ACACIA AURICULIFORMIS-EXTRACT SYNTHESIS PR-PROTEINS DEVELOPED POTENTIAL BIOMEDICINES-VACCINE AGAINST OKRA-DISEASES AND COVID-19: IMPROVED SCIENCE TECHNOLOGY COMMUNICATIONS BIO-ECONOMY APPLICATIONS

© 2020 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 249 ACACIA AURICULIFORMIS-EXTRACT SYNTHESIS PR-PROTEINS DEVELOPED POTENTIAL BIOMEDICINES-VACCINE AGAINST OKRA-DISEASES AND COVID-19: IMPROVED SCIENCE TECHNOLOGY COMMUNICATIONS BIO-ECONOMY APPLICATIONS


MORTALITY TEST
Two sets (for each test), of cavity blocks with 1 ml distilled water containing 50 larvae (J2) of M. incognita were taken; one set was treated as control and the other was treated as treatment set. To assess the direct effect of biomedicines; Acacia auriculiformis-extract, by treatment -test solution (prepared by 1mg/10ml distilled water), the water was removed by pipette from all the treatment sets, and immediately replaced by 1 ml of pretreatment -test solutions of Acacia auriculiformis-extract, were added respectively. To assess the direct effect of the control solution, the control set was received 1 ml of control solution and observed with every 30 minutes interval for a period of 12 hours exposure period at room temperature (25 ± 2°C). This mortality test was replicated five times. This mortality test was replicated five times [49], [50], [51], [52], [53], [54], [55], [56], [57], [58].

HARVESTING
Ninety-one days after plantation of germinated seeds of okra, all the plants were uprooted from each plot and the parameters of growth and nematode infection such as biomass of shoot and root, root gall number, nematode population per gram of root and 200 soil, fruit and root total protein fraction in each sample was estimated [50], [51], [55], [56], [57]. At thirty days after the last pretreatment, all the parameters of diseases were assessed again (after last pretreatments) for each group and all the data were used for statistical analysis by Student t-test [31], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47] and all the data were considered and were presented in Table 1.

PREPARATION OF ROOT PROTEINS
Fresh roots of okra plants of 4-groups were collected at random and root pieces were homogenized with distilled water using mortar and pestle followed by glass tube homogenizer [56], [57]. The extracts were then centrifuged at 3,500 rpm for 15 minutes at 40C in Remi C-24 refrigerated centrifuge and the supernatants were collected. All the samples were transferred into lyophilizer and after lyophilization, the powdery extract was stored (-200C) for protein separation [56], [57], [58], [59], [60].

DENSITOMETER SCANNING
Analysis of total soluble roots proteins separation was carried out essentially by the method of Laemmli [59], [60], by using one-dimensional vertical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with the modification as suggested by the LKB Instructional Manual (1986). A 10% separating gel and 5% stacking gel were used. Samples for the preparation of electrophoretic run were mixed with 2x treatment buffer. A sample of 65µl of root protein extract was loaded along with dye. The bands were scanned with recording electrophoretic scanner (Biomidi, 96-300 Densitometers, Pare de La Plaine, France). The observation was recorded from the densitometers scanning curve measuring the total number of root-proteins with molecular weight. Wide range markers (k) of Pharmacia United Ltd., Sweden, were used as the standard, for the molecular weight estimation of proteins [59], [60].

TOXICITY TEST ON MORTALITY
The biomedicines; Acacia auriculiformis extract, at extremely low doses, had no direct toxic effects on nematodes mortality respectively.

ANALYSIS OF RESIDUES TOXICITY
Okra leaves and fruits, collected fifteen days after the last pretreatment did not contain any toxic residue of the biomedicines; Acacia auriculiformis extract. Table 1 shows the pre-and post-treatment effects of high diluted biomedicines; Acacia auriculiformis extract, at extremely low doses, on Root-Knot (RK) disease, and Yellow Vein Mosaic Virus (YVMV) and Okra Enation Leaf Curl Virus (OELCV) foliar diseases of okra plants, assessed initially (Day-0) means before pretreatments and after a period of 30 days (Day -30) means after last posttreatment, in a field trial with the number of common-shared rootprotein in comparison to uninoculated untreated one (P<0.01 by 't'-test). All the pretreatments with biomedicines; Acacia auriculiformis-extract, significantly reduced the root-knot and foliar diseases in comparison to the first two plots; uninoculated untreated (control) and inoculated untreated ones. Though, all the plant's groups, average; the number of leaves, number of nematodes in soil, biomass of shoots, and protein of fruits, are significantly increased than inoculated untreated plants group, but average; the biomass of roots, number of root galls and nematodes populations in roots and root protein content percent, is higher in the inoculated untreated one. The highest number of common-shared root proteins are seven in healthy uninoculated untreated control plants and the lowest number is one in the inoculated untreated one (Table 1 and Fig. 1).

ROOT PROTEINS OF THE OKRA
'-' indicate no band Table 2 and Fig 1; shows the molecular weight (k) of the root proteins of the different groups of okra plants, pre-and post-treated with high diluted biomedicines; Acacia auriculiformis extract, at extremely low doses, on Root-Knot disease, and Yellow Vein Mosaic Virus (YVMV) and Okra Enation Leaf Curl Virus (OELCV) foliar diseases of okra plants. An analysis of root proteins of all groups by electrophoresis and densitometer scanning of all the test plants show that all the Acacia auriculiformis extract, treatments resulted in the increased number of proteins in the roots; the lowest number of protein is 11 in the uninoculated untreated roots and highest number of proteins, are 18 in the pretreated-and post-treated-Acacia auriculiformis extract, roots respectively (Table 2 and Fig. 1). The highest molecular weight of the protein is 280k (280,000kDa) and the lowest molecular weight of the protein is 12k (12,000kDa). The lowest number of the new pathogenesis-related protein (PR-proteins) is 4 in the uninoculated untreated roots and the highest number of the new pathogenesis-related proteins (PR-proteins) are 15 and 16, in the pretreated-and post-treated-Acacia auriculiformis extract, roots respectively (Table 2 and Fig. 1). Though, the PR-proteins of inoculated untreated is 14 (Table 2 and Fig. 1).

SCIENCE AND TECHNOLOGY COMMUNICATIONS APPLICATIONS
Achieved by; campaign, aware, discuss, arrange workshops and seminars, make news, and publish as abstract regarding the importance of "Pretreated-and Post-treated-Acacia auriculiformis extract, with High-Diluted, at Extremely Low Doses; Indicate the Potential Cost-Effective Biomedicines Against Various Pathogens, Including COVID-19?".

DEVELOPMENT OF IDEAS FOR PREPARATION OF A VACCINE
The results may fulfill the goal for the preparation of a vaccine for the treatment of coronavirus, with the present treatments with the high-diluted biomedicines; Acacia auriculiformis-extract, at extremely low doses, against COVID-19, need to develop future research of medicines after discussion.

FUTURE IDEAS IN RESEARCH
Here, the results fulfill the goal of a research title because the present treatments with biomedicines; Acacia auriculiformis-extract, at extremely low doses, with regular consumption of nutritious okra-vegetables of need to justify future research ideas.

DEFENSE RESPONSE ON PATHOGENESIS-RELATED (PR) PROTEINS
It is reported that a plant plasma membrane ATP binding cassette-type transporter [74] is involved in antifungal terpenoid, for this transporter in disease resistance or by the share of common antigens with its host plants [75] or by the pure compounds of acaciasides [76], [77], [78], [79], which use against pathogens.
Treatments with the biomedicines; highly-diluted Acacia auriculiformis-extract, at extremely low doses, all the test plants synthesis of various proteins (antigens), and lowest number of the new pathogenesis-related protein (PRproteins) is 4 in the uninoculated untreated roots and highest number of the new pathogenesis-related protein (PRproteins) is 16 in the Acacia auriculiformis-extract, post-treated roots, which induce defense responses involving several pathogenesis-related proteins, specially low molecular weight proteins, in which the naturally infected plant pathogens fail to tolerate, like the NE (nematode extract) [15], [20], [21], [22], [23], [48], [80], because during natural infection with pathogen, host plant showed minimum defense responses for the antigenic (proteins) similarity [75], [81] and it is proved from the inoculated untreated pretreatment okra plants and the high-diluted drugs; Acacia auriculiformis-extract-treated roots, produce highest number of new PR-proteins, 15 and 16 respectively. So, Acacia auriculiformis-extract treatments may be used directly against diseases of plants and animals, by improving immunity or defense responses with the help of new PR-proteins [81], [82], [83], [84], [85].

DEVELOPMENT OF IDEAS FOR PREPARATION OF VACCINE
• In Genome Biology: we're not completely human, at least when it comes to the genetic material inside our cells; 145-genes from bacteria, other unicellular organisms, and viruses with 17-as possible horizontal gene transfers [85], the genomic sequencing 96.2% identical to a bat coronavirus and shares a 79.5% sequence identity to SARS-CoV [86], [87], [88], [89] deal with the structure and function of genetic material underpinning all organisms [90], [92]. Approximately, ten percent of the human genome is made of bits of virus-DNA [90]. Human endogenous retroviruses are by far the most common virus-derived sequences in the human genome [91] which don't always require a body [92]. • In Genetic Resistance: it is reported that genetic resistance to coronavirus infection according to those three host resistance mechanisms: genetic control [16] and SARS-CoV-2 is the etiological agent responsible for the pandemic COVID-19 outbreak and the main protease (Mpro) of SARS-CoV-2 is a key enzyme that plays an important role in helping in viral replication and transcription-structure-based design of antiviral drug [93]. • In Immune System Blueprint: once the virus infects the host cell, it takes over the host cell's machinery to become a virus factory. When the human body is attacked by germs, the immune system kicks into gear to fight, by improving the immune system, creates a blueprint of the attacking agent, by which, the body effectively remembers the germ -enabling a person to fight for re-infection by the same or similar viruses [94]. • Traditional Medicine in Human History: in the evolution of human history shows the people are using traditional medicine for therapeutic purposes, and the 70%-80% population is primarily dependent on animals and plant-based [10], [95].
• Therapeutic Value of Traditional Medicines: it is reported that the biomedicine or phytomedicine-Acacia auriculiformis extract or Acaciasides (A&B) is being used traditionally to overcome various medical complications like sore eyes, aches, rheumatism, allergy, itching, and rashes. Besides, it has also been proven for many pharmacological activities like central nervous system depressant activity, antioxidant, antimicrobial, antimalarial, antifilarial, anticestocidal, antimutagenic, chemopreventive, spermicidal, wound healing, hepatoprotective and antidiabetic activity due to its low toxicity (LD50 = 3741.7 mg/kg) and high efficacy and the various phytochemical investigations reveal the presence of chief constituents as flavonoids and triterpenoid saponin glycosides. The low toxicity and the presence of major bioactive phytoconstituents like flavonoids and triterpenoid saponin glycosides are responsible for a therapeutic remedy for various diseases and pharmacological activities respectively. It has been used to treat several medical ailments due to its low toxicity and the presence of bioactive phytoconstituents [34]. Isolated saponins (acaciasides -A&-B acylated triterpenoid bisglycoside) from fruits (funicles) of A. auriculiformis were screened for their antifilarial activity and results were found to be significant [96]. A US patent claimed the potential of acaciasides (A&B) isolated from Acacia auriculiformis for the prevention of HIV infection and as a vaginal contraceptive [97]. And it has long been not only used in traditional medicine and but also as a food source for different functions [33]. Its essential oils has hepatoprotective-and hepatic non-toxic-effects, and as a natural source of hepatoprotective agent [98]. But, 4th May 2020, WHO considered as possible treatments for COVID-19 and should be tested for efficacy and adverse side effects? [98] • Therapeutic Approach: recently, the NovavaxInc, which contributed to the development of other epidemic vaccines, has announced it is currently in pre-clinical animal trials for several multiple nanoparticle COVID-19 vaccine candidates, by using their recombinant protein, the adjuvant is 'saponin-based' and it has shown a "potent and well-tolerated effect" [97], [99]. Scientists are trying to discover novel inhibitor molecules against enzymes Mpro and ACE2 by the use of phytochemicals, which be utilized for further innovation and development of antiviral compounds against Coronavirus [100]. • Vaccination: so, for successful vaccination requires four components. Human Immunomics Initiative (HII) aims to decode the underlying mechanisms and rules of how the human immune system fights disease with advances in computing and artificial intelligence, genomics, systems biology, and bioinformatics100. And should follow the guideline of WHO entitled "Vaccine-preventable diseases and vaccines" [10], [101], [102]. It reduces wait time for emergency vaccination [103] and one or more randomized trials will be needed to answer [104]. Then, with allopath is using trial and error method in some cases to treat COVID-19 [98], [105] and according to the World Health Organization's latest table of COVID-19 vaccines, 124-candidates with technologies or platforms. Merck's new investments focus on two different COVID-19 vaccines that are already in early clinical trials [106]. • Development of ideas for treatment: in this situation, it will be essential to inform public health expertise for moving academic research [107] with "Return to Work" [108] and a gradual, stepwise approach to reopening. And, the present results and discussion, fulfill the goal for the effective treatments methods, because the present treatments with the Acacia auriculiformis-extract, prepared from the funicles of Acacia auriculiformis, mixed with water at an extremely low dose [15], [19], [20], [21], [22], [23], [28], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48] need to justify [98] for immunomodulatory effect [109].

SITUATIONS FOR APPLICATIONS OF DRUGS AGAINST COVID-19
Recently, novel coronavirus infection shows new symptom, COVID-Toes among the kids [116] and on the other hand current pandemic locust attack in India causes the new crises of food securities [117], and substantial excess deaths in lower-income countries due to the poorer health care available [110], humanitarian crisis [111], the world has joined hands to find a vaccine for the novel coronavirus. At present, many groups (80) are working globally for vaccines for humans which normally take years to develop. Currently, there are [112] potential vaccines for the SARS-CoV-2 which are in different stages of clinical trials [118]. In this situations, it is thought that the personalized medicines, may improve medical decision-making individualized diagnosis, prevention, and treatment of diseases [119], because an institute of public organization of developing countries, with the support of WHO, was an example for the production of vaccines, and to distribute free for all the population in Brazil, succeeding to be an important producer, became the "bad example" [120]. So, the present treatments with the high-diluted Acacia auriculiformisextract, biomedical drugs, at an extremely low dose, should be immediately applied against pandemic COVID-19 for beneficial clinical treatments, with no side effects [121], [122], [123], [124].

Third emergency preparations of vaccine for all patients;
The adjuvant Acacia auriculiformis extract, [33], [98], [114], [115], may be used with recombinant protein nanoparticle antigens derived from the coronavirus spike protein and combine these antigens with its adjuvant Acacia auriculiformis-extract for the final formulation of the vaccine and it may be shown a "potent and well-tolerated effect" through stimulating the entry of antigen-presenting cells into the injection site and enhancing antigen presentation in local lymph nodes, boosting immune responses [31], [36], [37], [38], [39], [40], [42], [43], [44], [45], [46], [47], [97], [98], [99], [113], [114], [115], [133]. In a letter as an e-mail, the Science Advisory Board Net, at Express Cells, for their business of creating better knock-in cell lines for drug discovery, toxicology, and other biologic research and add for purchase SARS-CoV-2 Spike Protein (NC_045512.  [133] and the readily available coronavirus spike proteins may be helped to use for vaccine preparation which may fight against "COVID Toes among kids: New symptom of novel coronavirus infection" [116]. Here, vaccination or treatments, is the use of remedies against diseases either earlier in an epidemic or given routinely to prevent diseases. When the latter is used it involves mostly the users just like any conventional vaccination which administers the antigen in an inactive state to gain immunity towards the disease and is given before the onset of disease or disease symptoms in an individual as a prevention rather than cure [122] and in a clinical study shows the efficiency of triple antibiotic mixture and propolis as intracanal medication in revascularization process in immature apex [122], [134]. It is obligatory that information to the ClinicalTrials.gov, the U.S. National Library of Medicine, to the National Institutes of Health or other agencies of the U.S. Federal Government [95], [101], [102], [116], [122], [123], [125], [126], [127], and maybe easily emergency-applicable drugs with increasing immunity [128].

Science and Technology Communications Biomedicines Applications;
Recently, the Coronavirus pandemic has adverse effects on education, especially on school education and characteristics, including research, academic programs, staff professional development, and jobs also [129]. At present, many groups (80) are working globally for vaccines for a human which normally takes years to develop. Currently, there are [112] potential vaccines for the SARS-CoV-2 which are in different stages of clinical trials [118] and a committee that makes vaccine use recommendations to the U.S. Centers for Disease Control and Prevention (CDC), suggesting they should be high on the list [138]. Because, the behavioral scientists provide a road-map for the public to manage the trauma of COVID-19 and its aftermath successfully, learned from biomedical science [139], and gird for a war on vaccine misinformation [140].

Future Ideas in Research Vaccine;
Here, the results fulfill the goal of a research title because the present treatments with biomedicines; Acacia auriculiformis-extract, at extremely low doses, with regular consumption of okra vegetables of need to justify future research ideas. It is known that social vaccines resist and change unhealthy social and economic structures and useful metaphor for health promotion [130]. Okra plants itself may serve as a good "Eco-Friendly Highly Economical Plants as well as Biomedicines" thereby reducing diseases conserving our biodiversity contributing towards "Sustainable Climate Health and Development" and it may have important economic implications in agriculture to fulfill its food and nutrition requirement and improved midday meals by preventing malnutrition. And highly-trace-tolerance-okra may be used, in vaccine formulations or treatments or social vaccine, as one of the most powerful potential-biomedicine, improving natural immunity against COVID-19, enriching science and technology communication applications food security economy. It may be the most; costeffective, easily-available, safe-edible and prepare able as well as and safe alternative to live replicating COVID-19 social vaccines which restarts, a window of hope and opportunity open for nations to green their recovery the 21stcentury economy in ways that are clean, green, healthy, safe and more resilient, and a large number of people may be immune or resistant to SARS-COV-2 due to they have already been infected by other coronaviruses [141], [142], [143], [144], [145], [146] and Emergency Use Authorization of Covid Vaccines -Safety and Efficacy Follow-up Considerations [146]. And Okra maybe use as future biomedicine improved immunity against COVID-19 enriching science and technology communication applications food security economy [142], [146].

CONCLUSIONS
The biomedicines-Acacia auriculiformis-extract, at extremely low doses, not only used as a potentialbiomedical-drugs or vaccine against various plant-diseases, by synthesis of new PR-proteins, increasing naturaldefense-response, but also, it may be increased urgency to prepare safe and an effective emergency use for treatment for prevention coronavirus, the COVID-19, by increasing the immune system for the improvement of biomedicines economy applications science and technology communication cost-effectiveness and biodiversity conservations as well as bio-applications issues, reviving human civilizations in the old form. And in near future okra may itself be a 'Vaccine as well as Biomedicine', and safe alternative to live replicating COVID-19 vaccines which restarts, a window of hope and opportunity open for nations to green their recovery the 21st-century economy in ways that are clean, green, healthy, safe and more resilient.

SOURCES OF FUNDING
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.