|Year : 2022 | Volume
| Issue : 4 | Page : 514-521
Ginkgo biloba waste resources and waste treatment combined with modern progress and development model
Department of Traditional Chinese Medicine, Haiyuan College of Kunming Medical University, Kunming, Yunnan Province, China
|Date of Submission||18-Aug-2021|
|Date of Acceptance||16-Sep-2021|
|Date of Web Publication||09-Sep-2022|
Ms. Ying-Si Zhong
Haiyuan College of Kunming Medical University, No. 389 Haiyuan North Road, Hi-Tech Industrial Development Zone, Kunming, Yunnan Province
Source of Support: None, Conflict of Interest: None
Background: Ginkgo biloba L. is listed in the Red List of Endangered Species by the International Union for Conservation of Nature. G. biloba is an important medicinal plant in China and can be widely used in materials, gardens, and as a source of nutrients. With the large-scale planting of ginkgo plants, China, accounts for more than 70% of the world's total gingko output. Currently, G. biloba P. E. is the main extract under product development and application. However, G. biloba has been discarded as waste for a long time and has not been well developed and utilized. According to incomplete statistics, nearly 40,000 tons are discarded in China every year, which not only wastes resources but also pollutes the environment. Objective: This is an issue of great significance and adds value to scientific research. We aim to develop a key technology for resource recycling by combining G. biloba waste resources and waste treatment. Methods: Data were obtained by searching databases such as CNKI, and analyzing the herb application, modern application, main chemical components, utilization of waste parts, ways and modes combined with waste treatment, and safety of G. biloba. This systematic analysis can serve as a reference for the recycling of waste resources in other fields. Conclusion: The chemical constituents of the outer seed coat of G. biloba mainly include hydrophenols, phenolic acids, and biflavones, among which the phenolic acid of G. biloba can be used to extract glycolic acid, which can inhibit Mycobacterium tuberculosis. The domestic waste produced by tuberculosis (TB) patients contains a large number of bacilli, and the incidence of transmission can be reduced by using glycolic acid to inhibit the growth of TB bacilli in the waste.
Keywords: Chemical composition, Ginkgo biloba outer seed coat, Mycobacterium tuberculosis, pharmacological action, resource recycling
|How to cite this article:|
Zhong YS. Ginkgo biloba waste resources and waste treatment combined with modern progress and development model. World J Tradit Chin Med 2022;8:514-21
|How to cite this URL:|
Zhong YS. Ginkgo biloba waste resources and waste treatment combined with modern progress and development model. World J Tradit Chin Med [serial online] 2022 [cited 2022 Dec 1];8:514-21. Available from: https://www.wjtcm.net/text.asp?2022/8/4/514/355593
| Introduction|| |
Ginkgo biloba L. is a deciduous tree plant belonging to Ginkgoaceae and Ginkgo. It is also known as ginkgo, Gongsun Tree, duck foot tree, and duck palm tree. G. biloba is an important medicinal plant in China and can be widely used in materials, gardens, and as a source of nutrients. Ginkgo has a long history, spanning nearly 1000 years of cultivation, and is mainly distributed in China's Jiangsu, Guangxi, Guilin, Shandong, Zhejiang, and Hubei regions.
A large number of scientific studies have shown that G. biloba L. is full of resources. The ginkgo fruit, exoseed coat, and root contain a large number of active substances, which have a unique medicinal value.
In the Compendium of Materia Medica written by Li Shizhen in the Ming Dynasty, it is recorded that cooked food can warm lungs, nourish qi, calm asthma, reduce constipation, and stop white turbidity, while raw food can be used to reduce the phlegm and kill insects. In Modern Practical Chinese Medicine, it is mentioned that: “Kernel treats asthma, dizziness, tinnitus, chronic gonorrhea, and women's dysbiosis.” Ginkgolic can affect the respiratory system by relaxing the smooth muscle of the trachea, relieving cough and asthma, and clearing lungs and expectorant. Its effects on the circulatory system include dilating microvessels and preventing cardiovascular and cerebrovascular diseases.
Ginkgo mainly plays an important role in cardiovascular and cerebrovascular diseases, as it can improve circulation by dilating coronary arteries and improving atherosclerosis. Correspondingly, ginkgo can be used in hypertension, myocardial ischemia, and brain dysfunction. In addition, it can also remove free radicals and lower serum cholesterol.
The outer seed coat of G. biloba contains Hydrogenated Ginkgolic acid, phenols, flavonoids, polysaccharide compounds, and other effective components which function in bacteriostasis, sterilization, anti-inflammation, immunity enhancement, free radical scavenging, antitussive, anti-aging, and apoptosis of cancer cells.
In “Chongqing Herbal Medicine” gingko is reported to be used in the “qi tonifying weakness, treatment of leukorrhea, spermatorrhea, and other weak strain injury and other diseases.” “Chinese Medicine Sea” records that gingko is “used to fill deficiency and stop heritage.” “Xinhua Compendium of Materia Medica” says: “Gan, wei Ping.” The ginkgo root can be used to treat urinary system diseases, improve blood flow, lower blood pressure, lower blood lipids, prevent thrombosis, delay aging, improve memory decline in the elderly, relieve male impotence symptoms, and prevent hearing loss among other functions.
| Methods|| |
Applications of Ginkgo biloba L.
The age of the ancient ginkgo trees scattered around the area is presumed to be: G. biloba has been planted since the Shang and Zhou dynasties in China. After that, the planting of G. biloba has gradually increased and its scope has gradually expanded. During the Yuan Dynasty, gingko was widely used in medicine and first recorded under “Daily Herbs” in the annals of Traditional Chinese Medicine compiled by the Institute of Materia Medica, Chinese Academy of Medical Sciences (1984). It was named because G. biloba was solid and landed, with rotten flesh and only seed kernel. G. biloba L. was mentioned as a medicine in the Shaoxing Materia Medica, written by Wang Jixian, which studied the use of medicine in the Southern Song Dynasty. This indicates that at least in the Southern Song Dynasty, G. biloba was included in the pharmacopoeia and became a traditional Chinese medicine. The above results indicate that G. biloba has been cultivated since ancient times in China, and the G. biloba kernel and pericarp have medicinal properties.
Modern application of Ginkgo biloba L.
In modern times, G. biloba has been widely used in many fields. In terms of drug application, ginkgo seeds, nuts, and ectoderm have been used since ancient times. With the continuous development of science and technology, scientists have studied and explored its use in depth, and ginkgo and ginkgo pollen also have a high medicinal value. The planting of modern G. biloba is more extensive. According to the statistical data of the China Rural Technology Development Center in 1977, the annual output of G. biloba in China is about 15,000 tons, of which Jiangsu has the greatest production.
The main chemical constituents of the outer seed coat of Ginkgo biloba waste
At present, the main chemical components of the outer coat of G. biloba are flavonoids, lactones, phenolic acids, polysaccharides, and amino acids.
G. biloba contains more ginkgo flavonoids, which are bibrass chemical components, mainly concentrated in the outer seed coat.,, Yuexin et al.,, measured the flavonoid content at 1.3%, including G. biloba, iso G. biloba, jinpinbiflavone, 1-5-Methoxyleukophorin and Leukophorin. It can be concluded that G. biloba can be used in clinical practice. Modern pharmacological studies have found that the effective components of flavonoids are composed of monomers, which is the key to clinical efficacy.
Based on current reports, the outer coat of G. biloba contains lactones. Fengchang et al. separated eight crystals with the mixture of ginkgolides A and B and Ginkgolides C as the main chemical components by using medium limit solvent and nonlimit solvent, and the average content of total lactone was 5.6% measured by Wu.
Understanding and development of ginkgo phenolic acid have received increased attention in recent years. It has been found that Ginkgolic acid is abundant in the outer seed coat of G. biloba Xuewen and Moucheng reported that the content of glycolic acid monomers was highest, followed by hydrogenated glycolic acid. In modern pharmacological studies, ginkgo phenolic acid has good medicinal value, which provides a basis for clinical use. The gingko phenolic acid structure is shown in [Figure 1].
In recent years, the positive effect of plant polysaccharides on cancer treatment has been clinically confirmed. Mao determined by orthogonal design that the conditions for extraction of polysaccharides were a temperature of 100°C, material ratio 1:12, for 1 h, repeated three times. The extraction rate of polysaccharides from the outer seed coat reached 95.09% and the yield of crude polysaccharide was 23%. In terms of the significance and value of resource industrialization, G. biloba polysaccharide is rich and has high developmental value.
Biyu and Yumei determined 17 kinds of amino acids in the outer seed coat of G. biloba using an amino acid automatic analyzer. The outer seed coat had an amino acid content of 7.115%.
Overview of the discussion
In summary, Guoyan et al. isolated nine compounds from the main chemical constituents of the outer seed coat of G. biloba: ginnol [Figure 2], palmitone [Figure 3], β-sitosterol [Figure 4], stigmast-3,6-dione [Figure 5], stigmast-4-ene-3,6-dione [Figure 6], ginkgolic acid [Figure 7], ginkgoneolic acid [Figure 8], triacotanoic acid [Figure 9], and ginkgonine [[Figure 10], Note: The figure is a structural formula pieced together by Guoyan et al. based on the fragment inferred from it speak shape].
Analysis of the inhibitory effect of glycolic acid from the outer pericarp of Ginkgo biloba on Mycobacterium tuberculosis and fungi in domestic waste
Bacteriostasis, sterilization, insecticidal effect
The exocoat of G. biloba has bacteriostatic and bactericidal effects, which is also the basis for the combination of G. biloba waste resources and waste treatment in this paper. This effect is mainly related to Ginkgolic acid [Figure 7] and Ginkgoneolic acid [Figure 8] compounds isolated from the outer seed coat. Based on reports by Xuewen and Moucheng, Kubo et al., etc.,, we know that glycolic acid has strong bacteriostatic or bactericidal effects, especially against Staphylococcus aureus, dysentery bacillus, Pseudomonas aeruginosa, and other fungi and bacteria. The effective rate of fungi inhibition is 92%, which can inhibit tuberculosis (TB) bacilli.
The exocoat has the most outstanding inhibitory effect on pathogenic fungi, and the expression of acidic components in the outer coat after purification is significant. Chenglin an Jianfeng reported that G. biloba, a purified component of the outer seed coat, has a good inhibitory effect on 25 kinds of pathogenic fungi, and phenolic acids rich in components have a good prospect as plant antifungal agents. The daily household garbage can easily cause M. tuberculosis infection. The acidic components in the outer seed coat of G. biloba have a good inhibitory effect. Qiu and Ruhua reported that glycolic acid had inhibitory effects on human TB and Bovine TB in vitro, and ginkgo extract given via intragastric administration at 150–200mg/(kg·d) had significant therapeutic effects on guinea pigs infected with human TB.
Second, polysaccharides from the outer seed coat of G. biloba can also express good antibacterial effect. Ziheng et al. confirmed that the MIC of polysaccharides against S. aureus was 1.563mg/mL, showing good inhibitory activity.
In summary, it can be concluded that G. biloba phenolic acid can inhibit M. tuberculosis. The domestic waste produced by TB patients contains a large number of TB bacilli. Transmission can be reduced using glycolic acid to inhibit the growth of TB bacilli in the waste.
In addition, Xue-Wen et al. studied the inhibitory effects of ginkgo acid on maize big spot disease, barley stripe disease, and rice sheath blight. The inhibition rates were found to be above 70%. Jie et al. concluded that ginkgolic acid was the component with a significant inhibitory activity at a concentration of 1.90 × 10− 3, for which the corrected mortality rate reached 81.6%. Lichun et al. reported that petroleum ether extract and ethanol extracts of the G. biloba ectoderm had inhibitory effects on most experimental fungi (such as Microsporidium rusticolor, Tinea divaricum, and Microsporidium lanticulum), and their antibacterial efficiency was 81% for ethanol extract, 73% for petroleum ether extract, and 74% for clotrimazole. A concentration of 0.5% of G. biloba exfoliar intermediate had the same inhibitory effect on fungi as 0.5% of clotrimazole. Therefore, petroleum ether and ethanol extracts can be used in early scientific research experiments on a large number of fungi, to inhibit growth and to achieve the purpose of eliminating experimental interference.
Study on other functions of the outer seed coat of Ginkgo biloba in abandoned plant parts
Anti-tumor, free radical scavenging, anti-aging effect
It was reported that ginkgo acid, ginkgo phenol, and ginkgo diphenol could be isolated from the chloroform extract of the G. biloba ectoparum from the Liuqing et al., with a ratio of 3:2:2. These substances can inhibit the growth of human cells and sarcoma S180 cells in mice to a certain extent. It can be concluded that the outer seed coat possesses an anti-tumor effect. On the other hand, some scholars have proved through experiments that ginkgo acid can fully inhibit enzyme activity. The inhibition rate of hyaluronidase was reported to be more than 90%, which proves that the outer seed coat of G. biloba plays a role in scavenging free radicals.
Anti-inflammatory and anti-allergic effects
Hongquan et al. confirmed the anti-allergic effect of water-soluble components of G. biloba ectoparum at a concentration of 100 mg/kg or 200mg/kg by means of passive allergic skin reaction (PCA) in mice, degranulation of cranial perichondrium mast cells in rats, and sensitization of the ileum in guinea pigs. As shown in [Figure 11] (Data cited from Dongya), with the increase in concentration of G. biloba ectocarpus extract, the inhibitory effect of fungi was enhanced, which also proved that G. biloba ectocarpus extract had a significant inhibitory effect on fungi and an anti-inflammatory effect.
|Figure 11: Relationship between the concentration of the outer seed coat of Ginkgo biloba and the inhibitory fungal rate|
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Influence on immune function
Proteoglycans have always been of interest because of their anti-tumor activity as a natural substance. They have been widely used in the immune regulation mechanism. However, whether polysaccharides in the outer coat of G. biloba can promote the cellular immunity mediated by T-lymphocytes to achieve the anti-cancer effect is still unclear.
After reading a large number of reports, we learned from Liuqing et al. that not all polysaccharides from the G. biloba exocoat can improve the activity of immune cells. For example, the immune activity of deproteinized crude polysaccharides was greater than that of underproteinized polysaccharides. Therefore G. biloba can kill tumor cells and enhance immune function. Koch et al. injected G. biloba extract intraperitoneally into mice, and the lymphocyte tissue of the mice showed a hyperplastic reaction, proving the immunotoxic effect of the outer seed coat of G. biloba. However, in addition to the effect of polysaccharides on immune function, the role of other components, such as ginkgo phenolic acid, should not be underestimated. Xuewen and Moucheng reported that G. biloba has an inhibitory effect on both cellular and humoral immune functions.
Mutagenic and carcinogenic effects
A synthesis of what is known from reports by Xiangyang and Luqing et al., shows whether G. biloba can promote tumor growth in the body was determined using a mouse model. Experimental data show that G. biloba not only promotes tumor growth but also exhibits mutagenicity and carcinogenicity.
Ginkgo flavones have long been popular for their cardiovascular benefits. The main chemical constituents in G. biloba leaves are flavonoids and terpene lactones. Among them, flavonoids are effective for the treatment of cardiovascular and cerebrovascular diseases, which can be divided according to structure into: single flavonoids, such as quercetin; double flavonoids, such as ginkgo biflavonoids, etc.; and catechins. However, flavonoids and lipids from the outer coat of G. biloba leaf can also play this role.
Based on the reports of Xiangyang et al., Qiangqiang et al., and Ren,,, it was found that flavonoids and lipids in the outer pericardium of could be extracted and separated by macroporous resin adsorption, and the extracts could be used in the production of drugs for cardiovascular and cerebrovascular diseases [Figure 12].
|Figure 12: Extraction and separation process of the outer seed coat of Ginkgo biloba|
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Zhang reports that the chemical composition of G. biloba ectoparum is similar to that of G. biloba leaves, and also contains flavonoids. Experiments on mice also found that the ectoparum of G. biloba can significantly inhibit the cardiovascular function of mice, reduce oxygen consumption, and prolong the survival time of mice under hypoxia. It is well known that flavonoids are effective components for the treatment of cardiovascular diseases, thus proving that G. biloba can be used for the prevention and treatment of cardiovascular diseases.
Additives for daily chemical products
Xuewen and Moucheng reported that G. biloba contains ginkgo phenolic acid, which has been proven to inhibit the activity of hyaluronidase and remove free radicals to protect skin and increase whitening luster. Thus, it can be used as an additive for daily cosmetics.
| Conclusion|| |
Path analysis of the comprehensive development of G. biloba plant resources
This study comprehensively analyzed the herb application and modern application of all available parts of G. biloba (medicinal and nonmedicinal), and found that G. biloba seed kernel and G. biloba seed coat were mainly used as medicinal parts in ancient China. However, with the development of the times, a series of problems have been brought about. Modern studies have shown that the medicinal value of ginkgo and ginkgo pollen has been excavated, and the medicinal value of ginkgo pollen has replaced the medicinal value of the outer seed coat of ginkgo. As a result, ginkgo seeds harvested by humans have piled up, causing the outer seed coat of ginkgo to rot. This presents G. biloba as a waste, abandoned and unused status quo. As a result, during the ginkgo harvest season, the outer seed coat becomes a source of pollution in streams, ponds, and weirs in ginkgo production areas.
After a large number of literature search, a large number of scholars have found that the outer coat of G. biloba mainly contains flavonoids, lactones, phenolic acids, polysaccharides, amino acids, and other chemical components, which have antibacterial, bactericidal, and insecticidal effects; anti-tumor, free radical scavenging, anti-aging effects; anti-inflammatory, anti-allergic effects; and immune function effects.
In conclusion, the outer seed bark of G. biloba is not an unrecoverable resource. Because the author wants to collect and treat a large amount of garbage in a more green and efficient way, the outer seed coat of G. biloba has bacteriostatic and bactericidal effects, and the phenolic acid of G. biloba can be extracted to inhibit TB bacillus. In this way, the combination of the two, not only does not waste resources but also solves the dual problem of environmental pollution by reducing ginkgo pollution in streams, ponds, and weirs. This also brings more innovative and environmentally friendly ideas for sorting, collection, and disposal of waste, thus reducing the probability of the spread of infectious diseases. Therefore, we proposed that for the domestic waste produced by TB patients, we may use glycolic acid to inhibit the growth of TB bacilli to reduce the probability of transmission. Therefore, the ways to research and develop the technology and system of waste resource recycling, with more innovative and environmental protection ideas, can not only provide certain reference for the recycling of waste resources in other fields but also have great significance and value for promoting the “green” development of the country and realizing a “healthy China.”
Ginkgo biloba L. development and utilization patterns and safety considerations of plant waste resources
Development model based on main functional active compounds from waste resources of Ginkgo biloba
Based on the “green,” “sustainable use,” and “healthy China” idea, the authors report modern application of ginkgo waste resources. We put forward a model of ginkgo resources, waste resources, and recycling development from the exopleura of G. biloba L., which can reduce stream, pond, and weir pollution; and reduce the burden of living garbage classification and processing, improve air quality, and reduce the spread of infectious diseases. In addition, in combination with relevant references and research data from the “Exploration on other functions of the outer seed coat of G. biloba in abandoned parts” mentioned above, the development model is summarized in [Figure 13].
|Figure 13: Ginkgo biloba L. development and utilization mode of waste resources|
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Regarding the waste resources of G. biloba, this article mainly focuses on the development and utilization of G. biloba ectoparum, which has clear records of bacteriostatic, sterilization, insecticidal, anti-tumor, free radical scavenging, and anti-aging functions. At present, nine compounds, such as ginnol [Figure 2], palmitone [Figure 3], β-sitosterol [Figure 4], stigmast-3,6-dione [Figure 5], stigmast-4-ene-3,6-dione [Figure 6], ginkgolic acid [Figure 7], ginkgoneolic acid [Figure 8], triacotanoic acid [Figure 9]), and ginkgonine [Figure 10]). In this article, glycolic acid was applied to inhibit the growth of TB bacilli on the waste produced by TB infectious patients and reduce the spread of TB.
Development mode based on functional characteristics of different parts of ginkgo waste resources
Resource utilization of waste from the outer seed coat of G. biloba
The ginkgo outer seed coat is one of the components of ginkgo fruit (ginkgo). In the ginkgo fruit, the fleshy outer seed coat, bone in the seed coat, membrane inside the seed coat, kernel composition, and consumption of a large number of ginkgo fruit can cause poisoning. How does G. biloba become waste? Humans accumulate the collected ginkgo seeds for a long time, and when the amount accumulates to a certain extent, the outer seed coat rots. After washing, the outer seed coat is easily removed, leaving seeds, and the outer seed coat becomes waste. Through the comprehensive analysis of herb and modern applications, it was found that G. biloba can be used to inhibit and kill human TB bacillus, bovine TB bacillus, Bacillus subtilis, Escherichia More Details coli, yeast, S. aureus, dysentery bacillus, and Pseudomonas aeruginosa. Gingko mainly contains phenolic acids and hydrocarbon phenolic compounds, which have antibacterial, bactericidal, insecticidal, anti-tumor, free radical scavenging, anti-aging, and other effects.
Infectious waste refers to waste that can spread infectious diseases. It contains pathogens with sufficient pathogenic capacity to enter the body. This article is proposed for TB infection among infectious diseases. The waste produced by TB patients will be used as isolation waste, which will be treated by putting it into the container of “infectious waste” for high-pressure sterilization and incineration.
However, burning hurts both the environment and people by producing sulfur dioxide, suspended particulate matter, and nitrogen oxide which causes breathing difficulties, respiratory tract infection, and respiratory diseases. In addition, it produces toxic dioxins, which if pregnant women are exposed to in certain amounts in the environment, for a certain amount of time, may cause abortion or fetal abnormalities. Burning also releases a large number of toxic matter such as odor, sulfur, dust, and fine particles flying in the wind, resulting in suspended particles of sulfur dioxide in the air exceeding the standard, acid rain phenomenon, dust pollution, and other disasters occurring frequently.
Therefore, this article intends to excavate valuable waste resources to solve environmental problems, and proposes the use of G. biloba outer seed skin in inhibiting the growth of M. tuberculosis.
Resource utilization of Ginkgo biloba P. E. leaf residue and ginkgo leaf litter
The authors found that other waste resources of G. biloba L.(G. biloba P. E. leaf residue and G. biloba leaves) also have the value of transforming waste into treasure. Ostreatus ostreatus and cottonseed shells can be cultivated with G. biloba leaf residue. Chlorophyll can be separated and copper chlorophyll sodium salt can be prepared, which can be used as coloring agent in food, beverage, and medicine. It can be made into a broad-spectrum biological pesticide and insecticide, quick effect organic fertilizer, and be used in the control of aphids, cabbage worms, leek maggots, and prickly moth larvae by more than 80%. Under the leadership of Dong Chaoping, a group of Ningbo University students collected, extracted, compared, experimented, and extracted plant dyes from ginkgo leaves, and promoted the independently developed plant dye technology and technology to Hangzhou Zhongxin Printing and Dyeing Co., LTD., Hangzhou Yunwash Mountain Home Furnishing Co., LTD., and other enterprises.
Ginkgo biloba L. safety issues in the development of plant waste resources
To be rigorous and to ensure the safety of its development, the author made a search of relevant literature on the safety of G. biloba contacting human skin. The summary is shown in [Figure 14] (Refer to).
|Figure 14: Schematic diagram of the safety of ginkgolic acid purified solution|
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According to the acute transdermal toxicity test of ginkgolic acid A, it was judged that the purified ginkgolic acid was slightly toxic when the dosage was about 10–20 times the normal dosage. Several skin irritation tests done on ginkgo bilic acid B showed that purified ginkgo bilic acid causes light irritation when the dosage is about five times that of normal. Skin allergy tests of ginkgo bilic acid C showed that a dose of approximately five times that of the normal amount can cause light sensitization. It can be analyzed that the reaction dose caused by ginkgolic acid purified liquid is greatly different from the normal dose, and the reaction is mild, which can fully guarantee the health and safety of the human body.
The above figure fully illustrates the safety of G. biloba waste in this paper, which is harmless to the human body. In addition, this article is devoted to the treatment of household garbage generated by TB patients, which generally does not touch the human body, so it does not harm the human body.
| Discussion|| |
G. biloba L. is an important medicinal, garden, material, and nutritional plant in China. Only by excavating and utilizing waste resources can we better promote green development of the country and achieve the goal of a “healthy China.” This article aims to put forward the development of a strategy to address G. biloba waste resources and waste treatment, to provide more innovative environmental protection ideas for the recycling of waste resources in different fields.
Financial support and sponsorship
This study was financially supported by the National key entrepreneurship training project “Safe and Efficient Solutions and Services for Infectious Waste in Medical Waste--Artificial Intelligence Robot based on Big Data Statistics” (No.: 202013332016).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14]