After Treatment) Mean + SD Control 210.00 + 14.49 236.66 + 9.83 Standard 216.66 + 10.80 235.83 + 11.58 Trial I 211.66 + 14.71 246.66 + 9.83 Mean of Change in Bodyweight Groups Mean + SD Control 26.66 + 6.83 Standard 18.33 + 4.08 Trial I 35.00 + 6.32 Mean and Standard Deviation of Total Cholesterol (mg/dl) Before and After Treatment Groups Before Treatment After Treatment Control 53.60 + 4.48 55.13 + 4.57 Standard 53.81 + 2.69 41.36 + 2.66 Trial I 52.85 + 3.33 43.90 + 2.65 Mean and Standard Deviation of Triglycerides (mg/dl) Before and After Treatment Groups Before Treatment After Treatment Control 68.03 + 4.75 69.56 + 4.59 Standard 74.88 + 7.76 51.35 + 6.79 Trial I 71.66 + 8.85 52.27 + 8.58 Mean and Standard Deviation of HDL (mg/dl) Before and After Treatment Groups Before Treatment After Treatment Control 21.13 + 1.85 20.26 + 1.88 Standard 23.51 + 2.51 24.23 + 2.57

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[Find the meaning and references behind the names: Sri, Santosh, Matthias, Adam]

Vinod Katoch et al. An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) ISSN: 2456-3110 ORIGINAL ARTICLE October 2024 Journal of Ayurveda and Integrated Medical Sciences | October 2024 | Vol. 9 | Issue 10 31 Trial I 23.52 + 2.11 24.09 + 2.17 Mean and Standard Deviation of LDL (mg/dl) Before and After Treatment Groups Before Treatment After Treatment Control 18.86 + 3.72 20.98 + 4.04 Standard 15.31 + 1.55 6.69 + 1.59 Trial I 15.00 + 0.75 9.18 + 1.74 Mean and Standard Deviation of VLDL (mg/dl) Before and After Treatment Groups Before Treatment After Treatment Control 13.60 + 0.95 13.88 + 0.89 Standard 14.97 + 1.55 10.27 + 1.35 Trial I 12.87 + 1.83 10.45 + 1.71 DISCUSSION When bodyweight before and after treatment was compared, it was seen that increase in weight was maximum with Trial I being 35 gm while it was 19.17 gm, 34 gm and 26.66 gm respectively. When total cholesterol was attained, it was increased by 1.53 gm/dl for control group while for standard it got reduced, 12.45 gm/dl. For Trial drugs, quantity was reduced by 8.95 gm/dl. Total Triglycerides were observed increasing in control group slightly by 1.53 mg/dl while it was reduced by 23.53 mg/dl, 19.39 mg/dl respectively for standard, Trial drug I HDL was noted decreased by 0.87 mg/dl while it was marked increased by 0.72 mg/dl and 0.57 mg/dl in sequence for standard, Trial drugs I. CONCLUSION Mean of bodyweight changes before treatment and after treatment was 26.66, 18.33 and 35 respectively for control, standard and trial I groups. Mean of changes in total cholesterol before and after treatment in mg/dl for control, standard and trial I were respectively 1.53 (increased), 12.45 (reduced) and 8.95 (reduced). Mean of changes in triglycerides before and after treatment in mg/dl for control, standard and trial I were respectively 1.53 (increased), 23.53 (reduced) and 19.39 (reduced). Mean of changes in HDL before and after treatment in mg/dl for control, Standard and trial I were respectively 0.87 (reduced), 0.72 (increased) and 0.57 (increased). Mean of changes in LDL before and after treatment in mg/dl for control, Standard and trial I were respectively 2.12 (increased), 8.62 (reduced) and 5.82 (reduced). Mean of changes in VLDL before and after treatment in mg/dl for control, Standard and trial I were respectively 0.28 (increased), 4.70 (reduced) and 2.42 (reduced). Holoptelea bark was having better in action in case of HDL, LDL and VLDL. Statistical analysis of total cholesterol by means of ANOVA revealed Bark as significant after treatment. REFERENCES 1 https://wwwwho.int/data/gho/indicator-metadataregistry/ir-details/3236 2 Begunde DO, Mathers CD, Adam T, Ortegon M, Strong K. The burden and costs of chronic diseases in lowincome and middle-income countries. Lancet 2007; 370:1929-38. 3. Anonymous. Paraesthesia and neuropathy with hypolipidaemic agents. Aust Adverse Drug React Bull 1993; 12: 6 4 Susruta, Susruta Samhita with Nibandha Sangraha Commentary of Sri Dalhanacharya - Edited by Vaidya Yadavji Trikamji Acharya; Chowkhamba Krishnadas Academy, Varanasi – 2004 Dalhana on Shareera Sthana - 4/12,13 5 Susruta, Susruta Samhita with Nibandha Sangraha Commentary of Sri Dalhanacharya - Edited by Vaidya Yadavji Trikamji Acharya; Chowkhamba Krishnadas Academy, Varanasi, 2004, ShareeraSthana - 4/12,13. 6 Animal Experiments in Research, Lemmens Medien GmbH, Matthias-Grünew, Bonn,Germany. 7 CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals) Guidelines, India. 8 Santosh et al. (2012). ‘A Study of Anti -hyperlipidemia, Hypolipidemic, and Anti-antherogenic Activity of Fruit of

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[Find the meaning and references behind the names: Mar, Melissa, Nil, Med, Jan]

Vinod Katoch et al. An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) ISSN: 2456-3110 ORIGINAL ARTICLE October 2024 Journal of Ayurveda and Integrated Medical Sciences | October 2024 | Vol. 9 | Issue 10 32 Emblica officinalis (Amla) in High Fat Fed Albino Rats’. International Journal of Medical Research and Health Sciences . Volume 2, Issue 1 Jan-Mar 2013. 9 Zarei A et al. (2013) Effects of Melissa officinalis on thyroid hormones. Zahedan Journal of Research in Medical Sciences. 15 (8): 6-12. 10 Madhavi Jagtap et al. (2011) ‘A comparative evaluation of cardioprotective activity of two Makandi ( Coleus forskohlii Willd.) formulations against isoproterenol induced myocardial infarction in hyperlipidaemic rats’. Indian Journal of Natural Products and Resources. Volume 2(3), September 2011,pp.335-344. ******************************* How to cite this article: Vinod Katoch, Prashant Kumar Jha, Vikas Kumar. An experimental evaluation of bark of Chirbilva (Holoptelea integrifolia Planch.) for Hypercholesterolemia in Albino Rats. J Ayurveda Integr Med Sci 2024;10:26-32. http://dx.doi.org/10.21760/jaims.9.10.4 Source of Support: Nil, Conflict of Interest: None declared. Copyright © 2024 The Author(s); Published by Maharshi Charaka Ayurveda Organization, Vijayapur (Regd). This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by-nc-sa/4.0), which permits unrestricted use, distribution, and perform the work and make derivative works based on it only for non-commercial purposes, provided the original work is properly cited

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