UNFOLDING THE MYSTERIES OF GENETIC SCIENCES IN INCURABLE DISEASES LIKE CORONARY ARTERY DISEASES, CANCER, & RAREST OF THE RARE DISEASES LIKE MOYAMOYA, SLE, TAKAYASU ETC.

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Foundation on which building of applied human genetiC Engineering is erecteD

PREVENTION OF CORONARY ARTERY DISEASE, DIABETES and HYPERTENSION USING MEDICINAL PLANT EXTRACTS

The raw extracts obtained by mechanical extractions were fed to normal albino pigs and their weight, blood pressure and lipid profile were closely and carefully monitored.

     Materials and Methods:   

    Gnotobiotically grown albino pigs were selected as animal models for this study. All animals were kept under  careful observations of the investigators. De-worming and vaccination of animals were done regularly. They were hygienically fed, given antiseptic bath thrice a week. Routine blood examination after every 30days for hemoglobin estimation, total blood count and DLC were carried out.

     Growing albino pigs weighing 15.0-20.0kg were selected for the studies. Sixty five animals were divided into 13 groups of 5 animals in each group.

     Normal Diet of the animal models:

     All the 65 animals were fed with following diet:

    Breakfast at 7.00am-  Cooked unpolished Rice with mixed vegetables, lentils

    Lunch at 12.30pm- Same as breakfast.

   Dinner at 5.00pm- Cooked unpolished rice with mixed vegetables, lentils, and 100gm cooked beef/ 2eggs each.

            Plenty of sterile drinking water was provided Before starting the experimentation, fasting blood samples were collected from the ear veins of all the animal models for blood glucose, cholesterol, triglycerides, High Density lipoprotein, total protein, Calcium estimations. Blood pressure of every animal was monitored every alternate day using non-invasive techniques.

     Selection of medicinal plants-

    Our Cardiac Research Institute is located in a remote area surrounded by hills and lakes far away from the city. The local people are tribal and their usual diet is rice and curry made of  various seasonal herbs. It has been observed  that their diseases are mainly infectious in origin and occasionally of malignant type.  Coronary artery disease is found to be uncommon, although they are habitual drinkers. The alcohol is  made by them from rice. Smoking and chewing tobacco is not found to be uncommon. Fish and pork are their usual dishes along with herbs. Physically, they are hard working for their living. By observing their herbal food habits and absence of coronary artery disease, we have undertaken this research project of prevention and cure of coronary artery disease by using the medicinal plants those they eat normally.

     Type of extracts-

      Local name                             Botanical names

Plant Alpha                                                      -

Bor manimuni                                     Centella asiatica           

Shuklati                                                 (not known)

Durun                                                     Leucas ciliata

Sewali                                                     Nyctanthus arbortristis

Haldi-                                                      Black   Curcuma aromatica

Haldi-                                                      Yellow Curcuma domestica

Hibiscus–                                               FlowerHibiscus rosa-sinensis

Hibiscus-Leaves                                  Hibiscus rosa-sinensis

Arjun leaves                                          Terminalia arjuna

Bel leaves                                              Aegle marmelos

Plant Gamma                                                    -                                    

                                                                                                                                                Dr. Dhani Ram Baruh

Plant Beta                                                          -

    Cultivation- These identified herbs were cultivated within the campus of the institute. Most of the medicinal plants are seasonal. Availability of some medicinal plants are abundant in monsoon season. These are grown organically and harvested manually.

    Extraction- Fresh extraction of leaves/tubers/bark/ fruits of each herb were done manually and carefully in cryogenic condition at average of  2-4degree Celcius. The care was taken to avoid contamination.

     Doses- Fresh medicinal plant extracts of 30CC was fed to each animal model with feeding tube for 2weeks. After 2weeks, venous blood was collected from ear veins of each experimental animal and various parameters (as done earlier) were analysed in the laboratory to observe the effect of each extract.

To observe the delayed effect of each extract,  fasting blood samples were collected from ear veins after two weeks from each animal model.

     Observations& Results:

      All experimental animal models were carefully observed for their physical conditions and behavioral pattern. All animals were behaving normally and no abnormal cardio-vascular symptoms were observed except group 9, where animals died after feeding raw extracts after 1 week. They were showing tachycardia with heart rate of 200-250beats/minute. It was observed that animals of Group 3 were gaining weight rapidly. On the other hand, animals of Group 10 did not show any sign of weight gain. On the other hand, they were emaciated. Animals of Group 7 were showing signs of  dry skin.

Necropsy of animal models belonging to group 9 were carried out and following findings were obtained.

Chest: No external or internal abnormality was found.

Heart: There was no intra-pericardial fluid or hemorrhage.

Externally no abnormality was found. Right and left ventricles were normal in size and shape. No visible infarction was found. Both right and left Coronary arteries and their branches  were dissected and were found to be normal. All heart valves were normal. Aorta, Pulmonary arteries were normal in size. LV wall thickness was normal. No myocardial infarction was seen  in longitudinal and transverse section. Sections were made at 4mm interval. Externally and internally, RV wall did not reveal any abnormality or any infarction. RA, LA were found normal and SVC and IVC  were also found normal in size and shape.                    

   Lungs: Both pleural cavities were normal and there was no extra fluid in the cavity. Trachea was normal. Both right and left lungs were found in normal conditions, no infarction,  no collapse or no consolidation. Bronchi, bronchioles, pulmonary artery and its branches and pulmonary veins were normal. Cross sections were made at every 4mm intervals and there was no intra- or extra pulmonary infarction.

    Diaphragm: Normal.

    Thoracic aorta and oesophagus were normal.

   Liver: Contains 5 lobes, in normal shape and size. No extra and intra hepatic abnormality was detected.

   Stomach: Both outside and inside colour was normal. No intra-gastric ulcer  was found.

   Pylorus and Dueodenum : Normal

   Kidney: Normal

                           Gall bladder, common hepatic duct and portal veins were normal.

          It is observed from the necropsy that the cause of the death was due to severe tachycardia. In addition, biochemical studies revealed that all the animals were severely hypoglycemic, which might be one of the cause of their death.

      Group of animals belonging to 3 and 11 were showing weight gain. Animals belonging to group 3 were gaining weight rapidly. But animals of Group 11 were gaining more weight in comparison (2.5Kg in 10 days without providing any extra weight gaining diet). 

     All experimental animal models were weighed daily, which showed significant changes. The animals of  group 10, who were fed extract of  Arjun bark became cachectic showing signs of gastric irritation and vomited immediately after feeding the extract. Animals were continued to be observed  and found they were unable to eat and continued to be cachectic and died of severe haematemesis.

    Necropsy was performed to each animal models and following findings were obtained. Heart, liver and kidney were found normal. Stomach- multiple gastric ulcers were found along the greater curvature from where uncontrollable  bleeding took place and it was confirmed that death was due to gastric ulcers. This was partly contributed by hypoproteinemia and hypoglycemia. 

   Other group of animals were not showing any abnormal symptoms. Animals of Group 7 died suddenly after 12 days. Necropsy was performed and following findings were obtained.

  Heart- externally no anatomical change. Blood sample was collected from RA, RV, SV. It was found that LV was hardened and hypertrophied revealed by multiple transverse sections. RA,RV, LA were  normal in thickness.  Thickness of the LV wall was measured and found 2cm in thickness which makes the lumen narrower leading to insufficient filling  and thereby, inadequate ejection, probably  this was the cause of sudden death. There is no sign of myocardial infarction.

Liver- has 5 lobes anatomically and no infarction was detected. Macroscopically, liver was yellowish white in colour and enlarged by 1.5 times than normal. Hepatomegaly and discoloration   suggested that there was enough fatty deposition, which was the another contributory cause of death. Biochemical studies showed higher triglyceride  deposition in  liver tissue.

Stomach, kidney, lungs were normal. 

     Biochemical Assays:

Glucose (GOD/POD method), Triglycerides (GPO/PAP method), Cholesterol (CHOD/PAP method), protein (Biuret method), Calcium (OCPC method),  were carried out using kits supplied by Crest Biosystems, India, for in vitro studies. HDL-Cholesterol was precipitated using HDL cholesterol Ppt. Set (PEG Precipitation method) supplied by crest Biosystems, India.  

     Discussion:

    The medicinal plants cures certain diseases, but at the same time, exact mechanism is not fully known. Therefore, extraction, isolation and identification of the compound should be scientifically carried out, which we believe not done in the past. We have undertaken the extremely difficult task of cultivation, extraction, separation, isolation and identification of the compounds from medicinal plants which is selectively working on diabetes mellitus, hypertension, coronary artery disease, liver disease & kidney failures. We have observed that chemistry of medicinal plants are not as easy as synthetic organic chemistry. However, we have succeeded in getting new compounds from certain medicinal plants, which are edible and cures CAD, hypertension, diabetes mellitus, liver & kidney failures. No presently available synthetic drug selectively works on diastolic and systolic blood pressure. Animal models belonging to Group 1 showed drastic reduction in diastolic pressure without much change of systolic pressure, whereas animal models fed extract number 13 were showing reduction in systolic as well as diastolic pressure.

   Our aim was to find out whether which medicinal herb/plant was going to work on important parameters such as glucose and lipid profiles. We were interested to find out the plant which can be used to cure diabetes as well and we found that majority plant extracts reduce glucose profile except animal models of group no. 5. But at the same time, reducing glucose for curing diabetes is not our aim, reducing other risk factors such as triglycerides and cholesterol were equally important. But not majority plants were showing these properties hence we have discarded most of them. LDL-cholesterol, a major atherogenic factor was elevated in majority of  experimental animal models, with not much change in HDL- Cholesterol.    It was shown that LDL-Cholesterol,  (the major atherogenic constitutent  of the plaques) should be low and HDL-Cholesterol, (a protective factor) should be higher and it is probably a good indicator for absence of coronary artery disease. Animal models belonging to group 1, 12, & 13 showed elevated levels of HDL-cholesterol and reduced LDL-Cholesterol.

   It was interesting to see that increase in HDL-cholesterol with reduction in total cholesterol and triglycerides ultimately reduces LDL-cholesterol in those groups. An irreversible reduction of atherogenic factor, LDL-cholesterol is important as the percentage of this factor is generally increased in majority coronary artery diseased patients.  We have observed the reduction of glucose after feeding medicinal plant extracts in majority groups. But unfortunately, some of it shows either increase in total cholesterol or triglycerides. This situation did not encourage us to use these extracts in future. Hence, we have used the three noted medicinal plant extracts, which reduce glucose levels remarkably without increasing triglycerides or cholesterol in normal laboratory animals. These results encouraged us to study the effect of those three medicinal plant extracts to animal models feeding with highly atherogenic diet.

   Arjun bark extract is widely used as cardiac tonic in India, but gastric irritation and ulcers are found to be common problems associated with its use, which may have been ignored. Animal models fed this extract showed hypoproteinemia, cachexia and hypercholesteremia which persisted through out the period of studies and unable to reverse. The animals died after four weeks of starting the experimentation.

    In addition, loss of protein since feeding of the extract might be either due to decrease in protein synthesis or by increasing protein degradation. On the other hand, animal models fed with extract no.11 were showing rapid weight gain We have to further study whether this particular medicinal plant is having substance like anabolic steroids, which helps in weight gain or insulin like growth factor which enhances protein synthesis and suppresses protein degradation. Thus, it is clear that effect of medicinal plants on various parameters must be studied thoroughly. From our studies, we are confident that different compounds present in medicinal plants/any edible herbs are interacting and alters the number of vital metabolism.

   Calcium metabolism in coronary artery disease is crucial. Many investigators have studied calcification in atheromatous plaques, but its metabolism during initiation of atheroma is not thoroughly studied. Calcium channel blockers are used for coronary artery diseased patients, but still Calcium is found to be one of the content in atheroma. This question remains to be solved. In our experimental studies, some of the extract-fed animals showed increased plasma calcium levels, whereas in some cases, serum Calcium levels are reduced. It is assumed that extracts have interefered with extracellular and intra-cellular calcium ion exchange mechanism. Specially, 1,12,13 extracts helps in extracting calcium from deposition and increases serum Calcium levels. Hence, we are continuing our research to prevent Calcium deposition in atheromatous plaques.

      In conclusion, we have observed carefully and closely the safety and efficacy of 13 medicinal plants extracts using animal models, out of which only three are found to be safe and effective in coronary artery disease, essential hypertension and diabetes mellitus. Some plants are effective in reducing triglycerides, but effective in increasing cholesterol and some are effective in reducing blood glucose and effective in increasing triglycerides. Some are effecting in reducing systolic and diastolic blood pressure and some are increasing both. Therefore, in conclusion, only three plants are safe to use for efficient and effective outcome without any side effects and complications. We will continue to work on these three plant extracts for clinical use and results will be published in next paper. We have used pigs as experimental animal models as we found these are comparable to human anatomically and physiologically. We are convinced that coronary artery disease could be irradiated by preventing, recannalization can be achieved, so that efficient re-vascularization to myocardium could be fully obtained. We are also looking at the reactivation of beta cells of pancreas and production of native insulin to combat insulin dependent diabetes and activation of receptors to cure non-insulin dependent diabetes. We further confirmed by animal  experimental studies that coronary artery bypass surgery probably be kept permanently in cold storage.
 

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