Advances in Research of Pharmacological Action and Mechanism of Tibetan Medicine

Shuyan FU, Mengqi YAO, Ziqiang ZHOU, Fangyun SUN

Department of Medicine, Xizang Minzu University; Engineering Research Center for Tibetan Medicine Testing Technology of Ministry of Education, Xianyang 712082, China

Abstract Qishiwei Zhenzhu Wan (Ranasampel) was included in Chinese Pharmacopoeia and consists of 70 kinds of traditional Chinese medicines, including Zogta, Margarita, Croci Stigma, Santali Albi Lignum, and Dalbergiae Odoriferae Lignum, etc. At present, research on the pharmacological effects of Ranasampel is mainly focused on anti-Alzheimer’s disease, anti-cerebral ischemia-reperfusion injury, anti-Parkinson, improving learning and memory ability, and anti-myocardial ischemia. The mechanisms involved include nervous, cardiovascular and other systems. This article systematically expounded the pharmacological effects and mechanism of Ranasampel, in order to provide a certain theoretical reference for its future further research and clinical application.

Key words Qishiwei Zhenzhu Wan (Ranasampel), Alzheimer’s disease, Learning and memory, Nyocardial ischemia, Liver injury

1 Introduction

Qishiwei Zhenzhu Wan is a rare and precious Tibetan medicine that has been used in China with a long history. In Tibetan language, it is called Ranasampel. Ranasampel originated from the classic prescription Ershiwuwei Zhenzhu Wan recorded in the Chinese medical workFourMedicalTantrasin the 8thcentury. After the continuous efforts and improvements of several generations of Tibetan medicine scientists, it was finally developed by Tibetan medicine Suga Nidoje in the 15thcentury, and now included in theChinesePharmacopoeia(2020 edition)[1-2]. It is made up of 70 kinds of traditional Chinese medicines, such as Zogta, Margarita, Croci Stigma, Santali Albi Lignum, and Dalbergiae Odoriferae Lignum, including metals, minerals, animals, plants and other medicinal materials. It is aromatic in scent, sweet, astringent, and bitter in taste. It has multiple effects such as calming and tranquilizing, activating blood and dredging collaterals, regulating qi and blood, and rejuvenating the brain[2]. Ranasampel has obvious curative effects on Alzheimer’s disease[3], cerebral infarction[4-5], cerebral hemorrhage[6], stroke[7]and primary epilepsy[8]. In this study, we mainly summarize the pharmacological effects and mechanisms of Ranasampel on the nervous and cardiovascular systems, in order to provide a theoretical basis for further in-depth research.

2 Pharmacological effects on the nervous system and its mechanism

2.1 Anti-Alzheimer’s diseaseAlzheimer’s disease (AD) is a type of multifactorial neurodegenerative disease, characterized by cognitive system dysfunction and behavioral dysfunction[9]. The main pathological features include the accumulation of amyloid beta protein, hyperphosphorylated tau protein, and neurofibrillary tangles. These pathological features are mainly triggered and enhanced by oxidative stress which is an unbalanced state of oxidation and antioxidant. This imbalance may be caused by an increase in free radicals or a decrease in antioxidant properties[10]. At present, drugs for the treatment of Alzheimer’s disease only provide symptom relief, but can hardly solve the above-mentioned basic factors that lead to neurodegeneration and slow down the progression of the disease[11]. Therefore, the anti-oxidative stress properties of traditional Chinese medicine have attracted more and more attention and research. Ranasampel has obvious control effect on this feature.

According toinvitrostudy of Zhuetal[12], pretreatment of Ranasampel can significantly reduce the upregulation of 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, a marker of oxidative DNA damage) induced by H2O2in SH-SY5Y cells, and reverse H2O2related (100 μM) down-regulated repressor element silence transcription factor (REST) expression, and can significantly reduce the phosphorylation level of P38 MAPK and ERK1/2, and protect the neuronal damage induced by oxidative stress. This suggests that the mechanism for Ranasampel to improve SH-SY5Y cytotoxicity induced by H2O2is to exert various antioxidant properties by inhibiting the activation of MAPKs signaling pathway. Through animal experiment, Bai Zhenzhongetal.[13]proved that the aging AD model made by subcutaneous injection of D-galactose on the back of the neck and injection of Aβ25-35 into the lateral ventricle after continuously intragastric administration of Ranasampel for 8 weeks, it could significantly increase the activity of SOD in the serum of AD model rats and reduce the content of MDA, and the experimental results reveal that Ranasampel can treat AD by increasing the body’s ability to scavenge free radicals and effectively resist peroxidation damage. Another experiment used Western blot to detect mice given Ranasampel intragastrically and showed that the levels of Aβ1-42 and BACE1 lyase were significantly reduced, indicating that Ranasampel can treat AD by reducing the deposition of amyloid β protein in the brain[14].

2.2 Anti-cerebral ischemia reperfusion injuryIschemic stroke (IS) has a high risk of disability and even death to the patient, and the incidence accounts for about 80% of cerebrovascular diseases, it seriously endangers the health of the vulnerable people[15]. The IS is prone to secondary reperfusion injury, and induces oxygen free radical reaction, which destroys the integrity of the blood-brain barrier, causes brain edema, and causes more serious pathological damage, which is called cerebral ischemia-reperfusion injury (CIRI)[16]. Oxidative stress, autophagy, and apoptosis play an extremely important role in the pathogenesis of cerebral ischemia-reperfusion injury[17].

Studies have found that rats were given Ranasampel intragastric administration with suture method to build a cerebral ischemia model and reperfused after 2 h. After 24 h, it was observed that the cerebral infarction rate of the model rats was reduced and the brain tissue of the ischemic side Evans blue (EB) was reduced, and the neurobehavioral abnormalities were effectively reduced, and the number of Nissl bodies was increased, suggesting that Ranasampel can protect rat brain tissue from ischemia-reperfusion injury, and its mechanism may be related to reducing the damage of the blood-brain barrier, increasing the activity of SOD and reducing the level of MDA to a large extent[18-19]. A study also found that Ranasampel can protect rats from cerebral ischemia-reperfusion injury by increasing the ratio of Bcl-2/Bax and reducing the expression of caspase-3 protein, and the protective effect may be related to the anti-apoptotic mechanism of Ranasampel[20]. Specifically, after establishing the rat cerebral ischemia-reperfusion injury model, the study of the Ranasampel dose-time-effect relationship found that the effective dose of the treatment was 4.17-133.36 mg/kg, the maximum effective dose was 33.34 mg/kg, the optimal effective time point was 48 h[19,21]. According to reports, AQP4 is a selective aquaporin protein. When the brain edema occurs in rats, the expression of AQP4 will be up-regulated, causing astrocyte foot process edema and destroying the blood-brain barrier[22]. Zhu Minxiaetal.[23]prepared an MCAO model. After 40 min of ischemia and reperfusion for 24 h, they confirmed that the cerebral ischemic side had significant edema and the content of AQP4 increased, the administration of Ranasampel can significantly reduce the degree of cerebral edema and reduce the expression of AQP4, indicating that Ranasampel has the effect of preventing cerebral ischemia-reperfusion injury.

2.3 Improving the ability of learning and memoryStudies have shown that the changes of myelinated nerve fibers in the white matter of the brain are closely related to spatial learning and memory[24]. The greater the degree of nerve fiber damage, the greater the degree of white matter damage[25]. There is also a study showing that the mechanism of brain white matter damage is related to enhanced ketogenesis and mitochondrial function[26]. According to findings of Yan Qingweietal[27], Ranasampel can increase the FA value and MBP gene expression in the hippocampal white matter of APP/PS1 mice, and reduce the expression of c-PLA2, SCOT and MCTs proteins, indicating that Ranasampel can improve learning and memory ability by reducing white matter damage and enhancing the integrity of myelin sheath, and its mechanism may be related to the inhibition of hippocampal ketogenesis. Besides, Ranasampel can increase the mitochondrial fusion gene (mfn1, mfn2) and decrease the mRNA of the mitochondrial division gene (Opa1) by intragastric administration for 12 weeks, and it can be speculated that Ranasampel’s improvement in the learning and memory abilities of model mice is related to the fusion and division of mitochondria in the hippocampus[28]. Ranasampel can not only resist AD, but also improve the spatial learning and memory ability of patients. In the Morris water maze experiment, it was found that the incubation period, the number of times of crossing the platform, the swimming time of the target quadrant, the percentage of movement distance in the target quadrant, and the percentage of exercise time of the mice administered with Ranasampel were significantly improved compared with before treatment. In the opening experiment, mice given Ranasampel significantly increased the times of trying hole board. It proves that Ranasampel can significantly improve the learning, memory and space exploration ability of AD mice[13-14, 27,29]. The mechanism may be the same as the mechanism of reducing the generation of free radicals and resisting oxidative stress in the treatment of AD.

2.4 Anti-ParkinsonParkinson’s disease (PD) is a common degenerative disease of the nervous system in the elderly, mainly manifested by motor symptoms such as resting tremor, muscle rigidity, slow movement and uncoordinated movement. The loss of dopamine neurons produced in the dense parts of the substantia nigra is the basis of the core motor symptoms of progressive dyskinesia Parkinson’s disease[30].

Cui Haiyanetal.[31-32]used 6-hydroxydopamine (6-OHDA) injection into the right striatum to make a PD model. After intragastric administration with Ranasampel for 4 weeks, they found that the frequency of twisting on the contralateral side of PD rats was reduced and the balance bar incubation period and total time were shortened, and movement disorders were significantly alleviated; through immunohistochemical staining, they further observed a significant increase in TH-positive neurons in the administration group, and a significant increase in the amount of DA in the striatum by high performance liquid chromatography, indicating that Ranasampel treated rats with PD by the mechanism of protecting the dopaminergic neurons.

2.5 Resistance to traumatic brain injuryNeurotransmitters such as 5-HT, NA, β-EP participate in various physiological and biochemical processes of the brain, and are important neurotransmitters that cause secondary brain injury. Through opening the bone window one hour after the last administration of Ranasampel, Hai Pingetal.[33]used the gravity strike method to prepare the rat brain injury model, and took the brain tissue 4 h after the model was created, and it was determined that the pretreatment of Ranasampel could significantly reduce the expression of 5-HT and NA; and it was further found that Ranasampel can reduce the increase in β-EP content caused by brain injury[34]. Therefore, it can be speculated that Ranasampel may protect traumatic brain injury by regulating the mechanism of monoamine neurotransmitters in the brain.

2.6 Anti-convulsionConvulsions are characterized by body spasms and muscle twitches. They have rapid onset, rapid changes, and dangerous conditions, often endangering the lives of patients[35]. Clinically, diazepam is often used to treat convulsions, but long-term use will cause patients to develop drug resistance and dependence, and sudden withdrawal will cause withdrawal syndrome, which affects the prognosis[36]. However, the side effects of traditional Chinese medicine in the process of taking the medicine are relatively small, so the therapeutic effect of traditional Chinese medicine on convulsions has attracted more attention and research. Studies have shown that Ranasampel can delay the onset of thiosemicarbazide-induced running convulsions in mice, and can also reduce the convulsions rate of forced convulsions in mice induced by pharmacological and physiological multi-apparatus stimulation[37-38]. Through Ranasampel pretreatment, Hai Pingetal.[38]built a thiosemicarbazide-induced convulsive mouse model, measured the content of Glu and GABA with high pressure liquid chromatography, and found that Ranasampel can significantly reduce the ratio of Glu/GABA, and it is speculated that its anticonvulsant mechanism is achieved by balancing excitatory and inhibitory amino acids. Wen Qingjieetal.[39]found that Ranasampel can inhibit the autonomous activities of mice, cooperate with pentobarbital sodium subthreshold sleep and enhance the sleep effect of ether, and they speculated that the anticonvulsant mechanism of Ranasampel is related to the central inhibitory sedation.

3 Effect on the cardiovascular system and its mechanism

3.1 Anti-myocardial ischemiaMyocardial ischemia (MI) is insufficient blood supply to the myocardium and it can lead to serious complications and even life-threatening conditions[40]. Acute or persistent ischemia and hypoxia of myocardial tissue caused by coronary artery occlusion affect the cardiac conduction system and can cause severe myocardial infarction[41]. Acute myocardial infarction patients are closely related to peripheral blood over SOD, MDA,etc., so this indicator can be used to predict the condition of acute myocardial infarction[42]. Wu Qiongetal.[43]gave SD rats intragastrically administered Ranasampel for 7 d, and ligated the left anterior descending coronary artery as an acute myocardial infarction model and found that the serum levels of LDH and CK in the administration group were significantly reduced, SOD activity in the myocardial tissue was significantly increased, and the content of MDA is significantly reduced. They speculated that Ranasampel can improve the activity of antioxidant enzymes in the animal body, scavenge free radicals, and achieve a protective effect on acute myocardial ischemia.

3.2 Anti-thrombosisThrombosis is a multifactorial disease induced by promoting the combination of blood stasis and hypercoagulability. Thrombosis is closely related to platelet adhesion, aggregation, and activation of internal and external coagulation systems and fibrinolytic systems. At present, the drugs commonly used in clinical treatment of thrombosis mainly inhibit platelet aggregation, but patients often suffer from drug resistance and even bleeding and other serious complications[44]. Therefore, the antithrombotic properties of traditional Chinese medicines have attracted attention and research. By using internal carotid artery injection that the combined use of adenosine diphosphate, epinephrine and thrombin to establish cerebral thrombosis model rats, Zhen Lifangetal.[45]found that Ranasampel can significantly reduce the low-shear viscosity of whole blood, plasma viscosity, red blood cell aggregation index, and significantly reduce the content of TXB2, 6-keto-PGF1α in plasma and increase the ratio of TXB2/6-ketoPGF1α, indicating that Ranasampel can inhibit the increase of thromboxane A2 (TXA2) and prostacyclin (PGI2) caused by the compound thrombosis inducer, further regulate the dynamic balance between TXA2 and PGI2, reduce the risk of platelet aggregation and prevent thrombosis. There are also studies showing that after prophylactic administration of Ranasampel, rats were injected with adrenaline and ice-water bath to imitate the thrombosis model, and the administration of Ranasampel group could also significantly reduce fibrinogen, low-shear reduced viscosity of whole blood and low-shear viscosity of whole blood[46-47], showing that the anti-thrombotic mechanism of Ranasampel may be related to the dynamic balance of the content of TXA2 and PGI2 and the regulation of hemorheological indicators.

3.3 Anti-hypertensionHypertension is a relatively common chronic disease induced by a variety of factors in clinical practice, and its pathogenesis is characterized by increased systemic arterial blood pressure. Controlling hypertension can reduce the incidence of cardiovascular and cerebrovascular diseases and patient mortality[48]. A study found that with the increase of the age of the rats and the increase of salt intake, the blood pressure of the rats is increased, and Ranasampel has a good preventive effect on this hereditary hypertension[49]. Another study found that Ranasampel had a significant hypotensive effect on renal hypertension caused by ligating the renal artery of rats and loosening the ligation for 4 min[50]. The antihypertensive mechanism of Ranasampel still lacks data, and sill needs further exploration and research.

4 Liver protection and its mechanism

Liver injury may induce various liver diseases, and long-term liver damage is often the pathogenic factor of liver fibrosis, cirrhosis and even hepatocellular carcinoma[51]. Ranasampel and its components have a good liver-protecting effect[52]. CCl4enters the body and is activated by the cytochrome P450 system, by reducing phospholipid molecules, and combining with lipids and proteins on the liver cell membrane, it damages the normal structure and function of the liver cell membrane, accordingly leading to liver injury[53].

Using CCl4to induce an acute liver injury model, Nie Yuetal.[54]found that compared with the model group of mice, the pretreatment group with Ranasampel significantly reduced the pathological damage caused by CCl4such as hepatocyte swelling, and found that Ranasampel can reduce the elevated value of AST and ALT, reduce the expression of hepatotoxicity genes Gadd45 and Gadd153, and reduce the release of inflammatory factors MIP2 and IL-6. Compared with the normal group, the pretreatment group of Ranasampel can increase the expression of antioxidant-related genes MT-1 and GSH, suggesting that Ranasampel can protect against acute liver injury, the protective mechanism of Ranasampel may be achieved through activating the anti-oxidative damage pathway and reducing the content of inflammatory factors. According to findings of He Junnaetal[55], Ranasampel can significantly reduce the increase in the rate of caspase-3 and Fas-positive cells caused by phenytoin, and improve the morphology of liver cells, and it is speculated that the liver-protecting mechanism of Ranasampel may be related to the inhibition of Fas-mediated death receptor related to the apoptotic pathway.

5 Other pharmacological effects and mechanisms

In addition to the above effects, Ranasampel can also increase the content of Proteobacteria Enterobacteriaceae to regulate the intestinal flora of normal mice[56]; it can influence the effect of tissues to induce light and attenuation to regulate biophotons. The mechanism of Ranasampel’s pharmacological effects may be achieved through affecting tissue cell function by regulating the transmission of biomedical photon signals[57].

6 Conclusions and prospects

In summary, the Tibetan medicine Ranasampel can prevent and treat many diseases such as Alzheimer’s disease, cerebral ischemia-reperfusion injury, and Parkinson’s disease. Its pharmacological mechanism mainly involves the nervous and cardiovascular systems. This study is intended to provide some guidance for future study of Ranasampel.

However, there is still a lack of relevant research on the distribution of Ranasampel and their active components in the body and the targets of action, but this is a key point in the treatment of diseases. Besides, a study measured the content of five heavy metals, Cu, As, Cd, Pb, and Hg in Ranasampel, and concluded that the Hg content is the highest[58]. The main form of Hg is mercury sulfide[1]. The accumulation of Hg in the body is somewhat time-dose dependent[59]. The heavy metal components contained in Ranasampel greatly restrict the formulation of quality standards and the promotion of clinical application. In addition, different processing methods for Ranasampel may have different pharmacological effects. Improper or non-standard processing equipment and methods limit the development of the Tibetan medicine pharmaceutical industry. Therefore, it is necessary to make more in-depth research on the active components and key regulatory targets of Ranasampel in the treatment of various diseases. Furthermore, it is necessary to carry out a systematic safety evaluation of the metabolism and toxicity accumulation of heavy metals contained in the human body, and strictly control the processing equipment and production technology of Ranasampel, so as to formulate standardized quality standards, thereby expanding the wide range, safety and effectiveness of its clinical application, and promote the development of the Tibetan medicine industry.