Phytochemical Composition, Anti-Rheumatoid Arthritis Potential and Toxicity Profiles of Methanol Extracts of Zanthoxylum Usambarense Engl. And Rhamnus Prinoides L’hér
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Date
2023
Authors
James, Kamau Kimani
Journal Title
Journal ISSN
Volume Title
Publisher
Kenyatta University
Abstract
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune ailment marked by
synovial joint edema, stiffness and pain resulting in severe disability and sometimes
premature fatalities. A combination of glucocorticoids, non-steroidal anti-inflammatory
drugs and disease-modifying anti-rheumatic drugs are often prescribed to treat RA.
However, these medications are linked to severe effects, necessitating alternative medical
interventions. Herbal medicines are regarded as alternative therapeutics as they are
considered efficacious, easily biodegradable and have arguably minimal severe effects. In
Kenya, the Maasai Community uses the root bark of Zanthoxylum usamberense and the
stem bark of Rhamnus prinoides to treat RA. However, there is a paucity of scientific
evidence on quantitative phytochemical screening, anti-RA potential and mechanistic
effects, as well as toxicity profiles of the two acclaimed medicinal plants. The current study
objectives were to determine the qualitative and quantitative phytochemical composition,
anti-RA potential, expressions of selected RA biomarker genes as well as acute and subacute
toxicities profiles of MeOH extracts of the two medicinal plants. Plant samples were
collected in Narok County, Kenya. The two medicinal plant samples were identified by a
taxonomist, prepared, and extracted using MeOH. Liquid chromatography-mass
spectrometry (LC-MS) and GC-MS (Gas chromatography-mass spectrometry) were used in
qualitative and quantitative phytochemical analysis. In the anti-RA assay, rats were
assigned 6 groups of 5 rats each: normal control, disease control, positive control
(methotrexate), and 3 extract-treated groups (100, 200 and 300mg/kg body weight (bw)).
RA was induced using complete Freud’s adjuvant (CFA). Methotrexate (weekly) and
extracts (daily) at the three doses were orally administered from day 8 post-induction of RA.
The experimental rats were euthanized, and blood was drawn for hematological and
biochemical indices on the 29th day. The liver and tissue around the ankle joint were also
detached and used for antioxidant and gene expression assays, respectively. Selected RA
biomarker genes were assayed using quantitative Real-Time Polymerase Chain Reaction
assay. In vivo acute and sub-acute toxicological studies were performed according to the
OECD guidelines 425 and 407, respectively. The LC-MS analysis identified
phytochemicals of flavonoids, phenolic acids, tannins, stilbenes, phytosterols,
anthraquinones, and coumarins classes. Fatty acid methyl ester, fatty acid, diterpenes,
steroids, lignans, benzofuran, pyranone and triterpene were also detected using GC-MS.
The two extracts significantly attenuated body weight loss, alleviated ankle joint edema,
reduced arthritis scores, ameliorated increased thymus and spleen indices, attenuated
aberrant hematological and biochemical parameters, reduced levels of malonaldehyde and
enhanced enzymatic antioxidant activities following CFA-induced RA in rats. The two
extracts also significantly downregulated expression of STAT-3, MMP-3, NF-κB, RANKL,
COX-2, TNF-α, IL-1β and IL-6 and genes, as well as upregulated expression of IL-4, IL-10
and I-κBα genes following CFA-induced RA in rats. Zanthoxylum usambarense had better
anti-RA effects than R. prinoides. At the highest dosage of 2000mg/kg bw, the acute
toxicity tests of the two extracts neither caused toxicity signs nor fatalities in rats. The subacute
toxicity study of the two extracts did not adversely affect body weights, relative organ
weights, hematological parameters, liver function and renal function biomarkers, lipid
profiles and blood glucose levels, except for AST levels in rats administered with R.
prinoides extract dose of 900mg/kg bw. In conclusion, the two extracts possess
phytochemicals with anti-RA potential and can be used as potential leads in developing
anti-RA agents with minimal side effects. The selected RA biomarker genes can also be
used as targets in developing anti-RA agents. Therefore, the current study scientifically
validates the folkloric use of Z. usambarense and R. prinoides in managing RA. However,
the classes of phytocompounds with anti-RA activity should be isolated and their effects
determined.
Description
A Thesis Submitted in Fulfillment of the Requirements for the Award of the Degree of Doctor of Philosophy (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University June, 2023
Keywords
Phytochemical Composition, Anti-Rheumatoid Arthritis Potential, Toxicity Profiles, Methanol Extracts, Zanthoxylum Usambarense Engl., Rhamnus Prinoides L’hér