A method for increasing the biomass and medicinal ingredient content of artemisia annua

Abstract

The application belongs to the field of agricultural biotechnology, and provides an Agrobacterium sp. BA51, a preservation number of which is CCTCC NO: M 20251734, and a method for increasing the biomass and medicinal ingredient content of artemisia ann L. The strain is prepared into a microbial inoculant and inoculated into the rhizosphere soil of artemisia ann L., so that the plant growth promoting effect of the strain is utilized to effectively promote the root system development and plant growth of artemisia ann L., and the biomass and the content of medicinal ingredients such as artemisinin and flavonoids of artemisia ann L. are simultaneously improved. The method solves the problems in traditional cultivation, such as slow growth of artemisia ann L., insufficient and unstable accumulation of medicinal ingredients, excessive dependence on chemical fertilizers and the like, has the characteristics of simple operation, environmental friendliness and stable effect, and is suitable for large-scale green cultivation of artemisia ann L., thereby providing an effective way for high-yield and high-quality cultivation of artemisia ann L.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, and relates to Artemisia annua, specifically to a method for increasing the biomass and medicinal component content of Artemisia annua. Background Technology

[0002] Artemisia annua L., as the sole plant source of artemisinin, holds an irreplaceable position in the development of antimalarial drugs. Artemisinin and its derivatives are considered first-line antimalarial drugs due to their high efficacy and low toxicity. Furthermore, the flavonoids in Artemisia annua can synergistically enhance the bioactivity of artemisinin, further highlighting its medicinal value. However, the commercial cultivation of Artemisia annua faces significant challenges: firstly, the natural content of artemisinin in the plant is generally low, making it difficult to meet global market demand; secondly, its growth and the accumulation of secondary metabolites are easily constrained by environmental factors (such as light, temperature, and soil nutrients) and cultivation techniques, leading to significant fluctuations in yield and quality.

[0003] Currently, strategies to enhance the medicinal value of Artemisia annua mainly include genetic breeding, cultivation optimization, and chemical synthesis. While molecular breeding techniques can selectively breed varieties with high artemisinin content, their long cycle and potential trade-off with biomass reduction are significant challenges. Conventional cultivation techniques (such as fertilization regulation and water management) can promote biomass accumulation, but these are often negatively correlated with the content of secondary metabolites, making synergistic effects difficult to achieve. Although chemical synthesis of artemisinin is theoretically feasible, its industrial application is limited due to its complex synthetic routes, high costs, and difficulties in chiral purification. Furthermore, the over-reliance on chemical fertilizers and pesticides in traditional agriculture may lead to soil degradation and ecological risks, which does not meet the needs of sustainable development. Moreover, existing cultivation techniques cannot simultaneously achieve a synergistic increase in biomass and medicinal component content, and chemical synthesis of artemisinin faces challenges of high cost and complex procedures.

[0004] The application of microbial pesticides (such as plant rhizosphere growth promoters PGPR and arbuscular mycorrhizal fungi AMF) in improving crop quality and yield has attracted much attention. Studies have shown that specific microorganisms can simultaneously promote host growth and the accumulation of active ingredients by regulating plant hormone secretion, enhancing nutrient absorption, or inducing secondary metabolic pathways. However, the application of existing microbial technologies in the cultivation of Artemisia annua is still limited to single-function verification (such as promoting growth or increasing artemisinin levels), lacking a systematic approach to achieve synergistic enhancement of biomass and medicinal components.

[0005] Therefore, this invention provides a biological cultivation method that can effectively promote the growth of Artemisia annua and simultaneously increase its medicinal components, providing theoretical support for achieving synergistic effects between Artemisia annua yield and medicinal components, and promoting the application of microbial pesticides. Summary of the Invention

[0006] The purpose of this invention is to provide a method for increasing the biomass and medicinal component content of Artemisia annua by applying Agrobacterium BA51 to the cultivation process of Artemisia annua, thereby solving the technical problems of low biomass, unstable content of medicinal components (such as artemisinin), and dependence on chemical fertilizers or growth regulators in existing Artemisia annua cultivation.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: an Agrobacterium species for increasing the biomass and medicinal component content of Artemisia annua, wherein the Agrobacterium species is named BA51 and the preservation number is CCTCC NO: M20251734.

[0008] The present invention further provides a method for increasing the biomass and medicinal component content of Artemisia annua.

[0009] Preferably, this is achieved by inoculating Artemisia annua plants with Agrobacterium BA51.

[0010] Preferably, the steps include:

[0011] S1. The Agrobacterium BA51 is subjected to liquid fermentation culture to prepare a bacterial suspension;

[0012] S2. Apply the above-mentioned bacterial suspension to the rhizosphere soil of Artemisia annua by root irrigation.

[0013] Preferably, the final concentration of the bacterial suspension in step S1 is 1×10⁻⁶. 6 CFU / mL.

[0014] Preferably, the application operation in step S2 is carried out in stages during the seedling stage and the rapid growth stage of Artemisia annua.

[0015] Preferably, the specific steps for the application in multiple applications are as follows: the first application begins at the seedling stage, and the application is repeated every 10-15 days until harvest.

[0016] The present invention further provides the application of Agrobacterium BA51 in the above method.

[0017] Preferably, the Agrobacterium BA51 is used to prepare products that promote the growth of Artemisia annua and increase the content of medicinal components.

[0018] Preferably, the product is at least one of a reagent, a microbial agent, or a microbial fertilizer.

[0019] Preferably, the reagent further includes pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutically acceptable excipient is selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

[0021] The beneficial effects of this invention are:

[0022] 1. Significantly promotes plant vegetative growth: The key agronomic traits of Artemisia annua treated with BA51 strain, including plant height, aboveground fresh and dry weight, root length and root biomass, were significantly improved compared with the uninoculated control group, indicating that the strain can effectively enhance the photosynthetic and material accumulation capacity of Artemisia annua.

[0023] 2. Effectively shortens the growth cycle and optimizes the growth process: The plants in the treatment group showed more vigorous growth and their growth rate was significantly accelerated. They were able to enter the key physiological stage of synthesis and accumulation of medicinal active ingredients earlier, which may shorten the production cycle and improve cultivation efficiency.

[0024] 3. Synergistic Enhancement of Multiple Medicinal Components: Application of strain BA51 not only specifically increased the content of the target product artemisinin, but also simultaneously and significantly enhanced the levels of multiple medicinally valuable secondary metabolites in the leaves, including chlorogenic acid, rutin, isoquercitrin, quercetin, scopolamine, eugenol, and eugenol. This indicates that this strain has a broad positive regulatory effect on the secondary metabolic pathways of Artemisia annua, rather than inducing a single component.

[0025] 4. Providing a green and efficient cultivation method: This technical solution is environmentally friendly, has a simple operation process, and does not rely on exogenous chemically synthesized plant growth regulators, providing a sustainable microbial solution for the high-quality and high-yield cultivation of Artemisia annua. The widespread application of this method can provide solid technical support for the stable and efficient production of artemisinin and other related medicinal components, and has significant industrial application value. Attached Figure Description

[0026] Figure 1 This is the phylogenetic tree of the BA51 strain in this invention;

[0027] Figure 2 These are colony morphology photographs of the BA51 strain in this invention;

[0028] Figure 3 This is a comparison diagram of the growth-promoting effect of strain BA51 on Artemisia annua tissue culture seedlings in this invention;

[0029] Figure 4This is a key flavonoid metabolite spectrum of the BA51 strain and Artemisia annua tissue culture seedlings after 20 days of co-culture in this invention;

[0030] Figure 5 This is an artemisinin spectrum of the BA51 strain and Artemisia annua tissue culture seedlings after 20 days of co-culture.

[0031] Figure 6 This is a flowchart of the method for promoting the growth of Artemisia annua and increasing its medicinal component content in this invention;

[0032] Figure 7 This is a comparison diagram of the morphology of Artemisia annua plants in the soil-grown potted plants of the present invention between the control group and the BA51 treatment group.

[0033] Figure 8 This is a spectrum of key flavonoid metabolites in Artemisia annua from the control group and BA51 treatment group in soil-grown potted plants in this invention;

[0034] Figure 9 This is an artemisinin spectrum of Artemisia annua from the control group and the BA51 treatment group in soil-grown potted plants in this invention. Detailed Implementation

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] Example 1: Isolation, purification, and molecular identification of Agrobacterium spp.

[0039] 1. Method

[0040] Isolation and Purification: Intact roots of healthy Artemisia annua were collected, rinsed with running water for 60 min to remove soil, and surface sterilized in a laminar flow hood: sequentially shaken with 75% ethanol for 60 s, shaken with 0.1% mercuric chloride for 3 min, and finally rinsed 5 times with sterile water. The sterilized root segments were thoroughly ground in a sterile mortar with an appropriate amount of PBS buffer, serially diluted, and 100 µL of each solution was spread onto NA plates (5 g / L peptone, 3 g / L beef extract, 10 g / L sodium chloride, 18 g / L agar). Another 100 µL of the final rinse solution was spread onto another plate, and the sterilized root segments were imprinted on another NA plate as a control. All plates were incubated in the dark at 28 ℃. If no microbial growth was observed on the control plate, sterilization was considered complete. After the diluted plating showed bacterial growth, single colonies were picked and streaked repeatedly on LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar) for purification through a combination of liquid culture and serial dilutions, yielding strain BA51. The purified strain was finally cryopreserved at -80 °C using 25% glycerol as a cryoprotectant.

[0041] Molecular identification: Genomic DNA was extracted from BA51 bacteria using the Ezup column-based bacterial genomic DNA extraction kit (Sangon Biotech). The primers used for PCR amplification were as follows:

[0042] 7F: 5′-CAGAGTTTGATCCTGGCT-3′;

[0043] 1540R: 5′-AGGAGGGTGATCCAGCCGCA-3′.

[0044] The PCR reaction system (25 μL) contains: 0.2 μL Taq enzyme, 0.5 μL template DNA, 0.5 μL each of forward and reverse primers (10 μM), and 2.5 μL 10× Buffer (containing Mg). 2+ 1 μL of dNTPs were added, and ddH2O was added to make up the volume. The reaction program was: 94 ℃ pre-denaturation for 4 min; 30 cycles (94 ℃ 45 s, 55 ℃ 45 s, 72 ℃ 1 min); final extension at 72 ℃ for 10 min. The amplified products were sequenced after electrophoresis on a 1% agarose gel (150 V, 20 min). The obtained 16S rDNA sequence was BLAST-aligned in the NCBI database to determine the bacterial genus (http: / / www.ncbi.nlm.nih.gov). A phylogenetic tree was constructed using MEGA11 software. Figure 1 ).

[0045] 2. Results Analysis

[0046] BA51 is a bacterium belonging to the genus *Agrobacterium tumefaciens*, with accession number MH503856.1. The BA51 strain is round, milky white, with a smooth, moist surface, neat edges, a soft texture, and is opaque. Figure 2 After streak cultivation, it exhibits a fused, filamentous, spreading growth pattern.

[0047] The strain BA51 is classified as Agrobacterium tumefaciens BA51. It was deposited at the China Center for Type Culture Collection (CCTCC) on July 31, 2025, with accession number CCTCC NO: M 20251734. The deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China.

[0048] Example 2: Validation of co-culture of BA51 strain with Artemisia annua tissue culture seedlings

[0049] 1. Experimental Objective

[0050] Under strictly controlled aseptic conditions, the direct promoting effect of strain BA51 on the growth of Artemisia annua was verified, and its promoting effect on the synthesis of medicinal components was preliminarily explored. Thus, under the premise of eliminating interference from complex environmental factors such as soil, the core growth-promoting function of this strain was confirmed, providing a theoretical basis for subsequent potted and field applications.

[0051] 2. Materials and Methods

[0052] Aseptic seedlings of Artemisia annua were subcultured in MS medium supplemented with 0.5 mg / L NAA and 0.1 mg / L IAA. After 20 days of culture, seedlings with uniform growth were selected as inoculation material. Endophytic bacteria stored at low temperature were activated and prepared at a concentration of 7.5 × 10⁻⁶. 5 CFU / mL bacterial suspension. Each seedling in the BA51 treatment group was inoculated with 1 mL of bacterial suspension at the root, while the control group was inoculated with an equal volume of sterile water. All treatments were replicated five times and cultured for 20 days at 25 ℃ with a light intensity of 1500-2000 lux and a 12 h / 12 ​​h light / dark cycle.

[0053] After co-cultivation, growth indicators such as plant fresh weight, plant height, and root length were measured. Simultaneously, the contents of flavonoid components (including chlorogenic acid, rutin, isoquercitrin, quercetin, scopolamine, styraxanthin, and styraxanthin) and artemisinin in the leaves were measured.

[0054] Sample preparation: Accurately weigh 0.2 g of fresh leaves and place them in a brown vial. Add 4 mL of methanol and extract by sonication for 30 min. Filter the extract through a 0.22 μm organic microporous membrane, and use the filtrate as the flavonoid sample solution. Dilute another portion of the filtrate 200 times with methanol as the artemisinin sample solution. Calculate the concentration of each metabolite using the external standard method. Each treatment was performed in triplicate.

[0055] The flavonoid fraction was analyzed using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Shim-pack CLC-ODS column (150 × 6.0 mm, 5 µm), column temperature 40 ℃, detection wavelength 345 nm; mobile phase A was acetonitrile-methanol (11:5, v / v), and mobile phase B was 0.1% formic acid aqueous solution. The gradient elution program was set as follows: 6–18% A (0–8 min), 18–34% A (8–26 min), 34–40% A (26–35 min), 40–100% A (35–44 min), 100% A (44–53 min), 100–6% A (53–60 min). Flow rate: 1.4 mL / min. -1 (0-15 min), 0.8 mL·min -1 (15-35 min), 1.2 mL·min -1 (35-45 min), 1.4 mL·min -1 (45-60 min).

[0056] Artemisinin content was determined using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Chromatographic conditions: Thermo Scientific Hypersil GOLD column (50 × 2.1 mm, 5 μm); mobile phase A was 0.1% formic acid-acetonitrile solution, and mobile phase B was 0.1% formic acid aqueous solution; the elution program was 0–3 min, A:B = 4:1 (isocratic); the flow rate was 200 μL / min. -1 Mass spectrometry detection was performed using electrospray ionization (ESI) positive ion mode. The main parameters were: spray voltage 2500 V, sheath gas flow rate 35 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 270 ℃, and nebulization temperature 200 ℃.

[0057] Data analysis: Independent samples t-tests were performed using SPSS software to compare the statistical significance of differences between the treatment group and the control group (P < 0.05).

[0058] 3. Experimental Results

[0059] (1) Effects on the growth of Artemisia annua: The results of the growth-promoting effect of strain BA51 on Artemisia annua tissue culture seedlings showed that, compared with the control group, after 20 days of co-culture, all growth indicators of Artemisia annua tissue culture seedlings in the treatment group inoculated with strain BA51 were significantly improved. Figure 3 The growth and main medicinal component indicators are shown in Table 1. Plant height and aboveground fresh weight increased by 46.90% and 65.76%, respectively, indicating that BA51 significantly promoted the vegetative growth of the aboveground parts. Root length and root fresh weight increased by 48.09% and 69.17%, respectively, indicating that BA51 had a very significant promoting effect on the morphogenesis and biomass accumulation of Artemisia annua roots. At the same time, the aboveground dry weight of Artemisia annua increased by 98.72%, indicating that BA51 inoculation had a significant promoting effect on the biomass of Artemisia annua.

[0060] (2) Effects on medicinal components: HPLC chromatographic analysis showed that the levels of chlorogenic acid, rutin, isoquercitrin, quercetin, scopolamine, eugenol, and eugenol in the leaves of the treated group increased by 1026.34%, 550.90%, 127.67%, 1916.59%, 587.00%, 74.53%, and 229.67%, respectively. Simultaneously, the content of artemisinin also increased significantly, approximately 3.5 times that of the control group. Figure 4 , Figure 5 The specific parameters are shown in Table 1. This indicates that strain BA51 not only promotes growth but also promotes the accumulation of medicinal components in Artemisia annua.

[0061] Table 1. Growth and main medicinal components of strain BA51 after 20 days of co-culture with Artemisia annua tissue culture seedlings

[0062]

[0063] Note: Data are mean ± standard deviation (n=5); different lowercase letters in the same row indicate significant differences between different treatments (P < 0.05).

[0064] 4. Conclusion

[0065] This embodiment demonstrates, through a rigorous tissue culture co-culture system, that *Agrobacterium BA51* can directly and effectively promote the growth of sterile *Artemisia annua* seedlings without relying on the soil ecosystem. Simultaneously, this strain can preliminarily induce an increase in the content of key secondary metabolites and artemisinin in *Artemisia annua* leaves. These results confirm the plant growth-promoting and metabolic-inducing activities inherent in the BA51 strain, providing the intrinsic basis for its effectiveness in subsequent potted and field applications.

[0066] Application Example 1: Soil Effect Verification Experiment on BA51 Inoculant Promoting the Growth and Medicinal Component Accumulation of Artemisia annua.

[0067] 1. Materials

[0068] Plant materials: Seeds of superior varieties of Artemisia annua with consistent genetic background were selected.

[0069] Strains and Preparation: Agrobacterium BA51 with accession number CCTCC NO: M 20251734 was used. The bacteria were activated by streaking from a -80 ℃ storage tube. Single colonies were picked and inoculated into LB liquid medium, and cultured at 28 ℃ with shaking at 180 rpm until the late logarithmic phase (OD600≈1.0). The cells were collected by centrifugation, resuspended in sterile physiological saline, and diluted to a concentration of 1×10⁻⁶ before application. 6 CFU / mL.

[0070] Experimental design: Two treatment groups were set up: (1) Control group (CK): Irrigated with an equal volume of sterile LB medium diluted solution. (2) BA51 treatment group (T): Irrigated with BA51 bacterial suspension. Each group had at least 5 biological replicates.

[0071] Cultivation and Management: Sow seeds in sterilized potting soil for seedling raising. When seedlings have 4-6 true leaves (seedling stage), select seedlings with uniform growth and transplant them into individual pots. After the plants have stabilized, perform the first root drenching inoculation, watering each plant with 20 mL of bacterial suspension or control solution. Thereafter, repeat the inoculation every 10-15 days until the end of the vegetative growth stage. During this period, manage water and fertilizer as usual.

[0072] 2. Methods

[0073] A method for increasing the biomass and medicinal component content of Artemisia annua ( Figure 6 ), including the following steps:

[0074] Preliminary preparations:

[0075] (1) Treatment of seedling substrate

[0076] Select loose, nutrient-rich yellow soil as the seedling substrate. After natural air drying, crushing and removing plant residues, sterilize it with high-temperature steam at 121℃ for 100 minutes. After cooling, it is divided into seedling pots for later use.

[0077] (2) Cultivation of Artemisia annua seedlings

[0078] Sow the seeds of Artemisia annua in the seedling substrate at a depth of 2-3 cm, and lightly cover with fine soil. After sowing, keep the substrate moderately moist, avoid direct sunlight, and cultivate under suitable temperature conditions until seedlings emerge.

[0079] Operating steps:

[0080] S1. Preparation of bacterial suspension

[0081] Agrobacterium BA51 was inoculated onto LB solid medium and cultured at 28 °C for 3 days. Single colonies were then transferred to LB liquid medium and cultured with shaking at 28 °C and 180 rpm for 12 h to prepare a 1×10⁻⁶ colony. 6 CFU / mL bacterial suspension.

[0082] S2. Cultivation Management

[0083] When the Artemisia annua seedlings grow to 4-6 cm, start applying the microbial agent regularly. Spray the BA51 bacterial suspension prepared by S1 every 10-15 days, while carrying out routine water and fertilizer management until the plants mature and are harvested.

[0084] The determination of growth and medicinal component content is the same as that described in Example 2.

[0085] 3. Experimental Results

[0086] (1) Effects on the growth of Artemisia annua: The comparison of plant morphology between the control group and the BA51 treatment group in soil-grown potted plants showed that BA51 inoculant had a significant promoting effect on the biomass of Artemisia annua. Figure 7 The growth indicators and the content of the main medicinal components are shown in Table 2. The aboveground fresh weight and dry weight of the treatment group (T) increased by 246.44% and 224.07% respectively compared with the control group (CK), and the plant height increased significantly by 49.24%. At the same time, the root length and root fresh weight also increased by 22.67% and 177.50% respectively, indicating that BA51 inoculation has a significant promoting effect on the overall growth of Artemisia annua.

[0087] (2) Effects on medicinal components: HPLC chromatographic analysis showed that the levels of chlorogenic acid, rutin, isoquercitrin, quercetin, scopolamine, eugenol, and eugenol in the leaves of the treated group increased by 83.35%, 122.38%, 553.89%, 586.79%, 153.28%, 53.76%, and 84.40%, respectively. The artemisinin content increased from 1.28 mg / g to 2.91 mg / g, an increase of 128.19%. Figure 8 , Figure 9 This indicates that the quality of the medicinal materials has been significantly improved.

[0088] Table 2. Effects of BA51 inoculant on the growth and content of major medicinal components of soil-cultured Artemisia annua.

[0089]

[0090] Note: Data are mean ± standard deviation (n=5); different lowercase letters in the same row indicate significant differences between different treatments (P < 0.05).

[0091] In conclusion, inoculation with Agrobacterium BA51 effectively promotes the growth of Artemisia annua and significantly increases the accumulation of various medicinal active ingredients. This method is simple to operate, has good reproducibility, and shows promising prospects for widespread application.

[0092] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A strain of *Agrobacterium* for increasing the biomass and medicinal component content of *Artemisia annua*, characterized by: The Agrobacterium species was named BA51 and its accession number is CCTCC NO: M 20251734.

2. A method for increasing the biomass and medicinal component content of Artemisia annua, characterized in that: This is achieved by inoculating Artemisia annua plants with the Agrobacterium BA51 described in claim 1.

3. The method according to claim 2, characterized in that: Includes the following steps: S1. The Agrobacterium BA51 is subjected to liquid fermentation culture to prepare a bacterial suspension; S2. Apply the above-mentioned bacterial suspension to the rhizosphere soil of Artemisia annua by root irrigation.

4. The method according to claim 3, characterized in that: The final concentration of the bacterial suspension in step S1 is 1×10⁻⁶. 6 CFU / mL.

5. The method according to claim 3, characterized in that: The application in step S2 is carried out in stages during the seedling stage and the rapid growth stage of Artemisia annua.

6. The method according to claim 5, characterized in that: The specific steps for the application in stages are as follows: apply for the first time at the seedling stage, and repeat the application every 10-15 days until harvest.

7. The application of Agrobacterium BA51 according to claim 1, characterized in that: The Agrobacterium BA51 was used to prepare products that promote the growth of Artemisia annua and increase the content of medicinal components.

8. The application according to claim 7, characterized in that: The product is at least one of a reagent, a microbial agent, or a microbial fertilizer.

9. The application according to claim 8, characterized in that: The reagent also includes pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that: The pharmaceutically acceptable excipients are selected from at least one of the following pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and sustained-release materials.

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