CN111773275A

CN111773275A - Application of compound preparation for resisting novel coronavirus infection in preparation of medicine

Info

Publication number: CN111773275A
Application number: CN202010215824.6A
Authority: CN
Inventors: 李艳梅; 杨小生; 杨礼寿; 饶青; 宋晶睿; 邱剑飞; 杨珏
Original assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Current assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Priority date: 2020-03-24
Filing date: 2020-03-24
Publication date: 2020-10-16
Anticipated expiration: 2040-03-24
Also published as: CN111773275B

Abstract

The invention discloses an application of a compound preparation for preventing/treating novel coronavirus infection, wherein the compound preparation is prepared from coptis chinensis, rheum officinale and scutellaria baicalensis. The invention discloses a compound preparation which can inhibit the combination of novel coronavirus spike glycoprotein and a target cell receptor ACE2 and the expression of II-type transmembrane serine protease (TMPRSS2) to prevent the activity of virus entering target cells and can obviously inhibit the generation of inflammatory factors (IL-2, IL-6 and the like) by virus infected cells by constructing a drug screening model for blocking the novel coronavirus spike glycoprotein and the target cell receptor angiotensin converting enzyme 2(ACE2), can be used for preventing and treating novel coronavirus infectious diseases, provides medication selection for patients with novel coronavirus pneumonia, and has important effect and significance for preventing and controlling the current epidemic situation.

Description

Application of compound preparation for resisting novel coronavirus infection in preparation of medicine

Technical Field

The invention relates to a new application of a medicament, in particular to an application of a compound preparation in preparing a medicament for preventing/treating novel coronavirus infection, belonging to the technical field of Chinese medicament application.

Background

The Yiqing granules are a national medicine formula preparation and a Chinese patent medicine, and have the effects of clearing heat, purging fire and detoxifying; the traditional Chinese medicine composition is clinically used for treating conjunctival congestion and aphtha, sore throat and gum swelling and pain and constipation caused by fire toxin and blood heat; pharyngitis, tonsillitis, gingivitis, etc. The main raw material components comprise coptis chinensis, rhubarb and scutellaria baicalensis.

Coronavirus is a enveloped single-stranded positive-strand RNA virus widely found in human, mammalian and avian hosts and can cause respiratory, intestinal, liver and nervous system diseases. Six of the human coronaviruses are known, four of which generally cause only mild respiratory symptoms like the common cold, and the other two, severe acute respiratory syndrome coronavirus (SARS) and middle east respiratory syndrome coronavirus (MERS), which can cause severe respiratory diseases. The novel coronavirus (COVID-19) is the seventh of the human coronaviruses, belonging to the Beta coronavirus.

The new coronavirus COVID-19 infected pneumonia patients mainly show fever, hypodynamia and dry cough, a few patients have symptoms of nasal obstruction, watery nasal discharge, diarrhea and the like, the severe cases mostly have dyspnea after 1 week, severe patients rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct and blood coagulation dysfunction, and part of the light patients have no pulmonary inflammation. The blood routine suggests that white blood cell counts are normal or reduced, lymphocytes are reduced, and viral nucleic acid is detected positively. CT mostly shows double lung multiple wear glass density shadow (GGO), mainly refers to double lung pleural distribution, and can be accompanied with air bronchus sign, lobular space thickening and pleural thickening, and few or few pleural effusion or lymph node enlargement. Relevant studies concluded that its natural host may be a bat. Human respiratory epithelial cells are infected by a molecular mechanism of binding of the S-protein to angiotensin converting enzyme (ACE2) and activation of type II transmembrane serine protease (TMPRSS2) in the human body.

The possibility of using Reidesciclovir for the treatment of novel coronaviruses is reported in Science on a near day. The Wuhan virus institute of Chinese academy of sciences and the Beijing poison drug institute are reported to perform the research on the effect of the old drug on the novel coronavirus, which provides the opportunity for the novel coronavirus patients who can not be cured by the drug. National Weissenberg et al issued a notice of "treatment protocol for pneumonia with novel coronavirus infection printed (trial third edition)" in which antiviral treatment protocol recommended lopinavir and ritonavir as drug candidates. Additional studies have shown that based on the clinical value of ambroxol for the treatment of respiratory diseases, it may be beneficial to treat new types of coronavirus infections, which have entered the diagnostic protocol of the national Weijian Commission (third and fourth edition). However, no specific medicine for treating coronavirus infection exists at present.

According to the fact that a novel coronavirus gene sequence and ACE2 reported by scientists No. 1 and No. 7 in 2020 are receptors for the virus to enter target cells, and type II transmembrane serine protease (TMPRSS2) of the target cells can promote the novel coronavirus spike protein to be combined with ACE2 and enter the cells, the invention constructs a drug screening model for blocking the combination of the coronavirus spike protein and ACE2, and carries out in-vitro activity screening on a plurality of Guizhou national drug compound preparations by utilizing the model, and the 'Yiqing' compound preparation is found to have new application for the first time.

Disclosure of Invention

The invention aims to provide application of a compound preparation in preparing a medicament for preventing/treating novel coronavirus infection; a compound preparation can effectively inhibit the binding of novel coronavirus spike glycoprotein and a target cell receptor ACE2 and the expression of II-type transmembrane serine protease (TMPRSS2) to prevent viruses from entering target cells, and can effectively reduce the production of inflammatory factors (IL-2, IL-6, TNF-a, IL-8 and CXCL-10) by virus-infected cells.

The technical scheme of the invention is as follows:

the application of a compound preparation for preventing/treating novel coronavirus infection is prepared from 165 parts by weight of coptis chinensis, 500 parts by weight of rheum officinale and 250 parts by weight of scutellaria baicalensis.

In the application, the preparation method of the Yiqing compound preparation comprises the following steps: weighing the three raw material medicaments, respectively adding water to decoct for two times, the first time is 1.5 hours, the second time is 1 hour, merging decoction, filtering, decompressing and concentrating filtrate until the relative density is 1.25 at 70 ℃, spray-drying the filtrate into dry extract powder, merging the three extract powders, and then adding auxiliary materials or not adding the auxiliary materials to prepare various oral preparations.

In the application, the Yiqing compound preparation is granules prepared by adding a proper amount of sucrose and dextrin into the combined extract powder, uniformly mixing, preparing granules, drying and subpackaging.

The invention has the beneficial effects that: the invention constructs a drug screening model for blocking novel coronavirus spike glycoprotein and target cell receptor angiotensin converting enzyme 2(ACE2), screens a plurality of traditional Chinese medicine compound preparations, finds that the compound preparation has the activity of inhibiting the combination of the novel coronavirus spike glycoprotein and the target cell receptor (ACE2) and the expression of II-type transmembrane serine protease (TMPRSS2) to prevent viruses from entering target cells, can obviously inhibit the generation of inflammatory factors (IL-2, IL-6, TNF-a, IL-8 and CXCL-10) by virus infected cells, can be used for preventing and treating novel coronavirus infectious diseases, provides medication selection for patients with new coronary pneumonia, and has important effect and significance for preventing and controlling the current epidemic situation.

Drawings

FIG. 1 is a graph of the effect of Yiqing granule on inhibiting the binding of RBD-Fc to ACE 2; the binding inhibition effect of RBD-Fc on ACE2 expressed by 293T/ACE2 cells was determined by flow cytometry, the concentration of RBD-Fc was 1. mu.g/ml, and the concentration of Yiqing granules was 15. mu.g/ml, 30. mu.g/ml and 60. mu.g/ml, respectively, with DMSO as a control.

FIG. 2 is a graph of the effect of Yiqing particles on TMPRSS2 expression in infected viral cells; after treating the cells with different concentrations of primary particles for 24 hours, TMPRSS2 mRNA was measured using real-time quantitative qPCR. Error bars represent SD of the mean from 3 independent experiments.

FIG. 3-1 is a graph of the inhibitory effect of Monoclearn particles on VSV-COVID-19-St19/GFP entry into alveolar type II epithelial cells (AT 2); FIG. 3-2 is the percentage of virus infected cells after treatment with varying concentrations of Yiqing particles; (a) VSDVG was seeded on 293T cells expressing the indicated glycoproteins for 24 hours, and then culture supernatants were collected, filtered through a filter with a pore size of 0-22 mm, and seeded on AT2 cells, and the expression of GFP was examined under a fluorescence microscope; (b) the number of GFP positive cells after different concentrations of drug treatment was assessed by flow cytometry (n-7).

FIG. 4 is a graph of the inhibitory effect of primary particles on VSV-COVID-19-St19/GFP on virus infection of alveolar epithelial cells; after 24 hours of inoculating VSV-COVID-19-St19/GFP onto 293T cells expressing the indicated glycoproteins, the culture supernatant was collected, filtered through a filter having a pore size of 0 to 22mm and inoculated onto alveolar epithelial cells type II (AT2), and the VSV-COVID-19-St19/GFP virus titer, which was determined by the expression of EGFP gene (IU/mL), was obtained by the corresponding method of virus concentration.

FIG. 5 is a graph of the effect of Yiqing particles on the production of inflammatory factors following viral infection of alveolar type II cells; type II epithelial cells were cultured and inoculated with VSV-COVID-19-St19/GFP for 24 hours, and then the content of cytokines was quantified using ELSA, P <0.05, > P <0.01, > P <0.001, and the results were from 3 independent experiments.

The invention is further described with reference to the following figures and detailed description.

Detailed Description

Example 1: the application of the Yiqing granule comprises the following steps:

the Yiqing granule can be purchased from the market, can also be prepared according to the preparation method of the Yiqing granule in the 427 th page of the first part of the 'Chinese pharmacopoeia' 2015 edition, and the obtained granule can be directly used for preventing/treating novel coronavirus infectious diseases. The usage and dosage are as follows: orally administered at 6.5 g/time and 3 times/day.

Example 2: the application of a compound preparation:

weighing 165g of coptis chinensis, 500g of rheum officinale and 250g of scutellaria baicalensis, respectively adding water to decoct for two times, the first time is 1.5 hours, the second time is 1 hour, combining the decoctions, filtering, concentrating the filtrate under reduced pressure until the relative density is 1.25 at 70 ℃, spray-drying to obtain dry extract powder, combining the three extract powders, and then preparing various oral preparations by adopting the existing preparation forming process. The obtained Yiqing oral preparation can be used for preventing/treating novel coronavirus infectious diseases. The usage and dosage are as follows: orally administered at 6.5 g/time and 3 times/day.

Example 3: the application of a compound preparation:

165g of coptis chinensis, 500g of rhubarb and 250g of scutellaria baicalensis are weighed and prepared into various oral preparations by adopting a conventional extraction process and a preparation forming process. The obtained Yiqing oral preparation can be used for preventing/treating novel coronavirus infectious diseases. The usage and dosage are as follows: orally administered at 6.5 g/time and 3 times/day.

Experimental example:

the inventor constructs a stable COVID-19 RBD-Fc expression cell line, can secrete RBD spike protein into culture medium, and can be easily purified by protein A affinity chromatography; the constructed VSV-COVID-19-St19 pseudovirus can be combined with a receptor ACE2 to enter target cells but cannot generate progeny viruses, so that the system can be used for screening drugs for targeting VSV-COVID-19-S protein-mediated cell entry.

First, experimental material

1. Preparing a solution of a sample to be detected: appropriate mount of Yiqing granules are weighed according to the needs and prepared into solutions with different concentrations (50ug/ml,25ug/ml,12.5ug/ml,6.25ug/ml and 3.125ug/ml) by DMSO.

2. Cell line: human embryonic kidney 293T is used to produce Vesicular Stomatitis Virus (VSV) pseudotype virus carrying the COVID-19-S protein. Alveolar type II epithelial cells (AT2) were used for VSV pseudotype virus-infected target cells.

3. Modified Eagle Medium with high glucose concentration supplemented with 10% fetal calf serum (DMEM 10% FCS) was used to culture 293T and AT2 cells.

4. Autoclaved Phosphate Buffered Saline (PBS): 0.14. mu.M NaCl, 2. mu.M KCl, 3. mu.M Na₂HPO₄、1.5 μMKH₂PO₄，pH7.2。

5. Transfection reagent (QIAGEN, Germany).

6. A human expression plasmid encoding the COVID-19-S protein having 19 amino acids at the C-terminus. To generate a VSV pseudotype with high viral titer, we please prepare a plasmid encoding a C-terminally truncated form of the S protein, since a C-terminal 19 amino acid truncation has been shown to result in efficient incorporation of the S protein into the VSV particle, which then shows strong infection potential to the target cell.

VSVG-GFP is a VSV pseudotype with a VSV-G protein in which the VSV-G gene is replaced by the GFP gene. Pseudotypes with the VSV-G protein were used as "seed" viruses to produce S proteins with VSV prostheses. The VSVG-GFP system was purchased from America Biogen Inc.

An 8.0.22 μm pore size sterile filter.

9. Fluorescence microscopy for detection of GFP expression.

And 10, the ELISA kit is used for analyzing the content of the inflammatory cytokines in the cell culture solution.

Second, Experimental methods

1. Construction of recombinant plasmid

Amino acids 438-506 of the COVID-19 spike protein were located as the ACE2 binding domain (RBD). The RBD-encoding cDNA fragment was amplified by PCR using plasmid PUC18-S containing the coding sequence of human codon-optimized COVID-19 spike protein as a template, and primers (forward: 5'-GGCGCTAGCCATCACCAACCTGTGCCCC-3', containing a NheI recognition site; reverse: 5'-CGCGGATCCGTCACGGTGGCGGGGGCGTTC-3', containing a BamHI recognition site). The PCR product was digested with NheI and BamHI and then cloned downstream of the Peak13 expression vector CD5 antigen leader and upstream of the IgG1(Fc) Fc portion, and the Peak13 expression vector was also digested with NheI and BamHI. The resulting recombinant plasmid was named Peak 13-RBD-Fc.

2. Establishment of stable expression RBD-Fc cell line

One day prior to transfection, HEK293 cells were trypsinized at 1 × 10 per well⁵The density of cells was seeded into 6-well plates. The following day, 0.5 μ g Peak linearized with AvrII was treated with lipofectamine (TM) 2000(Invitrogen) according to the procedure described in the instructions of the reagents Ltd13-RBD-Fc plasmid or equal volume of H₂O was transfected into HEK293 cells. After 48 hours, aliquots of transfected cells were transferred to selection medium containing increasing concentrations of puromycin (0.3, 0.4, 0.5, 0.6 and 0.7. mu.g/ml). After 3 days, transfected HEK293 cells selected for the appropriate puromycin concentration at which the cells transfected with Peak13-RBD-Fc plasmid survived but were H-transfected₂The O-transfected cells all died, and the cells transfected with the Peak13-RBD-Fc plasmid were transferred to a 96-well plate by limiting dilution. After 10 days of culture, the cells expressing the highest amount of RBD-Fc were selected for the production of the recombinant protein RBD-Fc by ELISA assay using anti-human IgG peroxidase-conjugated antibody (Sigma).

3. Enzyme-linked immunosorbent assay (ELISA)

2 × 10 from each RBD-Fc-293 cell clone⁵Cells were seeded into 6-well plates and cultured with 2ml of medium (DMEM containing 10% FBS) for 72 hours. The concentration of RBD-Fc protein in the culture supernatants was quantified by ELISA assay using the kit BD OptEIA TM (BD Biosciences), following the procedure described in the instructions of the reagents Inc., briefly, 96-well flat-bottom EIA/RIA plates (Corning, N.Y.) were coated overnight with culture supernatants of each RBD-Fc-293 cell clone at 4 ℃ with 100. mu.l of medium and serial dilutions of human IgG standard in culture medium (10 ng to 30. mu.g/ml). The wells were then washed once with wash solution, blocked with assay diluent for 1 hour at room temperature, and then washed 3 times with wash solution. Next, anti-human IgG peroxidase-conjugated antibody (Sigma) diluted in Assay dilution (1: 5000) was added to each well and gently stirred at room temperature for 1 hour. After three washes, 100 μ l of substrate reagent was added to each well and the wells were incubated in the dark at room temperature for 30 minutes. Finally, the reaction was stopped using a stop solution and the color development was monitored at a wavelength of 450 nm.

4. Protein purification

Culture supernatants of RBD-Fc-293 cells were collected and dialyzed against solution (20mM sodium phosphate and 1mM EDTA, pH 7.0) over 8 hours. After centrifugation at 4000rpm for 30 minutes, the supernatant was filtered through a 0.45 μm dura filter (Millipore, irish). According to the steps of the instructionProcedure in a HiTrap Protein A HP 5ml column (GE Healthcare Amersham Biosciences AB) and Econo^TMPurification of RBD-Fc protein was performed by gradient pump tube kit (Bio-Rad, USA). Briefly, the column was first washed with 10 column volumes of binding buffer (20mM sodium phosphate, pH 7.0) at a flow rate of 5 ml/min. The cell culture supernatant was then pumped onto the column. Next, the column was washed with 20 column volumes of binding buffer and then with the supernatant. Finally, the RBD-Fc protein was eluted with 2-5 column volumes of elution buffer (0.1M glycine, pH 3.1). Neutralization buffer (60-200. mu.l, 1M Tris-HCl, pH 9.0) was added to each collection tube. The RBD-Fc Protein was further purified using a HiTrap Protein A HP 1ml chromatography column (GE Healthcare Amersham biosciences sAB). Protein concentration was determined using protein assay dye reagent concentrate (Bio-Rad) using Bovine Serum Albumin (BSA) as standard according to the protocol.

Binding inhibition assay for RBD-Fc to the receptor ACE2

The inhibitory effect of the drug on the binding of RBD-Fc to the cellular receptor ACE2 was measured by flow cytometry. Collection and separation 2X10⁶293T cells expressing ACE2 were washed with HBSS (Sigma-Aldrich). Cells were treated with 1. mu.g/ml RBD-Fc with or without 50. mu.g/ml drug, and then incubated for 30 minutes at room temperature. Cells were washed with HBSS and incubated with anti-human IgG-FITC at 1/50 dilution for 30 minutes at room temperature. After washing, cells were fixed with 1% formaldehyde in PBS and analyzed in a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software.

6. Establishment of ACE2 expressing cell line

AT2 cells were transfected with pTargeT plasmid (Promega) pcDNA carrying ACE2 gene, and then selected with G418-containing medium to obtain ACE 2-highly expressing cells (AT2-ACE 2). These cells were grown and stored in DMEM medium with 5% fetal bovine serum.

7. Construction of pseudovirus VSV-COVID-19-S protein pseudovirus

To construct VSV that produces the COVID-19-S protein pseudotype, a vector encoding the full-length COVID-19-S protein was first constructedExpression plasmid the cDNA of the COVID-19-S protein was amplified with the forward primer S-Bam-f (5'-GGATCCAAGTGATATTCTTGTTAACAAC-3') and the reverse primer S-Bam-r (5'-GGATCCAAGAGTAAAAAATTCCATCAT-3C) and cloned into the expression vector pKS336. the cDNA of the C-terminally truncated COVID-19-S protein was amplified from pKS-SARS-S using the forward primer S-Bam-f and the reverse primer S-Bam19r (5'-GGGATCCTTAGCAGCAAGAACCACAAGAGCATG-3') and then cloned into pKS336. the resulting plasmid pKS-COVID-19-St19, encoding the full length COVID-19-S protein in addition to the C-terminal 19' aa. after transfection of the expression plasmid pKS-COVID-19-St19 into 293T cells, expression of the S protein was detected on the cell membrane. in order to produce a pseudo-type VSV of the C-terminally truncated COVID-19-St19 protein, the VSV Δ G was inoculated into 293T cells with the expression plasmid pKS-19-St 19. the pKSS-19-St 293T 293 was used as a control for the efficient infection of lung cells, i.e.5-pKS19-19-STS-19-STS 5. the pKS19-19-STS-19, the expression of the pseudo-STS-pVSV-pKS19-STS-19⁵IU/ml。

Detection of entry of VSV-COVID-19-St19 into cells

To examine the ability of VSV-COVID-19-St19 to infect target cells, a time-dependent analysis of GFP expression was performed. A monolayer of alveolar cells on 24-well glass slides was infected with VSV-COVID-19-St 19. At different time points, the virus was infected and cells were photographed under a fluorescent microscope. The number of GFP expressing cells in the photographs was counted using ImageJ software (http:// rsb. info. nih. gov/ij /). Since pseudoviruses are unable to produce progeny viruses, this system can be used to evaluate the VSV-COVID-19-S protein-mediated cell entry mechanism. Time course analysis of the number of GFP positive cells indicated that VSV-COVID-19-St19 infection could be quantified 6 hours after viral infection.

mRNA determination of ACE2 and TMPRSS2

Total RNA was extracted from virus infected cells treated with different concentrations of drug using the kit according to the procedure described in the instructions of the reagents company using the quantitative qPCR apparatus LightCycler (Roche diagnostics) used. TMPRSS2 mRNA was amplified with primers 5'-CTCTACGGACCAAACTTCATC-3' and 5'-CCACTATTCCTTGGCTAGAGTAA-3'. ACE2 was amplified with primers 5'-CCGTATCAATGATGCTTTCCG-3' and 5'-CAGTGAAGATCAGGATGACAAT G-3'.

10. Determination of inflammatory cytokines

After the infected virus cells were treated with different concentrations of the drug for 24 hours, cell culture supernatants were collected. The contents of the cell inflammatory factors TNF-a, IL-6, IL-2, etc. in the cell culture solution were measured using a high sensitivity ELISA kit (QuantikineTM HS, R & D Systems) according to the procedures described in the instructions of the reagents Co. And drawing a standard curve to calculate the sensitivity.

Third, experimental results

1. The effect of Yiqing granules on the binding of the spike protein RBD of the novel coronavirus and the target cell receptor ACE2 is measured by a flow cytometer, and the result shows that the Yiqing granules can obviously inhibit the binding of the spike protein RBD of the novel coronavirus and the receptor ACE2 and have a dose-dependent effect (see figure 1). This indicates that the compound preparation can inhibit the novel coronavirus from entering cells.

2. The effect of the Yiqing particles on the expression of TMPRSS2 mRNA of a novel coronavirus target cell is determined by using real-time quantitative qPCR, and the result shows that the Yiqing particles have obvious inhibition effect on the expression of TMPRSS2 of infected virus cells and have dose-dependent effect (see figure 2). This indicates that a compound preparation can inhibit the binding of the novel coronavirus spike glycoprotein to the target cell receptor ACE2 by inhibiting the expression of type II transmembrane serine protease (TMPRSS2) to prevent the virus from entering the target cell.

3. Pseudotype virus VSV-COVID-19-St 19/GFP-infected alveolar type II epithelial cells (AT2) were treated with primary particles, and the amount of virus (GFP expression amount) was examined under a fluorescence microscope, and as a result, primary particles were found to significantly reduce the amount of virus entering the cells (see FIGS. 3-1 and 3-2). This indicates that the compound preparation can prevent virus from infecting cells.

4. The effect of the Yiqing particles on the entering of the novel coronavirus into cells is measured by a virus gradient method, and the result shows that the Yiqing particles have obvious inhibition effect on virus infection of alveolar epithelial cells and have dose-dependent effect (see figure 4). This shows that the compound preparation has obvious inhibiting effect on virus entering alveolar epithelial cell and may be used in preventing and treating new type coronavirus clinically.

5. The effect of the Yiqing granules on the production of inflammatory factors after the alveolar type II cells are infected with viruses is quantitatively determined by enzyme linked immunosorbent assay (ELSA), and the results show that the Yiqing granules can reduce the amount of the inflammatory factors (IL-2, IL-6, TNF-a, IL-8 and CXCL-10) produced by virus-infected cells and have dose-dependent effect (see figure 5). This shows that the compound preparation can be used for clinically treating the inflammation storm caused by the new coronavirus infection.

Claims

1. The application of a compound preparation for preventing/treating novel coronavirus infection is prepared from 165 parts by weight of coptis chinensis, 500 parts by weight of rheum officinale and 250 parts by weight of scutellaria baicalensis.

2. The use of the compound preparation according to claim 1, wherein: the preparation method of the Yiqing compound preparation comprises the following steps: weighing the three raw material medicaments, respectively adding water to decoct for two times, the first time is 1.5 hours, the second time is 1 hour, merging decoction, filtering, decompressing and concentrating filtrate until the relative density is 1.25 at 70 ℃, spray-drying into dry extract powder, merging the three extract powders, and then adding auxiliary materials or not adding the auxiliary materials to prepare various oral preparations.

3. The use of the compound preparation according to claim 2, wherein: the Yiqing compound preparation is prepared by adding a proper amount of sucrose and dextrin into the combined extract powder, uniformly mixing, granulating, drying and subpackaging.