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Viola odorata plant extracts are extensively used in indigenous medicine to treat infectious diseases. V. odorata's limited availability in nature, due to geographical conditions and adulteration, makes continuous supply of high-quality plant raw material challenging. This results in low-quality plant extracts that can have inconsistent and sometimes adverse effects when used in medicine. As an effective solution, in vitro plant biomass cultivation conserves plant diversity, ensuring high-quality raw materials with uniform quality and quantity. Toward this, the current study focuses on establishing bioprocess strategy to achieve maximum V. odorata biomass productivity up to bioreactor level. In this context, in vitro culture conditions were rationally optimised, focusing upon both statistical optimization and one-factor-at-a-time (OFAT) approach, for improved biomass productivity. In particular, basal medium, carbon and nitrogen source, inoculum density, macronutrient concentration, shaking speed, temperature, pH, light intensity, and plant growth regulators were systematically optimized in order to achieve the maximum biomass. This led to increased biomass production from 10.2 ± 0.8 g DW L-1 to 21.68 ± 0.82 g DW L-1 within 12 days of the batch cultivation period. Optimized culture conditions were implemented in three different types of bioreactors, namely, stirred tank, airlift, and bubble column, to cultivate V. odorata biomass. Of these three methods, cultivation in a stirred tank reactor produced ~19.7 g DW L-1 of maximum biomass within a 12-day batch cultivation period. Further, the metabolite profile of the bioreactor cultivated biomass (alcoholic extract) was compared with that of the natural plant biomass using gas chromatography-mass spectrometry based metabolomics, yielding around 18 key principles (metabolites) that exhibit resemblance with that in the natural source. The antibacterial activity of the plant extract was tested against three bacteria responsible for respiratory tract infections, and their antioxidant activity was compared with that of Vitamin E, wherein the results demonstrated comparable activity. The novelty of this study is in the fact that the V. odorata plant cells were cultivated in bioreactors for the first time for potential medicinal applications. It provides an alternative method for sustainable production of plant biomass, thereby conserving the natural plant sources.
Viola odorata plant extracts are extensively used in indigenous medicine to treat infectious diseases. V. odorata's limited availability in nature, due to geographical conditions and adulteration, makes continuous supply of high-quality plant raw material challenging. This results in low-quality plant extracts that can have inconsistent and sometimes adverse effects when used in medicine. As an effective solution, in vitro plant biomass cultivation conserves plant diversity, ensuring high-quality raw materials with uniform quality and quantity. Toward this, the current study focuses on establishing bioprocess strategy to achieve maximum V. odorata biomass productivity up to bioreactor level. In this context, in vitro culture conditions were rationally optimised, focusing upon both statistical optimization and one-factor-at-a-time (OFAT) approach, for improved biomass productivity. In particular, basal medium, carbon and nitrogen source, inoculum density, macronutrient concentration, shaking speed, temperature, pH, light intensity, and plant growth regulators were systematically optimized in order to achieve the maximum biomass. This led to increased biomass production from 10.2 ± 0.8 g DW L-1 to 21.68 ± 0.82 g DW L-1 within 12 days of the batch cultivation period. Optimized culture conditions were implemented in three different types of bioreactors, namely, stirred tank, airlift, and bubble column, to cultivate V. odorata biomass. Of these three methods, cultivation in a stirred tank reactor produced ~19.7 g DW L-1 of maximum biomass within a 12-day batch cultivation period. Further, the metabolite profile of the bioreactor cultivated biomass (alcoholic extract) was compared with that of the natural plant biomass using gas chromatography-mass spectrometry based metabolomics, yielding around 18 key principles (metabolites) that exhibit resemblance with that in the natural source. The antibacterial activity of the plant extract was tested against three bacteria responsible for respiratory tract infections, and their antioxidant activity was compared with that of Vitamin E, wherein the results demonstrated comparable activity. The novelty of this study is in the fact that the V. odorata plant cells were cultivated in bioreactors for the first time for potential medicinal applications. It provides an alternative method for sustainable production of plant biomass, thereby conserving the natural plant sources.
Turpinia arguta is an excellent medicinal plant mainly used for the treatment of pharyngitis, tonsillitis, and tonsillar abscesses. However, an efficient regeneration protocol using tissue cultures for T. arguta does not exist. Its main medicinal constituents are flavonoids, particularly ligustroflavone and rhoifolin. Here, we aimed to establish a tissue culture system for T. arguta for the first time using annual stem segments with axillary buds harvested from the field of the Jiangxi Academy of Forestry as explants by dynamically determining the accumulation of effective functional components in the tissue culture plantlets. Orthogonal tests were conducted to compare the effects of different explant disinfection times, media, and exogenous hormone ratios on the induction of the axillary bud growth, successional proliferation, and rooting of T. arguta stem segments. The best explant disinfection effect was achieved by disinfecting the T. arguta explant with 75% ethanol for 50 s, followed by 0.1% mercuric chloride (HgCl2) for 6 min, and the optimal media for successional proliferation and rooting were Murashige and Skoog (MS) + 0.2 mg/L of 6-benzyladenine (6-BA), + 0.03 mg/L of naphthaleneacetic acid (NAA), and ½ MS + 2.5 mg/L of indole-3-butyric acid + 0.5 mg/L of NAA, respectively. The detection of ligustroflavone and rhoifolin in tissue culture plantlets 0, 3, and 5 months after transplanting showed a significant increasing trend and eventually exceeded the content requirements of the 2020 Edition ofChinese Pharmacopoeia for T. arguta. Our findings provide, for the first time, an effective tissue culture system for T. arguta, thereby providing important information to support the germplasm preservation, innovation, and application of T. arguta in the future.
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