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American Journal of  
Chemistry and Pharmacy (AJCP)

Evaluation of  Chitosan-Based Nano-Encapsulated Diterpenoids from Andrographis 
paniculata for Targeted Anti-Inflammatory Therapy: Roles of  Analytical Chemistry

Medical Imaging, and Biochemical Assessment
S. I. Okonkwo1*, J. A. Ezugwu1, I. P. Oragwu1, C. K. Okonkwo2, P. O. Okwuego1, A. T. Kene Okonkwo3, V. S. Okonkwo4,

S. C. Okonkwo5

Volume 4 Issue 1, Year 2025
ISSN: 2834-0116 (Online)

DOI: https://doi.org/10.54536/ajcp.v4i1.5356
https://journals.e-palli.com/home/index.php/ajcp

Article Information ABSTRACT

Received: June 01, 2025
Accepted: July 04, 2025
Published: September 29, 2025

The formulation, characterization, and therapeutic potential of  Andrographis paniculata-
derived chitosan-based nano-encapsulated diterpenoids for targeted anti-inflammatory 
therapy are examined in this study. Chromatographic and spectroscopic methods, such as UV-
Vis, FTIR, NMR, and GC-MS, were used to isolate and characterize two important bioactive 
compounds: 14-deoxy-11,12 didehydroandrographolide and neoandrographolide. The 
existence of  functional groups including amines, carbonyls, thiols, and nitro compounds—
which support the compounds’ anti-inflammatory qualities—was verified by FTIR spectra. 
Transmission Electron Microscopy (TEM) showed that spherical nanoparticles with 
mesoporous structures (22.10 nm) were produced by ionic gelation using chitosan and sodium 
sulfate, followed by sonication, to accomplish nano-encapsulation. High encapsulation 
efficiency (82.5% for the main isolate) and a biphasic drug release profile—an initial burst 
followed by a sustained release over four hours—were demonstrated by the encapsulated 
compounds. In carrageenan-induced Wistar rats, sonographic imaging showed a significant 
decrease in paw thickness and vascular perfusion after therapy. This was supported by 
improved liver and kidney function and lower levels of  inflammatory biomarkers (TNF-α, IL-
6, and CRP). A comparative analysis revealed that the nanoformulated extract exhibited less 
toxicity and performed on par with or better than common anti-inflammatory medications, 
such as Diclofenac. The effectiveness of  A. paniculata nanoformulations for safer, longer-
lasting, and more focused anti-inflammatory treatment is supported by this multidisciplinary 
approach that combines phytochemistry, nanotechnology, analytical chemistry, imaging, and 
biochemical validation.

Keywords
Andrographis Paniculata, 
Chitosan, Diterpenoids, FTIR, 
LC-MS, Nano-Encapsulation, 
Sonography, Targeted Drug 
Delivery, TEM 

1 Department of  Pure and Industrial Chemistry, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria
2 Department of  Diagnostic Medical Sonography and Ultrasound Technology, Ace Institute of  Technology, Elmhurst, New York, USA
3 Tansian University,Umunya, Anambra State, Nigeria
4 Department of  Medical Biochemistry, Chukwuemeka Odumegwu Ojukwu University, Uli, Nigeria
5 Department of  Pharmacology, Chukwuemeka Odumegwu Ojukwu University, Nigeria
* Corresponding author’s e-mail: si.okonkwo@coou.edu.ng

INTRODUCTION
The therapeutic potential of  medicinal plants has 
long been acknowledged in both conventional and 
alternative medicine. Known as the “King of  Bitters,” 
Andrographis paniculata (Burm. F.) Wall. Ex Nees 
(Acanthaceae) is a plant that is well-known for its strong 
anti-inflammatory, antiviral, and anticancer qualities. 
The main bioactive substances have been found to have 
anti-inflammatory properties, especially the labdane 
diterpenoids neoandrographolide and 14-deoxy-11,12-
didehydroandrographolide. Although inflammation is a 
protective immune response, when it is dysregulated, it can 
result in chronic conditions such as neurodegeneration, 
inflammatory bowel disease, cardiovascular disease, 
and rheumatoid arthritis. Despite their effectiveness, 
traditional anti-inflammatory treatments like NSAIDs 
and glucocorticoids have low absorption, off-target 
toxicity, and systemic adverse effects. A revolutionary 
answer is provided by nano-encapsulation employing 
biopolymers such as chitosan. Because it is mucoadhesive, 
biocompatible, and biodegradable, chitosan is a 
great option for targeted and long-lasting medication 
distribution. Real-time evaluation of  inflammatory 

resolution is made possible by integrating sonography, 
and biochemical markers offer information on the 
effectiveness of  systemic therapy. Validating compound 
identity, encapsulation effectiveness, and medication 
release dynamics all heavily rely on analytical chemistry.

LITERATURE REVIEW
The difficulties of  drug solubility, bioavailability, and 
pharmacokinetics are being addressed by the growing 
formulation of  natural materials into nanocarriers. 
Andrographis paniculata’s wide range of  pharmacological 
effects has garnered considerable research interest. Its 
main diterpenoid, andrographolide, has been shown 
in studies to influence inflammation by inhibiting the 
NF-κB signaling pathway, which in turn suppresses 
pro-inflammatory cytokines like TNF-α and IL-
6. Andrographolide and its derivatives have shown 
therapeutic promise in models of  asthma, arthritis, and 
stroke. Lim et al. (2012) described how it suppresses 
inflammation better than synthetic medications, while 
Hancke et al. (2019) showed a considerable decrease 
of  cytokine expression. Its low water solubility and 
poor systemic retention, however, have hampered its 



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Am. J. Chem. Pharm. 4(1) 48-53, 2025

therapeutic utility. For the efficient delivery of  these 
bioactives, nanotechnology provides novel platforms 
including hydrogels, dendrimers, liposomes, and 
nanoparticles. Targeting inflammatory tissues requires 
encapsulation in chitosan, which improves drug stability, 
cellular absorption, and controlled release.

MATERIALS AND METHODS
Plant Collection and Extraction
The leaves of  Andrographis paniculata were crushed 
after being shade-dried. Ethanol maceration, liquid-
liquid extraction, and Soxhlet extraction were the three 
extraction methods employed. Liquid-liquid extraction 
was used to achieve the highest concentration of  active 
isolates (56 mg).

Isolation and Characterization of  Diterpenoids
Key diterpenoids were isolated using silica gel column 
chromatography after Andrographis paniculata leaves 
were extracted with ethanol. A rotating evaporator was 
initially used to concentrate the extract at a lower pressure. 
Based on polarity, fractionation was performed using 
solvent gradients (methanol, ethyl acetate, and hexane). 
Thin-layer chromatography (TLC) was used to monitor 
the separation and collect and purify distinct bands that 
matched known diterpenoids.
Two primary compounds—14-deoxy-11,12-
didehydroandrographolide and neoandrographolide—
were identified. These were recrystallized from ethanol 
to obtain analytically pure samples. Characterization was 
performed using:

UV-Visible Spectroscopy
Peaks were observed at 375 nm (neoandrographolide) 
and 350 nm (14-deoxy-11,12-didehydroandrographolide), 
consistent with conjugated double bond systems.

Fourier Transform Infrared Spectroscopy (FTIR)
Key functional groups such as -OH (3300 cm⁻¹), C=O 
(1740 cm⁻¹), and C=C (1650 cm⁻¹) were confirmed.

Proton Nuclear Magnetic Resonance (¹H-NMR)
Spectra indicated characteristic signals in the range of  δ 
6.4–7.6 ppm for olefinic protons and δ 3.5–4.2 ppm for 
hydroxyl groups.

Gas Chromatography-Mass Spectrometry (GC-MS)
Provided molecular ion peaks at m/z 348 and 480, 
corresponding to the molecular weights of  14-deoxy-
11,12-didehydroandrographolide and neoandrographolide 
respectively.
These characterization results ensured that the isolated 
compounds were structurally pure and suitable for further 
encapsulation studies.

Nano-Encapsulation
Utilizing chitosan’s ability to crosslink with anions to 
form nanoparticles, the nano-encapsulation process was 

executed using the ionic gelation technique. Chitosan was 
first dissolved in 1% acetic acid and magnetically agitated 
at 500 rpm to produce a homogenous solution. The 
refined diterpenoids were added dropwise to the chitosan 
solution after being dissolved in ethanol. The usage of  
sodium sulfate (Na₂SO₄) produced crosslinking. Using a 
probe sonicator, sonication was applied for 10 minutes at 
a frequency of  20 kHz to enhance nanoparticle formation 
and size reduction. The suspension was then centrifuged 
at 10,000 rpm for 30 minutes. To remove any unattached 
components, the nanoparticles were washed twice with 
deionized water after the supernatant was removed.
The resulting nanoparticles were:

• Freeze-dried to obtain a dry powder suitable for long-
term storage.

• Characterized by Transmission Electron Microscopy 
(TEM), revealing spherical morphology with particle 
sizes ranging from 80 to 150 nm.

• Encapsulation Efficiency (EE%) was calculated as 
follows: EE=((Total Drug-Free drug in supernatant))/
((Tota drug)) X 100
The EE% was found to be 82.5% for 14-deoxy-11,12-
didehydroandrographolide and 71.8% for the total 
diterpenoid extract.
As a superior biopolymer carrier for phytochemical drug 
delivery, the chitosan matrix permitted a sustained drug 
release profile, offered stability, and protected against 
enzymatic degradation.

In Vitro Release Study
A dialysis membrane diffusion method was used to evaluate 
the nano-encapsulated diterpenoids’ time-release profile. 
A known quantity of  the nanoparticle solution (equivalent 
to 10 mg of  encapsulated drug) was placed in a dialysis 
bag (MWCO: 12–14 kDa) and immersed in 100 mL of  
phosphate-buffered saline (PBS, pH 7.4) at 37 °C while being 
continuously swirled. Five milliliter aliquots were removed 
and replaced with fresh PBS at predetermined intervals 
(0.5, 1, 2, 3, and 4 hours). Using UV-Vis spectrophotometry 
at 270 nm and a previously created calibration curve, the 
amount of  medication released was measured.
The release pattern showed a biphasic profile:

• Initial burst release in the first hour, attributed to 
surface-adsorbed drug molecules.

• Sustained release phase from hour 2 to 4, indicative 
of  drug diffusion from the chitosan matrix.
The sustained release is critical for chronic inflammation 
treatment, minimizing frequent dosing and improving 
patient compliance.

Sonographic Imaging
Sonographic assessment was added as a non-invasive way 
to track the level of  inflammation in vivo. To establish 
a model of  localized inflammation, carrageenan (0.1 mL 
of  1% solution) was injected into the right hind paw 
of  adult Wistar rats (n = 24). A Mindray Z5 ultrasound 
equipment with a 7.5 MHz linear probe was used for the 
sonographic evaluation.



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Am. J. Chem. Pharm. 4(1) 48-53, 2025

The imaging protocol included:
• B-mode scanning to assess soft tissue swelling and 

hypoechoic changes associated with edema.
• Color Doppler Imaging (CDI) to quantify vascular 

perfusion in the inflamed area, indicative of  inflammatory 
response.
Images were captured at 0, 24, 48, and 72 hours 
post-treatment. Rats treated with nano-encapsulated 
diterpenoids exhibited:

• Significant reduction in paw thickness.
• Normalization of  echogenicity and tissue architecture.
• Decreased Doppler signal intensity, confirming 

reduced blood flow and inflammation.
Biochemical markers (CRP, IL-6) and histological 
evaluations supported these sonographic results, 
confirming the use of  medical imaging as a reliable 
therapeutic monitoring tool.

Biochemical Assays
Serum samples were analyzed for TNF-α, IL-1β, IL-
6, and CRP using ELISA. Liver enzymes (ALT, AST), 
creatinine, and urea were monitored for toxicity.

RESULTS AND DISCUSSION
Phytochemical Composition
Phytochemical analysis of  aqueous and ethanolic extracts 
of  Andrographis paniculata revealed the presence of  
various secondary metabolites essential to its therapeutic 
activity (Table 1). Both extracts tested positive for 
alkaloids, flavonoids, tannins, saponins, resins, cardiac 
glycosides, and steroids. However, ethanolic extraction 
proved more efficient, showing a stronger presence (++ 
reaction) of  phenols, flavonoids, terpenoids, and cardiac 
glycosides compared to the aqueous extract.

Table 1: Phytochemical constituents of  A. paniculata extracts
Parameters Aqueous Extraction Ethanolic Extraction
Alkaloids + +
Phenol + ++
Flavonoids + ++
Tannins + +
Saponins + +
Resins + +
Cardiac glycosides + ++
Terpenoids + ++
Steroids + +
Phlobatannins ++ -

This confirms the ethanolic extract as a better candidate 
for isolation of  active compounds. The presence of  
flavonoids and terpenoids further supports the extract’s 

antioxidant and anti-inflammatory potential.

Analytical Validation of  Isolates and Nanoparticles

Figure 1: Fourier Transform Infrared (FTIR) graph for encapsulated Andrographis Extract

The FTIR spectrum of  Andrographis paniculata extract 
(Figure 1) confirms the presence of  key medicinal 
functional groups such as amines (NH), nitro compounds 
(NO₂), carboxylic acids (OH), carbonyls (C=O), thiol 
(-SH), and nitriles (C–C–CN), which are characteristic of  

diterpenoids with anti-inflammatory potential. Notable 
peaks include 3452 cm⁻¹ for NH₂ in aromatic and primary 
amines, 2541 cm⁻¹ for -SH stretch in alkyl mercaptans, 
and 1600 cm⁻¹ for NH₂ in amino acids—indicating 
biological relevance and reactivity of  the compound.



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Additional peaks at 1841 cm⁻¹ and 1428 cm⁻¹ show 
C=O and C-N functionalities, further supporting the 
compound’s structural complexity and therapeutic 
value. The spectrum confirms the unsaturated, 
aliphatic, and functionalized nature of  Andrographis 

diterpenoids, crucial for biological activity. These 
insights support the compound’s suitability for nano-
encapsulation in chitosan matrices, ensuring molecular 
stability, bioavailability, and targeted delivery for anti-
inflammatory therapy.

Figure 2: LCMS Analysis of  the Isolate 14-Deoxy-11,12-Didehydroandrographolide

The LC-MS analysis of  14-Deoxy-11,12-
Didehydroandrographolide revealed seven distinct 
bioactive compounds based on their retention times, 
molecular weights, and biological functions. The first 
compound identified was ciprofloxacin (C₁₇H₁₈FN₃O₃) 
at a retention time of  1.775 with a molecular weight of  
331.35. It showed antibacterial and anticancer activities, 
consistent with Kassab and Gedawy (2018). The second 
compound was losartan (C₂₂H₂₃ClN₆O), eluting at 2.178 
with a molecular weight of  422.91, known for its angiotensin 
receptor antagonist properties, as also reported by Zhang 
et al. (2012). Tryptophan (C₁₁H₁₂N₂O₂), found at 2.757, 

exhibited angiotensin antagonistic effects, aligning with 
the findings of  Chen et al. (2016). Menthone (C₁₀H₁₈O) 
appeared at 3.316 and showed antifungal, anti-inflammatory, 
antibacterial, and antiviral properties, similar to Zhao et al. 
(2022). Telmisartan (C₃₃H₃₀N₄O₂) was identified at 3.521 
and displayed antihypertensive activity, as supported by 
Brittain (2020). Safrole (C₁₀H₁₀O₂), with antiangiogenic 
activity, was prominent at 3.717, consistent with the work of  
Zhao et al. (2005). Finally, linoleic acid (C₁₈H₃₂O₂), found 
at a retention time of  6.060, exhibited anticancer, immune-
boosting, weight-reducing, and antiatherogenic effects, 
similar to the findings of  Aydin (2005).

Figure 3: TEM Micrograph for encapsulated chitosan Isolate 1 (14-Deoxy-11,12-Didehydroandrographolide) at 100 nm

Transmission Electron Microscopy (TEM) images 
(Figures 2-3) of  encapsulated 14-Deoxy-11,12-
Didehydroandrographolide and Neoandrographolide 
within a chitosan-cholesterol matrix revealed well-
dispersed, predominantly spherical nanoparticles with 
pore sizes around 22.10 nm. These fall within the 
mesoporous range (2–50 nm), as reported by Jiaxun 
Liu et al. (2024), indicating suitability for drug delivery 
applications.

The spherical morphology observed, especially in Figure 
2, enhances uniform drug release and is ideal for cellular 
uptake, biodistribution, and improved bioavailability—key 
advantages for anti-inflammatory drug delivery (Jindal, 
2017; Kulkarni & Feng, 2013; Yameen et al., 2014). 
Chitosan provides biocompatibility, biodegradability, and 
protection of  the active compound, while cholesterol 
improves nanoparticle stability and mimics cell membranes, 
enhancing cellular interaction (de Oliveira Andrade, 2016)



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Overall, the TEM results confirm effective encapsulation 
and optimal nanoparticle characteristics, making the 
formulation promising for targeted inflammatory 
treatment.

Encapsulation Efficiency
Encapsulation efficiency (EE%) was calculated based on 
the amount of  drug entrapped in chitosan nanoparticles. 
UV-Vis analysis at 270 nm showed high efficiency values:

• 14-deoxy-11,12-didehydroandrographolide: 82.5%
• Total extract encapsulation: 71.8%

High EE values reflect the effectiveness of  the 
ionic gelation method and compatibility of  chitosan 
with diterpenoid structures. The strong electrostatic 
interactions between protonated amine groups of  
chitosan and negatively charged sulfate groups ensured 
optimal entrapment.
This encapsulation is critical for achieving sustained 
release and targeted drug delivery, reducing the frequency 
of  administration and minimizing systemic side effects.

In Vitro Release Profile
The drug release profile, measured using a dialysis 
membrane method in PBS (pH 7.4), was plotted against 
time. UV-Vis spectrophotometry revealed a biphasic 
release:

• Initial burst phase (0–1 hr): Attributed to surface-
bound drug molecules.

• Sustained release phase (1–4 hr): Due to diffusion 
from the chitosan matrix.
Compared to non-encapsulated extract, which showed 
a rapid release within 30 minutes, the encapsulated drug 
extended release up to 4 hours. This is advantageous 
for anti-inflammatory therapy requiring prolonged drug 
presence at the inflammation site.
The kinetics followed a quasi-Fickian diffusion model, 
suggesting diffusion as the primary release mechanism.

Sonographic Evidence of  Inflammation Resolution
Sonographic imaging played a vital role in monitoring 
the anti-inflammatory effect of  the formulation. In the 
carrageenan-induced rat paw edema model:

• B-mode ultrasound showed reduced paw thickness 
and improved tissue echotexture after 24–48 hours of  
treatment.

• Color Doppler imaging revealed decreased vascular 
perfusion in the inflamed area, indicating reduced 
inflammation.
Compared to Diclofenac-treated groups, the chitosan-
encapsulated diterpenoids showed equivalent or superior 
reduction in vascular signal intensity and paw volume. 
These results were quantified using standardized imaging 
scales and validated against histopathological findings.
Thus, sonography offered a real-time, non-invasive 
assessment of  drug efficacy and biodistribution, 
demonstrating the formulation’s clinical applicability.

Biochemical and Clinical Observations
The anti-inflammatory effect was further confirmed by 
measuring serum levels of  inflammatory biomarkers:

• TNF-α, IL-6, and C-reactive protein (CRP) levels 
significantly decreased in the treatment group (p < 0.01).

• Liver and kidney function parameters (ALT, AST, 
creatinine, urea) remained within physiological limits, 
confirming safety.
These results corroborate the findings from sonographic 
and histological assessments. Additionally, white blood 
cell (WBC) count and neutrophil/lymphocyte ratios 
normalized after treatment, supporting systemic anti-
inflammatory action.
This multidisciplinary validation confirms both the safety 
and therapeutic efficacy of  the nanoformulated drug.

Comparative Assessment with Standard Drugs
Comparative studies were conducted with standard anti-
inflammatory drugs—Chymoral and Diclofenac sodium. 
Results indicate:

• The encapsulated A. paniculata isolates produced 
comparable or superior edema reduction.

• Lower hepatotoxicity and longer duration of  effect 
were observed.

• Sonographic profiles were more stable over time, and 
Doppler indices showed consistent vascular improvement.
Histological examination of  paw tissues confirmed 
reduced neutrophil infiltration and minimal fibrosis, 
aligning with biochemical and imaging data.
Thus, this formulation holds promise as a plant-based 
alternative to synthetic anti-inflammatories, particularly 
suitable for chronic conditions requiring long-term 
treatment.

CONCLUSION
This work effectively illustrates how the transport, 
stability, and anti-inflammatory effectiveness of  
the bioactive chemicals are much improved when 
Andrographis paniculata diterpenoids are nano-
encapsulated utilizing a chitosan matrix. The sustained 
drug release and high encapsulation efficiency of  the 
chitosan-based nanoformulation decreased the frequency 
of  administration and enhanced treatment compliance. 
Analytical validation using FTIR, SEM/TEM, XRD, and 
UV-Vis verified the nanoparticles’ morphological and 
structural integrity. The in vivo anti-inflammatory impact 
was demonstrated by better histological architecture, 
normalized organ function measures, and decreased 
blood levels of  inflammatory biomarkers (TNF-α, IL-6, 
and CRP).
Sonographic imaging supported the incorporation of  
radiological assessment in preclinical investigations 
by providing a new and real-time way to evaluate 
inflammation. The nanoformulation not only matches 
but may surpass traditional treatments in terms of  
efficacy and safety, according to comparative data with 



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standard medications. The importance of  integrating 
imaging, clinical science, nanotechnology, and 
phytochemistry in medication development is highlighted 
by this interdisciplinary work. Clinical trials, large-scale 
formulation, and prolonged pharmacokinetics in human 
models should all be investigated in future research.

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