In vitro propagation of Codonopsis pilosula (Franch.) Nannf. using apical shoot segments and phytochemical assessments of the maternal and regenerated plants.

BACKGROUND: Codonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC-MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared. RESULTS: Full-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L-1) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L-1) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants. CONCLUSIONS: The proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand.

Influence of various temperatures, seed priming treatments and durations on germination and growth of the medicinal plant Aspilia africana.

For millennia, Aspilia africana has been used across Africa to treat various diseases including malaria, wounds, and diabetes. In this study, temperature influenced the in vitro germination of A. africana with highest final germination percentage (FGP) and germination index (GI) of 65.0 ± 7.64% and 2.26 ± 0.223, respectively, at 19.8 °C. Priming seeds with H2O, KNO3, and GA3 (gibberellic acid 3) improved both in vitro germination and ex vitro emergence of A. africana seeds. Seed priming with [Formula: see text] M GA3 produced overall highest in vitro FGP (from 90.0 ± 4.08% to 100 ± 0.00%) and GI (from 2.97 ± 0.385 to 3.80 ± 0.239) across all priming durations. Seeds primed with KNO3 had better germination parameters for 6 and 12 h compared to 18 and 24 h. Furthermore, the highest in vitro FGP (100 ± 0.00%) was observed in seeds primed for 12 h with [Formula: see text] M GA3. Ex vitro A. africana seed emergence was significantly enhanced by GA3 priming. Priming A. africana seeds with H2O, KNO3, and GA3 improved their growth after 3 months, with the overall best growth for seeds primed with [Formula: see text] M GA3. Seed priming of A. africana is a feasible approach for improving germination and seed emergence, and enhancing plant growth.

GC-MS and LC-TOF-MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark.

Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and ß-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1ß secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and ß-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p < 0.001) TRAP activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited significant (p < 0.05) BMM cell viability. NO production was significantly inhibited (all p < 0.0001) by the sample. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and ß-sitosterol (p < 0.0001) and further, chlorogenic acid and naringenin remarkably inhibited IL-1ß production. The P. africana methanolic extract significantly inhibited RANKL-induced TRAP activity. The phytochemical study of P. africana stem bark revealed a number of chemical compounds with anti-osteoporosis activity. There was no observed hepatocyte apoptosis in the liver of zebrafish larvae. In conclusion, the stem bark of P. africana is non-toxic to the liver and its inhibition of TRAP activity makes it an important source for future anti-osteoporosis drug development.

Root Extract of a Micropropagated Prunus africana Medicinal Plant Induced Apoptosis in Human Prostate Cancer Cells (PC-3) via Caspase-3 Activation.

Prostate cancer is one of the major causes of cancer-related deaths among men globally. Medicinal plants have been explored as alternative treatment options. Herein, we assessed the in vitro cytotoxic effects of 70% ethanolic root extracts of six-month-old micropropagated Prunus africana (PIR) on PC-3 prostate cancer cells as an alternative to the traditionally used P. africana stem-bark extract (PWS) treatment. In vitro assays on PC-3 cells included annexin-V and propidium iodide staining, DAPI staining, and caspase-3 activity analysis through western blotting. PC-3 cells were exposed to PWS and PIR at different concentrations, and dose-dependent antiprostate cancer effects were observed. PC-3 cell viability was determined using CCK-8 assay, which yielded IC50 values of 52.30 and 82.40 µg/mL for PWS and PIR, respectively. Annexin-V and PI staining showed dose-dependent apoptosis of PC-3 cells. Significant (p < 0.001) percent of DAPI-stained apoptotic PC-3 cells were observed in PWS, PIR, and doxorubicin treatment compared with the negative control. PWS treatment substantially elevated cleaved caspase-3 levels in PC-3 cells compared with the PIR treatment. These results provide evidence for the antiprostate cancer potential of PIR and sets a basis for further research to enhance future utilization of roots of young micropropagated P. africana for prostate cancer treatment as an alternative to stem bark. Moreover, micropropagation approach may help provide the required raw materials and hence reduce the demand for P. africana from endangered wild population.

Antioxidant Activity, Polyphenolic Content, and FT-NIR Analysis of Different Aspilia africana Medicinal Plant Tissues.

Aspilia africana has been used for generations to treat many diseases in Africa. Its biological activities, including antioxidant and anti-inflammatory potential, are attributed to a number of secondary metabolites, including alkaloids and polyphenolics. The antioxidant activities of A. africana callus (CA), juvenile in vitro leaf (IL) and root (IR), ex vitro root (SR) and leaf (SL), and wild leaf (WL) dried samples were assessed based on their diphenylpicrylhydrazyl (DPPH) free radical scavenging abilities. The total phenolic and flavonoid content of different plant samples was compared. Further, high-pressure liquid chromatography (HPLC) was used to quantitatively determine chlorogenic acid content in the A. africana plant samples. Fourier transform near-infrared (FT-NIR) analysis was also carried out to compare the antioxidant phytochemical content in the A. africana plant tissues. Among the samples, IR, with the highest total phenolic content (167.84 ± 1.057 mg GAE/g), total flavonoid content (135.06 ± 0.786 mg RUE/g), and chlorogenic acid (5.23 ± 0.298 mg/g) content, had the most potent antioxidant activity (IC50 = 27.25 ± 5.028 µg/mL), followed by WL. The lowest polyphenolic content and antioxidant activity were observed in SR. The antioxidant activities of A. africana tissues were positively correlated with the total phenolic and flavonoid content in the samples. The differences in antioxidant activities of A. africana tissues could be attributed to the difference in their polyphenolic content. Our study reports, for the first time, the antioxidant activities of A. africana callus and roots (in vitro and ex vitro). The A. africana samples IR, CA, and WL could be valuable natural sources of antioxidants that could be further exploited for the development of useful pharmaceutical products.

An in vitro Propagation of Aspilia africana (Pers.) C. D. Adams, and Evaluation of Its Anatomy and Physiology of Acclimatized Plants.

Aspilia africana (Pers.) C. D. Adams is an important medicinal plant, that has been used as traditional medicine in many African countries for the treatment of various health problems, including inflammatory conditions, osteoporosis, tuberculosis, cough, measles, diabetes, diarrhea, malaria, and wounds. We developed an efficient and reproducible protocol for in vitro regeneration of A. africana from nodes. We assessed the effects of plant tissue culture media on A. africana growth, cytokinins for in vitro shoot regeneration and proliferation, and auxins for the rooting of regenerated shoots. Furthermore, chlorophyll content, photosynthetic rates, anatomy (leaves, stems, and roots), and Fourier transform near-infrared (FT-NIR) spectra (leaves, stems, and roots) of the in vitro regenerated and maternal A. africana plants were compared. Murashige and Skoog media, containing vitamins fortified with benzylaminopurine (BA, 1.0 mg/l), regenerated the highest number of shoots (13.0 ± 0.424) from A. africana nodal segments. 1-naphthaleneacetic acid (NAA, 0.1 mg/l) produced up to 13.10 ± 0.873 roots, 136.35 ± 4.316 mm length, and was the most efficient for rooting. During acclimatization, the in vitro regenerated A. africana plants had a survival rate of 95.7%, displaying normal morphology and growth features. In vitro regenerated and mother A. africana plants had similar chlorophyll contents, photosynthetic rates, stem and root anatomies, and FT-NIR spectra of the leaf, stem, and roots. The established regeneration protocol could be used for large-scale multiplication of the plant within a short time, thus substantially contributing to its rapid propagation and germplasm preservation, in addition to providing a basis for the domestication of this useful, high-value medicinal plant.

Indirect in vitro Regeneration of the Medicinal Plant, Aspilia africana, and Histological Assessment at Different Developmental Stages.

The medicinal plant, Aspilia africana, has been traditionally used in several African countries to treat many diseases such as tuberculosis, cough, inflammation, malaria, osteoporosis, and diabetes. In this study, we developed a protocol for in vitro propagation of A. africana using indirect shoot organogenesis from leaf and root explants of in vitro-grown seedlings and assessed the tissues at different developmental stages. The highest callus induction (91.9 ± 2.96%) from leaf explants was in the Murashige and Skoog (MS) medium augmented with 1.0 mg/L 6-Benzylaminopurine (BAP) and 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) while from root explants, the highest callus induction (92.6 ± 2.80%) was in the same plant tissue culture medium augmented with 0.5 mg/L BAP and 1.0 mg/L 2,4-D. The best shoot regeneration capacity from leaf-derived calli (i.e., 80.0 ± 6.23% regeneration percentage and 12.0 ± 6.23 shoots per callus) was obtained in medium augmented with 1.0 mg/L BAP and 0.05 mg/L α-Naphthaleneacetic acid (NAA); the best regeneration capacity for root-derived calli (i.e., 86.7 ± 6.24% shoot regeneration percentage and 14.7 ± 1.11 shoots per callus) was obtained in the MS medium augmented with 1.0 mg/L BAP, 0.05 mg/L NAA, and 0.1 mg/L Thidiazuron (TDZ). Regenerated plantlets developed a robust root system in 1/2 MS medium augmented with 0.1 mg/L NAA and had a survival rate of 93.6% at acclimatization. The in vitro regenerated stem tissue was fully differentiated, while the young leaf tissue consisted of largely unorganized and poorly differentiated cells with large intercellular airspaces typical of in vitro leaf tissues. Our study established a protocol for the indirect regeneration of A. africana and offers a basis for its domestication, large-scale multiplication, and germplasm preservation. To the best of our knowledge, this is the first study to develop an indirect regeneration protocol for A. africana and conduct anatomical assessment through the different stages of development from callus to a fully developed plantlet.

Ethnopharmacological Potentials of Warburgia ugandensis on Antimicrobial Activities.

Warburgia ugandensis (W. ugandensis) is known by various names, including the East African greenheart, pepper bark tree, and Ugandan greenheart, and has a rich history of extensive use in the treatment of a host of human diseases in many African countries. This review is based on the botany and ethnopharmacological potentials of W. ugandensis for the treatment of pneumonia, asthma, malaria, candidiasis, skin infections, human immunodeficiency virus opportunistic infections, diarrhea, and measles given the common use in the management of these diseases. Extracts from W. ugandensis have strong antimicrobial activities against a broad spectrum of pathogens mainly because of the presence of abundant terpenoids, drimane, and coloratane type sesquditerpenoids amongst which are ugandensial, warburganal, mukaadial, and other secondary metabolites, such as tannins, flavonoids, saponins, steroids, and mannitol. This group of compounds gives the plant a high therapeutic value. Based on the review, there is a need for identification and isolation of the highly therapeutic phytochemical constituents and a drive for more preclinical and clinical trials to validate the safety and efficacy of the extracts. This gives basis for the potential development of new therapeutic drugs from the plant.

A Micropropagation Protocol for the Endangered Medicinal Tree Prunus africana (Hook f.) Kalkman: Genetic Fidelity and Physiological Parameter Assessment.

Prunus africana is an endangered medicinal plant and hence new propagation methods are urgently required to increase its populations. Unfortunately, propagation through seeds is challenging due to its long flowering cycle and recalcitrant seeds. We developed a protocol for micropropagation using nodal segment explants. A woody plant medium supplemented with vitamins, 15 g L-1 sucrose, and 1.0 mg L-1 6-benzylaminopurine (BAP) supported the optimum rate (100%) of axillary shoot initiation. Supplementation with 15 g L-1 sucrose and 1.5 mg L-1 indole-3-acetic acid (IAA) provided the optimum rate (75%) of root initiation. Rooted plantlets were successfully planted in sterilized horticultural soil containing perlite (2:1 v/v) and the survival rate was 98% following acclimatization. The photosynthetic rate assessed using FlourPen FP110 series showed that the ratio of variable fluorescence to maximum fluorescence mean value for in vitro regenerated P. africana (0.830 ± 0.0008) was similar to that of the maternal P. africana plant (0.825 ± 0.005), indicating similarity in their photosynthetic performance; a pivotal process for growth and development. The Fourier transform near-IR (FT-NIR) spectrometer analysis of the in vitro regenerated and the maternal P. africana plant samples exhibited homogeneity in the absorbance peaks at 8,273, 6,344, and 4,938-4,500 cm-1 associated with lipids, starch, and proteins. The genetic fidelity of regenerated plants was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Our protocol is suitable for use in large-scale P. africana to meet the increasing demands for it in the global market.

Ethnopharmacological Potential of Aspilia africana for the Treatment of Inflammatory Diseases.

Inflammatory diseases are major health concerns affecting millions of people worldwide. Aspilia africana has been used for centuries by many African communities in the treatment of a wide range of health conditions, including inflammatory diseases, osteoporosis, rheumatic pains, and wounds. Analysis of the phytochemical composition of A. africana indicated that the plant is rich in a broad range of secondary metabolites, including flavonoids, alkaloids, tannins, saponins, terpenoids, sterols, phenolic compounds, and glycosides. This explains the efficacy of the plant in treating inflammation-related diseases, as well as several other health conditions affecting different African communities. The mechanisms of action of the anti-inflammatory phytochemical compounds in A. africana include inhibition of a number of physiological processes involved in the inflammatory process and synthesis or action of proinflammatory enzymes. The phytochemicals enhance anti-inflammatory biological responses such as inhibition of a number of chemical mediators including histamine, prostanoids and kinins, 5-lipoxygenase. and cyclooxygenase and activation of phosphodiesterase and transcriptase. Currently used anti-inflammatory medications are associated with several disadvantages such as drug toxicity and iatrogenic reactions, thereby complicating the treatment process. The adverse effects related to the use of these conventional synthetic drugs have been the driving force behind consideration of natural remedies, and efforts are being made toward the development of anti-inflammatory agents based on natural extracts. A. africana is rich in secondary metabolites, and its use as a traditional medicine for treating inflammatory diseases has been validated through in vitro and in vivo studies. Therefore, the plant could be further explored for potential development of novel anti-inflammatory therapeutics.

Exploring Antimalarial Herbal Plants across Communities in Uganda Based on Electronic Data.

Malaria is one of the most rampant diseases today not only in Uganda but also throughout Africa. Hence, it needs very close attention as it can be severe, causing many deaths, especially due to the rising prevalence of pathogenic resistance to current antimalarial drugs. The majority of the Ugandan population relies on traditional herbal medicines for various health issues. Thus, herein, we review various plant resources used to treat malaria across communities in Uganda so as to provide comprehensive and valuable ethnobotanical data about these plants. Approximately 182 plant species from 63 different plant families are used for malaria treatment across several communities in Uganda, of which 112 plant species have been investigated for antimalarial activities and 96% of the plant species showing positive results. Some plants showed very strong antimalarial activities and could be investigated further for the identification and validation of potentially therapeutic antimalarial compounds. There is no record of an investigation of antimalarial activity for approximately 39% of the plant species used for malaria treatment, yet these plants could be potential sources for potent antimalarial remedies. Thus, the review provides guidance for areas of further research on potential plant resources that could be sources of compounds with therapeutic properties for the treatment of malaria. Some of the plants were investigated for antimalarial activities, and their efficacy, toxicity, and safety aspects still need to be studied.