Tu Youyou discovered artemisinin
On March 18, 1978, the "National Science Congress" was held in Beijing. During the meeting, Comrade Deng Xiaoping delivered a speech of the times entitled "Building Ambitions, Setting Great Aspirations, and Marching Toward the Modernization of Science and Technology." Guo Moruo, president of the Chinese Academy of Sciences, delivered a written speech entitled "The Spring of Science". Since then, China has clearly established the basic Marxist viewpoint of "science and technology is a productive force" and ushered in the spring of new China's scientific undertakings. Ms. Tu Youyou, the inventor of artemisinin, attended this important meeting as the first inventor of the first invention unit, and her achievements in the discovery of artemisinin won the "National Science Congress Award".
Tu Youyou attended the National Science Congress
Tu Youyou was admitted to the Department of Pharmacy of Peking University in 1951, and the major he chose was pharmacognosy, which was not of interest to ordinary people at that time. Because the pharmacy profession is most likely to be closest to exploring the field of traditional Chinese medicine with a long history, in line with her own interests and ideals, Tu Youyou chose the major of pharmacognosy.
In 1955, the state established the Institute of Traditional Chinese Medicine (now the Chinese Academy of Traditional Chinese Medicine), and a group of well-known old Chinese medicine doctors were drawn from all over the country to Beijing to enrich the expert strength of Traditional Chinese medicine research and form a national team for traditional Chinese medicine research. It was also in this year that Tu Youyou graduated from university and was assigned to this Institute of Traditional Chinese Medicine directly under the Ministry of Health. At the beginning of her work, Tu Youyou was mainly engaged in biopharmacy research. In 1956, the country set off a climax in the prevention and treatment of schistosomiasis, and she conducted pharmacognosical research on the effective drug hemitrix, and later, completed the pharmacognosy research of the more complex varieties of Chinese medicine Yinchaihu. These two achievements have been successively included in the Journal of Chinese Medicine.
In 1958, Chairman Mao Zedong pointed out: "Chinese medicine is a great treasure house, and efforts should be made to explore and improve it." Subsequently, a class on the study of Traditional Chinese medicine in Western medicine was widely held throughout the country, and the chairman said in his instructions on the "Western Learning" study class: "This is a major event, we cannot wait to ignore it, please actively handle it", "Western Learning" gradually formed a climax, and the study of Western medicine in Chinese medicine also became a common practice.
In 1959, Tu Youyou participated in the "National Third Session of the National Western Medicine Departure to Learn Chinese Medicine Class" held by the Ministry of Health, and systematically learned the knowledge of Chinese medicine. In the 2 and a half years of study, she not only mastered theoretical knowledge, but also participated in clinical studies. Through this study, Tu Youyou deeply felt the importance of combining TCM theory and clinical practice. She also went deep into the medicinal materials company according to her profession, to the old medicine
He studied the identification of Chinese medicine and the processing technology of Chinese medicine, and participated in the summary of the experience of Chinese medicine processing in Beijing, so as to have a further perceptual understanding of the authenticity and authentic quality of the medicinal materials, as well as the processing technology. Later, Tu Youyou participated in the research work on the preparation of traditional Chinese medicine issued by the Ministry of Health, and was one of the main authors of the book "Integration of Experience in Chinese Medicine Cannon Burning". Tu Youyou herself did not think that her background in the study and practice of these combinations of Traditional Chinese and Western medicine would lay an important foundation for her discovery of artemisinin a few years later. (Tu Youyou in 1956, see picture)
She immersed herself in the study of traditional Chinese medicine and began a career that she had worked for all her life. At that time, the fledgling Institute of Traditional Chinese Medicine had poor working conditions, rudimentary equipment, and insufficient scientific research personnel. However, the party's policy of "inheriting and carrying forward the treasure house of traditional Chinese medicine and actively developing the cause of traditional Chinese medicine" has become the goal of the majority of traditional Chinese medicine workers. It has added strength and confidence to Tu Youyou, who has just entered the workplace. Although Tu Youyou suffers from chronic diseases such as tuberculosis, she still insists on working, whether it is collecting specimens in the field or conducting experimental research indoors, she has actively completed it. For her outstanding performance in her work, in 1958, she was named an active member of socialist construction in the Ministry of Health.
In 1967, the "523" office began to organize seven provinces and cities and dozens of units across the country to jointly tackle key problems, screening more than 40,000 kinds of compounds and Chinese herbal medicines without satisfactory results. Since the 1960s, the United States has screened 300,000 compounds in response to the urgent needs of the war, and has also achieved no breakthrough results.
Under such a difficult circumstance, Bai Bingqiu and Zhang Jianfang, two directors and deputy directors of the "523" office, who had been established for two years on January 21, 1969, accompanied by Comrade Tian Xin of the office, went to the China Academy of Traditional Chinese Medicine. At that time, Gao Henian, vice president of the China Academy of Traditional Chinese Medicine, and Zhang Guozhen, deputy director of the Institute of Chinese Medicine, were responsible for the reception. The responsible comrade of the "523" office said that "the central leadership attaches great importance to the research work of antimalarial prevention and treatment, but the antimalarial of traditional Chinese medicine has done a lot of work, has not solved the problem, we have little experience, few methods, I hope you can participate in this task." The leaders of the institute immediately said that although it is still in the national cultural revolution and all the scientific research work of the China Academy of Traditional Chinese Medicine has come to a standstill, we cannot shirk our resignation and should do our best to undertake the task."
After accepting the task, the China Academy of Traditional Chinese Medicine set up a scientific research team, and the controversial comrade Tu Youyou was selected to be included (at that time, one of her uncles surnamed Yao settled in Hong Kong). On January 21, 1969, Tu Youyou of the Institute of Traditional Chinese Medicine, as the leader of the scientific research team, officially participated in the "523" project. Tu Youyou, with the foundation of modern medicine and the knowledge of traditional Chinese medicine that she has just acquired, has actively devoted herself to her work. She systematically collected and sorted out the medical records and Materia Medica of the past, went through the letters of the people since the establishment of the hospital, and consulted the famous old doctors hired from all over the country when the hospital was established. The famous Chinese medicine doctor Pu Fuzhou recommended "lightning strike scatter" and "holy scatter"; Yue Meizhong recommended "wood thief frying" and "guizhi white tiger soup". In a full two months, on the basis of bringing together more than 2,000 prescription drugs for internal and external use, including plants, animals, minerals, etc., the "Antimalarial Single Test Formula Collection" (see figure below) based on more than 640 prescription drugs was sorted out, mimeographed into a book, and sent to the National 523 Office in April 1969, please transfer it to the seven provinces and cities that undertake the task for joint excavation. In this "Antimalarial Single Test Formula Collection", there are many places of Artemisia annua, and on page 15 Tu Youyou writes:
Prescription: Artemisia annua five dollars to half a catty
Usage: Mash the juice or decoct or grind finely, boiling water and serving.
Source: Fujian, Guizhou, Yunnan, Guangxi, Hunan, Jiangxi.
Remarks: There are still other medicines in various places to treat malaria: (1) Artemisia annua three two, Ma Sha root three two (also known as false sesame) water decoction, it is said that the effect is very good. ......
Remarks on another prescription (page 14):
Seven peppers, seven almond tips, one jujube, seven sheep feces egg tips, put into dates and cooked and pounded into pills, one or two hours before malaria, one pill per serving (Shandong).
At the same time, the scientific research team led by Tu Youyou carried out experimental research on the screening of traditional Chinese medicine in the model of rat malaria, and in the first round of screening and experimental research of more than 100 samples, it was found that the inhibition rate of pepper extract on malaria parasite was as high as 84%, and the inhibition rate of Artemisia annua extract on malaria was 68%, which was not as effective as pepper. Tu Youyou started the second round of experiments, in this round of experiments, Artemisia annua only had a 40% or even 12% inhibition rate, the results were still not good, so it was temporarily put down.
The knowledge accumulated in the Western Middle School Study Class and the compilation of the "Antimalarial Single Test Formula Collection" allowed Tu Youyou to grasp a lot of information. Artemisia annua was first found in the Book of Fifty-Two Sick Remedies excavated from the Mawangdui No. 3 Han Tomb (around 168 BC), and was later included in the Shennong Materia Medica. Artemisia annua antimalariality first appeared in ge hong's "Elbow Reserve Emergency Fang" of the Eastern Jin Dynasty in 340 AD. Subsequent generations of medical texts have also been recorded. For example, in the "Compendium of Materia Medica", Li Shizhen is also called the treatment of "malaria cold and fever". From the records of successive generations of Materia Medica and Fangshu medical records, artemisia annua is affirmed by long-term clinical practice experience. Tu Youyou re-studied the "Elbow Reserve Emergency Formula", from "artemisia annua a grip, a liter of water stains, twisting juice, full service" malaria, let her realize that there may be avoidance of high temperature or enzymatic solutions and other related ideas, and the previous extraction method may be problematic. ("Fifty-two Sick Prescriptions" and Ge Hong's "Elbow Reserve Emergency Formula", see right photo) And this scientific analysis and speculative inspiration became a key part of Tu Youyou's breakthrough in artemisinin extraction.
Low temperature extraction method
The experiment, which was put aside for the time being, was restarted by Tu Youyou, this time trying to switch to a low boiling point solvent, ether, for extraction. Ether is a colorless, transparent, volatile liquid with an aromatic pungent odor. The melting point is –116.3°C. Boiling point 34.6 °C. After 384 trials, she finally found on October 4, 1971, that sample No. 191, which had been isolated, showed a surprising 100% inhibition rate against Plasmodium murine, and repeated the results on monkey malaria with consistent results. The effective site of artemisinin may be determined. Tu Youyou divides the extract into neutral and acidic parts. After testing, Tu Youyou found that the proportion of acidic parts was large and ineffective, and the toxicity was relatively concentrated; the neutral part was the effective part of antimalarial efficacy, small dose and good safety.
At this time, Tu Youyou realized that artemisinin has many other coexistence components in the raw drug, and under the condition of conventional extraction, the interference destroys the functional group with antimalarial activity in artemisinin - the oxygen bridge, resulting in the loss of antimalarial activity, resulting in the failure of screening. In addition, as far as Artemisia annua herbs are concerned, ineffective stems account for the vast majority, and only a small number of finely fragmented leaves contain artemisinin, the active ingredient for antimalarials. The relationship between the harvest season of artemisinin and the efficacy of artemisinin is also very large, and the plant only biosyntheticizes the active ingredient artemisinin in the body in the late stage of growth.
These processes were documented in the "523 Group Summary" of the Beijing Institute of Chinese Medicine on December 5, 1971, and these findings were also reported at the Nanjing Conference on March 8, 1972.
Tu Youyou's group experimented with a plasmodium berghei model. Healthy mice, weighing 18 to 22 g, randomly grouped, blood was taken by the eye extraction method of malaria rats, diluted with normal saline containing anticoagulants, and inoculated with 1×107 malaria parasites per mouse intraperitoneal injection. The drug is started 24 hours after inoculation, for 3 consecutive days, and 24 hours after the last dose, the blood smear is taken at the tail, the malaria parasite inhibition and negative transformation are observed, and the inhibition rate and half of the negative amount (ED50) are calculated. The results showed that the neutral part of Artemisia annua was given a gastric dose of 1g/kg × 3 days, which could turn the malaria parasite into a negative, and its ED50 was 2646mg/kg.
In December of that year, Tu Youyou's group experimented with plasmodium cynomolgi (plasmodium cynomolgi) model. Blood transsemination, when the parasitism rate reaches more than 10 ‰ after 8 days of inoculation, is administered orally, once a day, 8 to 9 times a day, and the blood tablets are applied every 24 hours on the day of discontinuation; or sporozoite inoculation, after the appearance of protozoa, by gastric administration, once a day, for 3 days, to observe the antimalarial efficacy of asexual bodies in the internal stage of monkey malaria red. The results showed that the neutral part had a significant antimalarial effect on monkey malaria. Both dose groups can make monkey malaria parasite yin.
Artemisinin species
Artemisia annua plant and name
A large number of experiments made Tu Youyou understand that artemisia annua varieties are also very important, and different varieties have different levels of artemisinin. When Mr. Zhao Yihuang (during Tu Youyou University, Mr. Zhao Yihuang was a professor of pharmacognosy, tu youyou used to listen to Mr. Zhao's teachings) was working at the Peking Research Institute (1934-1937), the original plant of artemisia annua in Beijing was "Artemisia annua L. f. genuine Pamp.), and pointed out that it was a foreign body with the same name as Artemisia annua L. f. genuine Pamp., which was the same name as Artemisia annua. He did not echo the erroneous conclusion reported by Japanese scholars that "artemisia annua described in the Compendium of The Compendium is quite consistent with the plant Artemisia apiacea Hance", and clearly pointed out that Artemisia annua is the "Artemisia annua " of the Materia Medica " artemisia annua " .
The Pharmacopoeia of the People's Republic of China (1963, 1977 editions, etc.) recognize Artemisia apiacea hance as the Chinese medicine Artemisia annua, which is a mistake. After the research and development of artemisinin, a new antimalarial drug from Artemisia artemisia annua L, the 1985 edition of the Pharmacopoeia of the People's Republic of China has been amended. However, the 1985 edition to the 2005 edition of the Pharmacopoeia of the People's Republic of China has always called the Chinese medicine Artemisia annua L. the Asteracea plant Artemisia arnua L., especially the 2002 edition of the "New Compilation of Chinese Medicines" has also changed the original plant Artemisia Artemisia annua L. as defined in the original 1988 edition of the "Chinese Medicine History" article on The original plant Artemisia Artemisia annua L. and then changed to the plant Artemisia arnua L., which is called to maintain the same as the 2000 edition of the "Artemisia annua L.". The Pharmacopoeia of the People's Republic of China (hereinafter referred to as the Pharmacopoeia) is consistent, also known as "medicinal artemisia annua". The chinese medicine Artemisia annua should be the plant Artemisia Artemisia annua L, and the fallacy caused by Japanese scholars should be corrected. As for Artemisia apiacea hance, Chen Chongming and others have long been examined as evil artemisia.
Artemisia annua variety and content:
There are six varieties of Artemisia annua, which are described below.
Artemisia Artemisia annua L., aliases: Artemisia annua, Artemisia annua, Artemisia annua, Artemisia annua, Artemisia annua, Artemisia annua, Artemisia annua, Artemisia vulgaris. It is an annual herb, 40 to 150 cm tall, and has a unique aroma throughout the plant. Artemisia annua is distributed throughout the country and is rich in resources, growing in wilderness, hillside roadsides, and river banks. (Artemisia annua, see right)
Artemisia artemisia apiacea Hance, aliases: Artemisia vulgaris, Artemisia annua. Annual or biennial herb, 40 to 150 cm tall, smooth and glabrous, with a green aroma. This species usually grows mostly on river banks, sandy lands and seashores; it is distributed in northeast China to south China and southwest China.
Artemisia scoparia waldst et ktiaib. One or two years old to perennial herb. The root spindle is or conical, more vertical. It grows on ditch edges, hillsides, gravel and saline land. It is found throughout the country.
Artemisia artemisia capillaris thunb. It is a perennial herb with branches of the roots, often obliquely or multi-conical and straight, but not spindle-shaped. It grows on sandy soils along beaches and coastal rivers, and rarely on hillsides in offshore areas. It is distributed in the eastern and southern coastal provinces and regions of China, and is distributed in Shandong, Jiangsu, Zhejiang, Fujian and other provinces.
Artemisia artemisia japonica Thunb. It is a perennial herb with stout rhizomes. The stem is erect, often clustered, 50 to 150 cm tall, with outstretched or erect branches in the upper part.
Artemisia artemisia eriopoda Bunge. It is a perennial herb with an erect stem, 30 to 70 cm tall, solitary or several clusters, nearly glabrous but covered with villi at the base, with inflorescence branches in the upper or lower parts.
In the process of extracting artemisinin, Tu Youyou used the authentic plant Artemisia annua from Beijing. Among the six varieties of commercial Artemisia annua, only the traditional Chinese medicine genuine Artemisia Annua L. contains artemisinin, the active ingredient for antimalarials; the origin of the same variety of Artemisia annua is also critical, and the content of artemisinin in the leaves of Artemisia annua in Beijing is extremely low, only a few in 10,000. The combination of the above factors makes the probability of the discovery of artemisinin small and small, which also shows that the discovery is not easy and precious. The wisdom of Comrade Tu Youyou and her colleagues has made outstanding contributions to the discovery of a new antimalarial drug, artemisinin.
Tu Youyou found that the artemisia annua larvae do not contain artemisinin, so there is no antimalarial activity, and the amount of artemisinic acid contained in it is gradually converted into a variety of sesquiterpene compounds with the growth time. Therefore, Artemisia annua should not be harvested early, and young plants should not be used medicinally, especially as raw materials for antimalarial drugs. The medicinal parts of Artemisia annua antimalarial are mainly leaves, and the stems and roots are ineffective, but after experimental studies, in terms of traditional antipyretic effects, leaves and stems are effective, and the effect of leaves is stronger than that of stems. In the harvest season, through the sample experiments in Beijing, Shandong, Sichuan and other places, it is advisable to take the leaf maortification period before flowering in July and August in autumn, although the flower buds contain a higher amount of artemisinin, but the buds are in full bloom, the leaves have been scarce, and the proportion of flower buds is after all small, so it is appropriate for Artemisia annua to harvest before flowering. The content of artemisinin in the young tip of the leaves is higher than that of the stem and leaf, which is consistent with the ancient literature that the use of leaf tips to treat malaria. As the storage time increases, the content decreases, so it is better to harvest and use it in the current year.
Regional collection time site artemisinin content Beijing July 12 leaf 0.10% Beijing August 10 (pre-flowering) leaf 0.12% Beijing August 14 (bud stage) leaf 0.16% Beijing August 21 (flowering stage) leaf 0.24% Beijing August 29 (flowering period) leaf 0.12% Shandong early June leaf trace Shandong early July leaf 0.02% Shandong early August leaf 0.08% Shandong early September leaf (mixed with buds) 0.12% Sichuan May 30 leaf 0.38% Sichuan June 30 leaf 0.43% Sichuan July 15 leaf 0.65% Sichuan August 30 leaf 0.56%
In fact, these areas are also at different altitudes, Sun Nianxi and others believe that Artemisia annua is widely distributed in the world, and China is distributed from the coastal zone at an altitude of 50m to the Qinghai-Tibet Plateau at an altitude of 3650m, with a variety of ecological types. In the artificially controlled environment, as long as the basic growth nutrient conditions of Artemisia annua are met, the nutrient content and growth matrix in the growth environment do not have much effect on the content of artemisinin, but the content of artemisinin in different ecological conditions in different regions is different. Different altitudes and climatic conditions vary greatly, and their changes can affect the growth and development of plants, material metabolism, functional structure, etc.
Different altitudes also had a great influence on artemisinin content, artemisinin yield and dry leaf yield, and the content and yield of artemisinin above 1000m above sea level were significantly lower than that at low altitude; the maximum period of artemisinin content accumulation above 1600m was also later than that at low altitude, which delayed the harvesting period. The suitable altitude for the growth of Artemisia annua is within 1000m, and the high temperature and low humidity environment are more conducive to the accumulation of artemisinin.
Toxicity trials and clinical trials
Toxicity tests
As a drug scientist, Tu Youyou first considered conducting an acute toxicity test, she used mice to do experiments, and obtained test data, LD50 is 7425 mg/kg. Secondly, she considered cardiotoxicity, which used mice, cats and dogs to do experiments, observing changes in heart rhythm, blood pressure and ECG ratio before and after administration, and found no significant changes. Later, she conducted liver and kidney experiments on 10 dogs, and the results showed little effect. Pathological tests on 8 dogs also showed no poisoning lesions.
Next, more critical human trials were conducted. A total of two batches of trials were organized during the experimental phase. The first batch of 3 people led by Tu Youyou, approved by the leadership, were admitted to the Dongzhimen Hospital affiliated to the College of Traditional Chinese Medicine, which was called "pathfinding trial service" at that time. The dose starts at 0.35 g per person and is increased sequentially to 0.5 g, 1.0 g, 2.0 g, 3.0 g, 4.0 g, 5.0 g once daily for seven days. In order to catch up with the malaria season, in August 1972, Tu Youyou, together with the medical team of the Chinese Medicine Research Institute led by Dai Shaode, rushed to the Changjiang Malaria District of Hainan Province to conduct clinical research. Considering the flexibility of the clinical drug regimen, in order to fully display its antimalarial efficacy and strive for the purpose of winning the first battle, a second trial of increasing the dose was organized, a total of 5 people, the dose was 3.0g each time, 2 times a day, and served for 3 days. Before, during and after taking the drug, ecglyctic, liver function, kidney function, chest X-ray, blood routine, urine routine, fecal routine and other examinations are carried out.
The results showed that after taking the drug, the blood and urine routine were normal, the kidney function was in the normal range, the urea nitrogen was normal, the chest X-ray, the electrocardiogram were normal during and after taking the drug, there was no obvious change in blood pressure, the fundus was normal, the visual field did not change much from the control before taking the drug, the body temperature and pulse were normal, and no symptoms of the respiratory system, urinary system and central nervous system were found clinically. The toxic side effects are: mild gastrointestinal symptoms, two cases of abdominal pain occurred 1 hour after taking the drug, but it was not severe, and it healed without treatment. Animal toxicity experiments and human trial observations showed that the neutral part of Artemisia annua had no significant effect on other organs except for a few animals with mild or transient effects on the activity of liver transaminases.
clinical trial
After the successful excavation of artemisinin antimalarial in 1971, in accordance with the arrangement of the "523 Office" on March 8, 1972, at the National Malaria Prevention and Control Drug Professional Conference in Nanjing, Comrade Tu Youyou, on behalf of the research group, reported the comprehensive results with the title of "Using Mao Zedong Thought to Guide the Work of Antimalarial Chinese Herbal Medicine", and the whole audience was excited, and the meeting asked the Institute of Chinese Medicine to apply artemisinin to the clinic and comprehensively observe the efficacy.
In the process of clinical trials, Comrade Tu Youyou took the lead in trial administration, observed toxicity, and personally carried the drug to the scene of Changjiang Malaria District in Hainan Province, verified 11 cases of vivax malaria, 9 cases of falciparum malaria, and l cases of mixed infection, and as a result, after the patient took the drug, the high fever of 40 degrees quickly dropped to normal, and the malaria parasite was greatly killed to yin, and the efficacy was significantly better than chloroquine. In the same year, 9 cases were verified in Beijing 302 Hospital, and all of them achieved good efficacy. At the national "523" conference held in Beijing on November 17 of the same year, Comrade Tu Youyou reported for the first time that all the effective efficacy of 30 cases of Artemisia annua antimalarial was summarized.
On 5 November 1972, the "Exchange of Research On Malaria Control" was also devoted to this.
Since then, Tu Youyou's group has caused a national climax in the study of Artemisia annua antimalarial. In particular, In Shandong and Yunnan, in early 1973, the two institutes sent letters to the Institute of Chinese Medicine of the China Academy of Traditional Chinese Medicine, requesting further clarification of the original plant varieties of Artemisia annua, and to understand the types of antimalarial active ingredients, clinical efficacy, toxic side effects, etc. The Institute of Chinese Medicine has given selfless support, and on the basis of the reply letter, it has further carried out the anti-malarial work of Artemisia annua, and the research has gradually deepened.
In 1973, the Shandong Institute for the Prevention and Control of Parasitic Diseases issued an official letter to the Institute of Traditional Chinese Medicine, which read as follows.
Institute of Pharmacology, Beijing Academy of Traditional Chinese Medicine:
I have learned from your advanced experience, last year the application of artemol ether and alcohol extracts produced in this province to treat malaria, through animal tests, to obtain good results, this year is ready to continue to do a small number of clinical trials for observation. According to the introduction of the Office of the National Malaria Research Leading Group, your institute has used Artemisia annua for clinical trials in 72 years, and has made new progress in the research of the drug. In order to further learn from you, the National Malaria Research Office introduces us directly to you, please inform you of your valuable experience in animal toxicity tests, ingredient analysis and clinical trial preparations, dosages, effects, etc., and your experience will blossom and bear fruit in our province.
Sincerely
Revolutionary salute
Shandong Institute for Parasitic Disease Control
March 14, 1973
In August 1973, during the malaria season, Tu Youyou once again brought the artemisia monomer isolated and purified from the effective site to the Nanjiang Malaria District of Hainan for clinical experiments, confirming that artemisinin was an active ingredient in the antimalarial component of Artemisia annua (this time the clinic encountered pre-contraction of the heart, reducing the original plan of 14 cases to 8 cases).
In April 1973, Luo Zeyuan, Zhan Eryi and others of yunnan pharmaceutical institute also learned from the Institute of Traditional Chinese Medicine, using the local artemisia brassica, and extracted and analyzed the effective monomers with ether. In 1975, Li Guoqiao of the Guangdong Acupuncture and Moxibustion Group coordinated by the national "523 Office" to conduct clinical observation of 18 cases with extracts from Yunnan in gengma area, of which 14 cases were falciparum malaria, and the efficacy was very good.
Shandong Institute of Parasitic Disease Control and Shandong Institute of Traditional Chinese Medicine, through learning, they also proposed effective monomers in November 1973 and participated in the subsequent collaboration. The two units worked together to observe the clinical use of 30 cases of vivax malaria patients in Juye County from August to September 1973, which had a strong killing effect on malaria parasites, and the effect of controlling clinical symptoms was rapid, and no obvious side effects were seen. Zhu Hai and Wei Zhenxing of the institute have done a lot of work in the future industrialization.
Scientific truth and its evidence must be able to withstand experimental repetition, and both Shandong and Yunnan were extracted with ether, and not only were the experiments repeated, but the same results were obtained.
Active parts and active ingredients
The research on artemisinin in the Institute of Chinese Medicine of the Chinese Academy of Chinese Medicine first conducts a study of the effective part, followed by the study of the active ingredient. The active ingredient is the extraction of artemisinin crystals, and Tu Youyou's team isolated and determined the active ingredient on the basis of the original. The specific method is to use ether neutral part of the extract mixed with polymillamide, with 47% ethanol filtration, leachate concentrated under reduced pressure, concentrate with ether extraction, the resulting ether extract, through silica gel column chromatography, first with petroleum ether, and then with 10% ethyl acetate - petroleum ether, 15% ethyl acetate - petroleum ether elution, successively to obtain yellow oil, clear color oil, artemisinin. The potency of artemisinin, artemisinin, black oil and other components shows that only artemisinin (dose 50 to 100 mg/kg) is effective, and artemisinin (dose 100 mg/kg) and artemisinin (dose 400 mg/kg) have no effect. However, artemisinin is matched with the same amount of artemisinin, and the dose of artemisinin is reduced by half, which can make the malaria parasite turn negative. But it has not been tried for clinical use.
Due to the large number of impurities, complex extraction methods and large amounts of ether used in Beijing, it is not suitable for mass production, and Tu Youyou's team also used "dilute alcohol extraction method" and "solvent gasoline extraction method".
On November 8, 1972, it was isolated and purified from the neutral effective part of Artemisia annua to obtain an effective antimalarial monomer, which is a white needle crystal, with a melting point of 156~157 °C, and 50~100mg/kg can turn the plasmodium into the yin, named artemisinin. After animal safety experiments, human trials, in August 1973 it was made into a tablet, to The Nanchang Jiang malaria area of Hainan for clinical verification, the initial trial of 5 cases, although 3 cases are effective, but the effect is not ideal, after timely investigation of the cause, found that the tablet disintegration degree is problematic. At that time, during the Cultural Revolution, the preparation room of the Institute of Chinese Medicines did not resume business, so it was sent to an outside unit for production, and the degree of disintegration was not measured. Immediately with artemisinin raw powder into the capsule, by the deputy director of the firm's business Zhang Guozheng again to Hainan Changjiang Malaria District for clinical efficacy observation, for the treatment of 3 cases, the total dose of 3 ~ 3.5g, all effective, the average protozoa yin time of 18 • 5h, the average antipyretic time of 30h, confirming that artemisinin is the active ingredient of Artemisia annua antimalarial. From 1978, the Artemisinin Identification Association completed the verification of 529 clinical cases. After returning to Beijing in October, the results were reported to the "523 Office" in a timely manner, and the "523 Office" wrote to the Institute of Traditional Chinese Medicine on November 2, 1973, informing the convening of the "Professional Conference on the Research of Malaria Prevention and Control Drugs (Including Chemical Synthesis)" to discuss the "problem of finding new drugs in combination of traditional Chinese and Western medicine", and especially proposed that "Artemisia annua is a key drug, please bring relevant information to the exchange". Tu Youyou of the Institute of Traditional Chinese Medicine reported again on the research of artemisinin as required.
At the end of 1972, after attending the annual meeting of heads of the 523 offices in various regions in Beijing, Fu Liangshu, director of the Kunming District 523 Office, conveyed this information to the Yunnan Provincial Institute of Materia Medica about the discovery of artemisinin in the Beijing Institute of Traditional Chinese Medicine, and suggested that they take advantage of the favorable conditions of the abundant local plant resources to conduct extensive screening of artemisia annua plants. In April 1973, Luo Zeyuan, a researcher at the institute, used "Artemisia annua large-headed" to extract ether to isolate effective monomers, named "artemisinin". The monomer was found to have rapidly disappeared through the institute's rat malaria suppression test. In October 1973, they completed preliminary studies on the corresponding drug burial and toxicity. In addition, they also found that the content of Artemisia annua in Youyang, Sichuan, was 0.2%, which was 10 times higher than that of Yunnan.
In November 1973, the Shandong Institute of Traditional Chinese Medicine extracted an effective monomer from the local artemisia arbusus and named it "Artemisinin", and its melting point was initially determined to be 149~151 °C. In 1974, in the clinical trial in Juye County, it showed high efficiency, and compiled the "Preliminary Observation of Artemisinin and Artemisia Acetonin Extracted Simple Dosage Forms for the Treatment of Patients with Vivas malaria".
Nanjing Conference and Great Collaboration
On March 8, 1972, Tu Youyou, on behalf of the research group, reported the comprehensive results with the title of "Using Mao Zedong Thought to Guide the Work of Antimalarial Chinese Herbal Medicine", the whole audience was excited, the report used a large number of facts and experimental data, the antimalarial effect of Artemisia annua was affirmed and recognized, and October 4, 1971 became a memorable day in history. The following is an excerpt from the original text.
"Screening for effective antimalarial drugs in the laboratory is not a smooth sailing for us, but a process of repeated practice and continuous improvement."
"Our work has screened nearly 100 kinds of Chinese herbal medicines since mid-July, but the potency is not high, the slightly better inhibition rate is only 30 to 40%, and many of them have negative results, which makes us think deeply."
Chairman Mao's teaching on "practice, understanding, re-practicing, and re-understanding" has given us great encouragement and enlightenment, prompting us to further energize us in the difficulty of screening "failures.", and the facts have proved that "a correct understanding often needs to go from material to spiritual, from spirit to matter, that is, from practice to understanding, from understanding to practice." It can only be done after many iterations." ”
"Through the observation of antimony gluconate sodium gluconate with relatively high efficacy and relatively pure composition of rat malaria, it was found that the effective dose had a great relationship with the titer, and the inhibition rate of 10 mg/only ·times was 99%, while the result of giving 0.5 mg/only ·time was 17%, the result of giving 2 mg/only ·time was 35%, and the result of 5 mg (half) was 40%. This shows that even a drug with a relatively high potency. When the dose is insufficient, it can also have bad results, or even complete ineffectiveness, with negative results. Generally speaking, don't say negative results, that is, the inhibition rate of 30 to 40% is also easy to ignore the past and be screened out. This is still the case with chemical drugs of higher purity, not to mention Chinese herbal medicine. The composition is complex, there are many impurities, when we have not yet mastered its objective law, it is likely that due to improper methods, the active ingredients cannot be well concentrated, so that they cannot reach the level of sufficient to fully display the potency, which makes the screening results not reflect the titer as they should be. Through this enlightenment, we recognize the inefficient and ineffective results of the treatment experiment. It should be analyzed from a dialectical point of view, under certain conditions, inefficient or even ineffective results may have factors that are transformed into efficient. We therefore conducted a further analysis of the drugs that showed inefficiency and ineffectiveness in the previous work. Is it possible to mask the properties of drugs because we can't work at home? Is it possible to further increase the potency of a drug by exploring the objective law of its efficacy through our repeated experiments, too careful work? ”
"Through multi-host analysis, we selected a part of the drug and further re-screened. When re-screening, we refer to the experience of folk medicine, improve the extraction method and add multi-dose groups to explore the relationship between drug dosage and titer. After repeated practice, the animal titer of Artemisia annua was finally increased from 30 to 40% to 100%, and the decoction of Artemisia annua was ineffective. The potency of 95% ethanol extract is also not good. Only about 30 to 40%, and later inspired by the use of Materia Medica and folk "stranded juice", we took into account that the active ingredient may have temperature interference. Therefore, it was switched to ether extraction. In this way, the animal potency has increased significantly. After comparison, it was found that although the ethanol extract also contained the substance extracted from ether, the impurities were about 2/3 more, which greatly affected the active ingredient to fully display the due potency. In addition, the harvest season of the drug also has an impact on the potency, and we have taken a detour in this regard. At the beginning, we only paid attention to the variety problem, and we learned that the artemisia annua sold in Beijing is a close-produced artemisia annua in Beijing, and there is no variety problem, so we are very relieved. Later, according to the amount of raw drug, the potency is unstable, only to notice that the original harvest season is different, the extract content is different, so there is a problem in measuring according to the raw drug. Instead, the potency was stabilized by the amount of the extract. All these show that there are many factors that affect the valence, and we need to carry out meticulous and thorough work. ”
"The titer of artemisin ether extract is increased, but the toxicity is still large, and the safety factor is still relatively low." This is the main contradiction in further work, and we must make every effort to grasp this contradiction and resolve this contradiction before clinical application. We recognize that there is often a dialectical relationship between the efficacy and toxicity of a drug, especially insecticidal drugs, which are more likely to encounter the problem of toxicity. However, the composition of Chinese herbal medicine is complex, and through further detoxification, it is possible to reduce toxicity and continue to improve the efficacy. After the exploration of separation and extraction, we found that a large number of acidic parts removed with sodium hydroxide are exactly the parts that are ineffective and toxic. The neutral part that remains is not only significantly less toxic, but also more expensive per unit. Further removal of drug toxicity provides more favorable conditions for the transition from laboratory work to clinical practice. ”
"Our work is still very little, it is only preliminary, and laboratory work must be closely integrated with the clinic. For a drug, the real test is clinical. ”
After the good clinical results of Changjiang in August 1972, the status of the Institute of Chinese Medicine of the Chinese Medicine Research Institute gradually improved, coupled with the active participation of various localities, and exciting results were achieved. In the autumn of 1973, Zhou Keding, the head of the National 523 Office, and Zhang Yanzhen of the Beijing Institute of Chinese Medicine learned about the work in Yunnan; in November 1973, Shi Rinrong of the National 523 Office and meng Guangrong of the Beijing Institute of Chinese Medicine learned about the work in Shandong.
From February 28 to March 1, 1974, the "523 Office" entrusted the Institute of Chinese Medicine to preside over the National Artemisinin Antimalarial Collaboration Conference, and according to the requirements of the "523 Office", the laboratories of artemisinin research of the Institute of Chinese Medicine of the Chinese Academy of Chinese Medicine were disclosed, and the laboratories of the Institute of Chinese Medicine of the Chinese Academy of Chinese Medicine were disclosed for detailed introduction and field visits, forming a national cooperation situation.
In 1974, after the Institute of Traditional Chinese Medicine hosted the cooperation, the team continued to expand, in addition to Shandong, Yunnan, Sichuan, Guangxi, Guangdong and other provinces continue to participate, according to the 1978 artemisinin identification meeting data, as many as 40 participating units, there are 10 provinces, municipalities and autonomous regions involved in the clinic, a total of 6555 cases of verified cases, artemisinin preparations have 2099 cases.
From the launch of the "523 Task" in 1967 to the end of the "523 Task" in 1980, the research of artemisinin has achieved fruitful results: in 1967, the research and development of new antimalarial drugs was launched; in 1971, Tu Youyou's group found that Artemisia annua antimalarial has a special effect; in 1972, the group extracted artemisinin monomer crystals; in 1973, Shandong and Yunnan extracted artemisinin monomer crystals; in 1974, the clinical confirmation of artemisinin antimalarial efficacy is remarkable, artemisinin structure discovery; 1976, In 1977, the research results of artemisinin antimalarial drugs were announced to the world; in 1979, the State Science and Technology Commission issued invention certificates to six units, including the Institute of Chinese Medicine of the Chinese Academy of Traditional Chinese Medicine and the Institute of Traditional Chinese Medicine of Shandong Province.
The characters involved are: Tu Youyou, who discovered artemisinin; Li Guoqiao, who further confirmed the antimalarial efficacy of artemisinin; Liang Li and Zhou Weishan, who measured the structure; and Li Ying, who synthesized artemether. In addition, there are Zhou Yiqing, Wei Zhenxing, Luo Zeyuan, Liu Xu, Deng Rongxian, Teng Qihe, and so on, who have done a lot of work in other aspects.
Although generally speaking, in modern society, large scientific research projects must adopt the organizational mode of collective cooperation, and the management mode of large projects and the teamwork spirit of relevant personnel will inevitably become important factors affecting the research and development results, but for the research and development of artemisinin, the role played by the "523 task" in a specific medical research project in a specific environment must still be highly evaluated and realistically affirmed.
In the process of the discovery of artemisinin, the group of scientists did not distinguish between each other, did not fortify each other, exchanged information, helped each other, divided labor and cooperation, and fully demonstrated the collectivist spirit of public and selflessness, "seeing the cause as heavy as a mountain, and seeing fame and fortune as light as water". Outstanding talents in various disciplines and fields have broken the research mode of each discipline being closed to each other and fighting for each other, and formed a strong research and development synergy.
Under the background of the "Cultural Revolution", the relatively independent, capable, and cross-departmental leadership forces gave organizational guarantees and coordination guarantees, making government decrees smooth and responsive.
"Breakthrough" innovation has led to countless "cumulative" innovations, which is also a major feature of engineering innovation. After the discovery of artemisinin, the process of extracting artemisinin crystals has been continuously transformed, and Yunnan and Shandong have actually become rising stars in artemisinin extraction technology and technology. Various derivative studies and compound drug studies have sprung up like mushrooms.
(Author Zhang Wenhu)