In Vitro and In-Vivo Analysis of the Combined Extract of Tabernaemontana divaricata and Mangifera indica Leaves for Enhanced Anti-Inflammatory and Antioxidant Properties

doi:10.21203/rs.3.rs-5344453/v1

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Research Article

In Vitro and In-Vivo Analysis of the Combined Extract of Tabernaemontana divaricata and Mangifera indica Leaves for Enhanced Anti-Inflammatory and Antioxidant Properties

https://doi.org/10.21203/rs.3.rs-5344453/v1

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Ethnopharmacological Relevance: Tabernaemontana divaricata

(T. divaricata) and Mangifera indica (M. indica) are plants traditionally used in Ayurvedic medicine for their anti-inflammatory and antioxidant properties. T. divaricata is known for its analgesic and wound-healing effects, while M. indica is celebrated for its role in managing inflammation and oxidative stress.

Aim of the Study:

The study aimed to evaluate the synergistic effects of combined hydroalcoholic extracts from T. divaricata and M. indica leaves, focusing on their anti-inflammatory and antioxidant properties.

Materials and Methods

The leaves were collected, authenticated, and subjected to hydroalcoholic extraction. The extracts were then combined in various ratios and analyzed for phytochemical content. The antioxidant activity was evaluated using the DPPH assay, while anti-inflammatory effects were assessed through the carrageenan-induced rat paw edema model.

Results

The combined extracts exhibited significantly enhanced antioxidant and anti-inflammatory activities compared to the individual extracts. The most potent combination, a 2:1 ratio of M. indica to T. divaricata, showed the highest DPPH inhibition (94.37%) and maximum edema reduction in the animal model.

Conclusion

The combination of T. divaricata and M. indica extracts offers a synergistic therapeutic potential, enhancing both antioxidant and anti-inflammatory effects, which supports their traditional use and suggests potential for developing new natural therapeutics.

Clinical Pharmacology

Anti-inflammatory

Anti-oxidant

Tabernaemontana divaricate

Mangifera indica

Inflammation and oxidative stress are two pivotal processes implicated in the pathogenesis of various chronic diseases, including cardiovascular disorders, cancer, diabetes, and neurodegenerative conditions (Leyane, Jere, and Houreld 2022). The increasing prevalence of these conditions has heightened the search for natural remedies that can offer effective therapeutic interventions with minimal side effects. In this context, plants and their bioactive compounds have garnered significant attention due to their historical use in traditional medicine and their potential in modern therapeutics (Pandey et al. 2011).

Tabernaemontana divaricata, commonly known as the crepe jasmine (Ghosh, Poddar, and Chatterjee 2021). Tabernaemontana divaricata belongs to the family Apocynaceae. This family is commonly known as the dogbane family, and it includes a wide variety of flowering plants, many of which have medicinal properties. Tabernaemontana divaricata is widely used in Ayurvedic medicine, primarily for its anti-inflammatory, analgesic, and wound-healing properties. The plant is rich in alkaloids, flavonoids, and terpenoids, which contribute to its therapeutic effects.

Mangifera indica, known as the mango tree (Ghosh, Poddar, and Chatterjee 2021) is the plants traditionally recognized for their medicinal properties. Mangifera indica belongs to the family Anacardiaceae, which is known for including several economically important fruit-bearing trees and plants. Mangifera indica, on the other hand, is renowned not only for its nutritional value but also for its extensive medicinal applications (Ediriweera, Tennekoon, and Samarakoon 2017), particularly in treating inflammation, infections, and oxidative stress-related conditions. The leaves of Mangifera indica are known to contain polyphenols, flavonoids, and other potent antioxidants (Mirza et al. 2021).

Given the individual therapeutic properties of these plants, combining the extracts of Tabernaemontana divaricata and Mangifera indica leaves presents a promising approach to enhancing their anti-inflammatory and antioxidant effects. The rationale behind this combination is to leverage the synergistic effects of the bioactive compounds present in both plants, which may result in a more potent therapeutic outcome than the individual extracts.

This study aims to explore the anti-inflammatory and antioxidant properties of the combined extract of Tabernaemontana divaricata and Mangifera indica leaves. By evaluating these effects through various in vitro and in vivo models, the research seeks to establish a scientific basis for the traditional use of these plants and potentially contribute to the development of new natural therapeutics for managing inflammatory and oxidative stress-related conditions.

2.1 Collection and identification of plant material

Tabernaemontana divaricata and Mangifera indica leaves were gathered from the surrounding area of Bhopal, Madhya Pradesh, and verified at RB Science, Bhopal. leaves were gathered from a nearby farm in Bhopal, Madhya Pradesh, and verified at RB Science, Bhopal.

2.2 Chemicals and reagents

Analytical grade compounds were all that were used. Reagents are chemicals used to prepare buffers, analytical solutions, and other things for experiments. The leaves of Tabernaemontana divaricata and Mangifera indica were freshly collected from the local area, providing the primary plant materials for the study. Several chemicals were sourced from well-known suppliers: ethanol was procured from Loba Chemie (P) Ltd, Mumbai, while gallic acid and vanillin were obtained from CDH, New Delhi. Folin-Ciocalteu reagent was sourced from Avra, Hyderabad, and sodium carbonate and glacial acetic acid were acquired from Oxford Fine Chemicals, Mumbai. Methanol was provided by S.D. Fine Chemicals, Mumbai, and petroleum ether and chloroform were purchased from Rankem, Mumbai. Additionally, sulfuric acid was also supplied by Rankem, Mumbai. For experiments requiring water, freshly distilled water was prepared in the laboratory to ensure purity.

2.3 Extraction Process of leaves of Taebermontana divaricata and Mangifera indica

Powdered leaves (91 g, Taebermontana divaricata; 102 g Mangifera indica) were separately sealed in the Soxhlet apparatus's extractor and defatted with petroleum ether utilizing heated continuous extraction process until colorless siphoning solution is obtained (5–6 h). The extraction After the solvent was eliminated, the marc was dried, and then extracted with a blend of ethanol-water (70:30) as the solvent. The extracts were filtered hot to remove impurities and the solvent was evaporated using rotary vacuum evaporator. To eliminate the oleo-resinous extract, it was gathered and put in a water bath remaining solvent and finally placed in desiccator maximum drying. The dried/semidried extracts were stored in desiccator for further experimental procedures.

2.4 Qualitative Phytochemical Screening

Both thehydro-alcoholic extracts were assessed using phytochemical qualitative responses to determine whether typical plant secondary metabolites are present or absent. Many classes, including triterpenes/steroids, alkaloids, glycosides, flavonoids, saponins, tannins, and phenolics, were screened for. As analytical reactions to these tests, the color intensity or the precipitate formation were employed (Banu and Cathrine 2015).

2.4.1 Test for Alkaloids

Mayer’s test: To a few ml of plant sample extract, two drops of Mayer’s reagent was added along the sides of test tube.
Wagner's test: Test tube walls were lined with a few drops of Wagner's reagent mixed with a few milliliters of plant extract.
Hager's test: In the test tube, a small amount of plant extract and a few drops of Hager's reagent are applied along the sides.
Dragendroff’s Test: Each extract was mixed with one milliliter (ml) and a few drops of Dragendroff's solution.

2.4.2 Test for Glycosides

Froth test: A test tube containing 1ml of the extract in water was filled and given a good shaking.
Borntrager's test: The extract and 1.0 milliliter of diluted sulfuric acid were combined in a test tube and brought to a boil for five minutes. Following the chilling and shaking of the filtrate with an equivalent volume of dichloromethane, the bottom layer (dichloromethane) was separated and shaken with half its volume of diluted ammonia.
Kedde’s test: After extracting the material using hloroform, evaporation dries it out. One drop of 90% alcohol and two drops of 2% 3, 5-dinitro benzoic acid (3, 5-dinitro benzene carboxylic acid, Kedde's reagent) in 90% alcohol should be added to the previously indicated residue. The solution becomes alkaline when 20% sodium hydroxide solution is added.
Keller killiani test (Test for deoxy sugars): Following a chloroform extraction, the extract is evaporated until it is completely dry. 0.4 milliliters of glacial acetic acid with a tiny amount of ferric chloride solution were added to the residual. After moving the mixture into a test tube, 0.5 ml of concentrated sulfuric acid was applied along the test tube's wall.

2.4.3 Test for Tannins and phenolic compounds

Gelatin test: To the extract, a 1% gelatin solution containing 10% sodium chloride was added.
Ferric chloride test: Iron chloride solution that had been newly made was added to the extract.
Vanillin hydrochloride test: A few drops of vanillin hydrochloride reagent were added to the extract test solution for treatment.
Alkaline reagent test: Sodium hydroxide solution was used to treat the extract test solution.

2.4.4 Test for flavonoids

Shinoda test: To Conc. hydrochloric acid was added dropwise to the extract test solution along with a few pieces of magnesium ribbon.
Zinc hydrochloride reduction test: A mixture of concentrated hydrochloric acid and zinc dust was added to the test solution.
Alkaline reagent test: The test solution received a few drops of sodium hydroxide solution added to it. A small amount of strong hydrochloric acid was added. Later, if color showed.

2.4.5 Proteins and amino acids

Millons test: The extract's Two milliliters of Millon's reagent (mercuric nitrate in nitric acid with traces of nitrous acid) were mixed with the test solution, and left to react.
Ninhydrin test: A 0.2% ninhydrin solution was added to the extract solution and brought to a boil.

2.4.5 Sterols and terpenoids

Libermann-Burchard test: After adding a few drops of acetic anhydride to the extract, it was heated and chilled. Concentrated sulfuric acid was poured into the test tube from the sides.
Salkowski test: By dissolving the extract in chloroform, a little amount of concentrated sulfuric acid was included. After giving the mixture a good shake, it was allowed to stand for some time.

2.5 Chromatographic analysis of the extracts

The extracts were dissolved in ethanol by sonication and filtered through a 0.45µ nylon syringe filter prior to injecting in the sample loop of HPLC instrument.

Acetonitrile-buffer (pH 2.5) (15:85) was the solvent system used to extract Mangifera indica; the C18 column (chromosil, 5µ) was used to measure the flow rate, which was set at 1.0 mL per minute. The extract's mangiferin content was found using a 253 wavelength (KUMAR, SRIVASTAVA, and KUMAR¹ 2008). After injecting a 20µL sample into the sample loop, the chromatogram was acquired. Using the aforementioned technique, a chromatogram of standard mangiferin (5µg/mL) was also produced. Acetonitrile-water (pH 2.5) (95:5) was the solvent system used to extract Taebermontana divaricata. A C8 column (chromosil, 5µ) was used to measure the flow rate, which was set at 1.5 mL per minute. The presence of ß-sitosterol in the extract was determined using a wavelength of 202 (Shah et al. 2010). An example 20µL was injected into the sample loop and the chromatogram was obtained. A chromatogram of standard ß-sitosterol (5µg/mL) was also chromatographed using the above method.

2.6 FT-IR analysis of the extracts

The dried extracts were scanned in the range of 400 to 4000 cm-1 using a FT-IR spectrophotometer and the stretching and bending vibrations were observed.

2.7 Total Phenolic Content

An adaptation of the procedure described served as the foundation for the extraction of phenolic chemicals. One dried extract (0.1 g) was dissolved in 5 mL of ethanol to determine the total phenolic content. The fixes served as the stock solutions for further studies and were kept in amber-colored bottles at 4°C (Mishra and Jain 2021).

In order to ascertain the overall phenolic content One milliliter (200 µL) of the extract sample was combined with 1.4 milliliters Folin-Ciocalteu reagent (100 µL). After two minutes, add 300 µL of a 20% Na2CO3 aqueous solution, and let the mixture remain for two hours. A UV-Vis spectrophotometer was used to measure the absorbance at 765 nm. To construct the calibration curve, standard solutions containing 10–100 ppm of gallic acid were treated in a similar manner. With the same chemicals and 200 µL of methanol, the control solution was prepared and incubated similarly to the other samples. The results were expressed in milligrams per 100 mg of the dry material using the gallic acid equivalent (GAE).

2.8 Preparation the combined extracts for the antioxidant and anti-inflammatory effect

The hydro-alcoholic extracts from Mangifera indica and Taebermontana divaricata were combined in three different ratios (1:1, 1:2, and 2:1), respectively, and the antioxidant and anti-inflammatory effect was assessed using the techniques described in the following sections. The statistical significance of the combined extracts' antioxidant and anti-inflammatory properties was examined by comparing them to those of the individual extracts.

2.9 Evaluation of Anti-oxidant Activity

The steady free radical DPPH was used to gauge the test solution's free radical scavenging activity in terms of its capacity to donate hydrogen or scavenge radicals. The previously described approach was used to determine the DPPH radical scavenging activity (Amreen and Chaurey 2021). A 1 mM DPPH solution and an extract solution (100 µg/mL) were produced separately in ethanol. A 1.5 ml DPPH solution was mixed with 1.5 ml of the test solution. The equivalent blank solution, made with 3 mL of ethanol, was used to measure the absorbance at 517 nm. 3 mL of DPPH was the control sample that was used. Three duplicates of the assay were run. The following formula was used to determine the percentage inhibition of the free radical DPPH based on the control reading.

$$\:\:\varvec{D}\varvec{P}\varvec{P}\varvec{H}\:\varvec{s}\varvec{c}\varvec{a}\varvec{v}\varvec{e}\varvec{n}\varvec{g}\varvec{e}\varvec{d}\:\left(\varvec{\%}\right)=\frac{Acom-Atest}{Acom}\times\:1000$$

Where, A _con - is the absorbance of the control reaction, A _test - is the absorbance in the presence of the test solution.

2.10 Animal

Adult male Wistar rats weighing 250–300g were maintained in the Institute of Biological Science of the Federal University of Rio Grande at 22 ± 2ºC, with a relative humidity of 50–60% under a 12–12 h light-dark cycle with food and water ad libitum. The experiments were performed after approval of the protocol by the Institutional Ethics Committee (approval number P021/2013).

2.11 Experimental protocol

Animals were distributed in six groups and received the following treatments:

Group I - Control - treated with vehicle (normal saline)

Group II– Taebermontana divaricata extract (200 mg/kg)

Group III – Mangifera indica extract (200 mg/kg)

Group IV – Combined extract 1:1 (100 mg/kg)

Group V – Combined extract 1:2 (100 mg/kg)

Group VI – Combined extract 2:1 (100 mg/kg)

2.12 Evaluation of anti-inflammatory action

The anti-inflammatory activity of the extracts was assessed using the rat paw edema technique caused by carrageenan (Kemisetti and Manda 2018).

To induce paw oedema, 0.1 mL (1% solution) of carrageenan was subcutaneously injected into the plantar surface of the rat's right hind paw. Thirty minutes prior to the carrageenan injection, each animal group received a dosage of extracts at a rate of 100 mg/kg. The following groups (n = 6) of animals were formed.

Using a vernier caliper, the Paw diameters were measured immediately prior to the administration of carrageenan, and then at 1, 2, 4, and 6 hours later. The outcomes were contrasted with those of the control group. The following formula was used to determine each group's % suppression of paw inflammation:

$$\:\varvec{\%}\:\varvec{i}\varvec{n}\varvec{h}\varvec{i}\varvec{b}\varvec{t}\varvec{i}\varvec{o}\varvec{n}\:=\:\frac{C-T}{C}\times\:100$$

Where, C = Paw volume (mL) in vehicle treated group (control), T = Paw volume (mL) in drug treated group

3.1 Extraction yield

The defatting of the leaves was done by petroleum ether and the extraction of desired phytoconstituents by ethanol-water (70:30) as the solvent blend (Tables 1 and 2).

Table 1

Extract yield and features of *Mangifera indica*
Extraction solvent	Marc weight	Extract Weight	Yield of extract	Color of extract
Petroleum Ether	102 g	1.4 g	1.37%	Light Green
Ethanol-water (70:30)	98.4 g	8.6 g	8.74%	Dark Brown

Table 2

Extract yield and features of *Taebermonatana divaricata*
Extraction solvent	Marc weight	Extract Weight	Yield of extract	Color of extract
Petroleum Ether	91 g	1.1 g	1.21%	Light Brown
Ethanol-water (70:30)	88.7 g	9.1 g	10.26%	Dark Green

It was found that the hydro-alcoholic solvent blend was able to extract more contents from T. divaricata as compared to M. indica while the fatty material in both the plants was almost similar.

3.2 Preliminary phytochemical Screening

The extracts included alkaloids, flavonoids, triterpenoids, phenols, tannins, and saponins, as shown by the qualitative phytochemical screening.

Table 3

Qualitative phytochemical screening observations.
Chemical Tests	Observation	M. indica	T. divaricata
Test for Alkaloids
Mayer’s reagent	Cream colour precipitate	+	+
Hager’s reagent	Yellow colour precipitate	-	+
Wagner’s reagent	Reddish brown precipitate	+	+
Dragendorff’s reagent	Reddish brown precipitate	+	+
Test for Glycosides
Froth test	Frothing is seen	+	+
Kedde's Test	No color	-	-
Bontrager's Test	Rose pink or red color in the ammonical layer not found	-	-
Keller-Kiliani	No color in acetic acid layer	-	-
Test for Phenolics and Tannins
Ferric chloride	Blue green color	-	+
Gelatin Solution	White precipitate	+	+
Alkaline reagent test	Yellow to red precipitate	+	+
Vanillin HCl test	Purplish red color	+	+
Test for Flavonoids
Shinoda test	Red color	+	+
Alkaline reagent test	Yellow color that turns red on acidification	+	+
Zinc HCl reduction test	Red color	+	+
Test for Proteins
Millon's Test	White precipitate, turns red on heating	-	-
Ninhydrin Test	Voilet color	-	-
Test for Sterols/triterpenoids
Liberman-Burchard Test	Brown ring at junction Upper layer turns green	-	+
Salkowski Test	Yellow color in lower layer	-	-

3.3 Chromatographic and spectral analysis of extracts

The hydro-alcoholic extracts of both the plants were analyzed by HPLC for the presence of specific phytoconstituent (qualitatively). The M. indica extract was found to contain Mangiferin with retention time of 2.819 (Figs. 2 and 3), while the T. divaricata extract exhibited the presence of ß-sitosterol at retention time 5.82 minutes (Figs. 4 and 5).

3.4 FTIR results

A broad peak around 3400 − 3200 cm⁻¹, which may correspond to O-H stretching vibrations, indicating the presence of hydroxyl groups (alcohols, phenols), 1700 cm⁻¹ suggests the presence of carbonyl groups, which could indicate the presence of ketones, aldehydes, or carboxylic acids, 2850–3000 cm⁻¹ suggest C-H stretching, which is typical in alkanes and other hydrocarbon chains, 1600 − 1500 cm⁻¹, which may indicate C = C stretching in aromatic rings, 1050–1250 cm⁻¹ is typical of C-O stretching vibrations, which can be found in alcohols, ethers, esters, and carboxylic acids (Fig. 6).

The FTIR spectrum of Tabernaemontana divaricata extract presents distinct peaks indicative of various functional groups. A broad peak around 3400 − 3200 cm⁻¹ corresponds to O-H stretching, suggesting the presence of hydroxyl groups commonly found in alcohols and phenols. The peak near 1700 cm⁻¹ indicates C = O stretching, pointing to the presence of carbonyl groups, which may be associated with ketones, aldehydes, or carboxylic acids. Peaks around 2850–3000 cm⁻¹ represent C-H stretching vibrations typical of aliphatic hydrocarbons. The peak near 1600 cm⁻¹ is likely due to C = C stretching, often associated with aromatic rings. Additionally, the region around 1050–1250 cm⁻¹ suggests C-O stretching vibrations, indicating the presence of alcohols, ethers, or ester functional groups. This analysis highlights the diverse chemical nature of the T. divaricata extract, confirming the presence of multiple functional groups, including hydroxyl, carbonyl, and aromatic structures (Fig. 7).

3.5 Total Phenolic Content

As one of the most stable and reasonably priced natural phenols, gallic acid was utilized as the benchmark for determining the total phenolic content. Using the blue color absorption data collected in the concentration range of 10 to 100 ppm, the standard curve for gallic acid was created (Table 4 and Fig. 8). Regression coefficient and Beer's law range were ascertained from this. Gallic acid's linearity equation was determined to be Abs = 0.006x − 0.0042. GAE mg/100 mg was the determined total Phenolic content of the samples

Table 4

Calibration curve data for TPC.
Concentration ppm	Absorbance at 765 nm
0	0
10	0.051
20	0.118
30	0.173
40	0.233
50	0.291
60	0.348
70	0.422
80	0.475
90	0.550
100	0.598

The amount of phenolics present (total) in the M. indica and T. divaricata extracts were found to be 62.5 GAE mg/100 mg and GAE 55 mg/100 mg respectively.

3.6 Pharmacological evaluation

The anti-oxidant and anti-inflammatory properties, extracts of M. indica and T. divaricata were combined in three different ratios. These combined extracts were labelled as C1, C2 and C3 containing 1:1, 1:2 and 2:1 ratio of M. indica and T. divaricata respectively.

3.6.1 DPPH radical scavenging assay

Using the DPPH radical scavenging assay method, the combined extracts' antioxidant activity was ascertained. When appropriate reducing chemicals are used to react with DPPH radicals, the radicals lose color stoichiometric to the number of electrons consumed, which may be detected using a 517 nm using spectrophotometer. If DPPH has antioxidant properties, the deep purple color of the molecule becomes less intense. The antioxidant activity was found to be synergistically improved on combining the extracts (Table 5 and Fig. 9).

Table 5

Inhibition of DPPH radical.
Test solution	C1	C2	C3	M. indica	T. divaricata
% DPPH inhibition	65.8 ± 0.361	82.9 ± 1.513	94.37 ± 0.702	44.8 ± 1.127	35.87 ± 1.464

It was found from the result that combining the extracts resulted in synergizing the antioxidant activity when compared to the individual extracts. The highest amount of DPPH inhibition was witnessed in C3 (94.37 ± 0.702%) where M. indica and T. divaricata were mixed in the ratio 2:1 (Fig. 9).

3.6.2 Anti-inflammatory activity

The anti-inflammatory characteristics of the combined extracts were examined using the rat carrageenan-induced paw edema approach. Acute inflammation caused by carrageenan is one of the finest test methods for anti-inflammatory medication evaluation. Having the capacity to extracts and the mixed extracts to inhibit edema on pretreatment was calculated from the paw diameter obtained at different times post treatment (Table 6 and Fig. 10).

Table 6

Rat paw diameter and edema inhibition.
Group	Paw thickness (mm)/Edema inhibition (%)
Group	1h	2h	3h	4h
Control	0.488 ± 0.032	0.674 ± 0.030	0.794 ± 0.017	0.832 ± 0.025
Ibuprofen	0.272 ± 0.036 (44.26)	0.352 ± 0.016 (47.77)	0.362 ± 0.041 (54.41)	0.216 ± 0.023 (74.04)
M. indica	0.420 ± 0.056 (13.93)	0.446 ± 0.036 (33.83)	0.450 ± 0.037 (43.32)	0.414 ± 0.026 (50.24)
T. divaricata	0.452 ± 0.024 (7.38)	0.508 ± 0.016 (24.63)	0.524 ± 0.009 (34.01)	0.442 ± 0.019 (46.88)
C1	0.392 ± 0.020 (19.67)	0.434 ± 0.023 (35.61)	0.436 ± 0.019 (45.09)	0.402 ± 0.048 (51.68)
C2	0.372 ± 0.027 (23.77)	0.422 ± 0.024 (37.39)	0.428 ± 0.016 (46.10)	0.376 ± 0.030 (54.81)
C3	0.352 ± 0.029 (27.87)	0.406 ± 0.032 (39.76)	0.418 ± 0.013 (47.36)	0.326 ± 0.015 (60.82)

The study concludes that the combination of Mangifera indica and Tabernaemontana divaricata leaf extracts significantly enhances their antioxidant and anti-inflammatory properties compared to the individual extracts. The observed synergistic effects, particularly in the 2:1 ratio of M. indica to T. divaricata, suggest that these plant extracts could be effectively utilized in developing natural therapeutics for managing conditions associated with oxidative stress and inflammation. The presence of bioactive compounds such as flavonoids and phenolics likely contributes to these enhanced activities, reinforcing the potential of these plant combinations in therapeutic applications. Research results led to the conclusion that combining the extracts greatly enhanced their anti-inflammatory and antioxidant properties. The greatest increase in activity was specifically observed when two parts M. indica extract and one part T. divaricata extract were used together.

Author Contributions

Ganesh Mangesh Walanj, Pranjali Thakare and Nasir Ahmad Sarwary: Conceptualization, methodology, Data collection, analysis, writing – original draft.
Anand Kumar, Dilip KumarTiwari, Abhishek Sharma and Devesh Kumar: Data collection, project administration, writing – review & editing.

Competing Interests

The authors declare that there are no competing interests related to this publication.

Ethics approval

This study was conducted in accordance with the ethical standards of the Institutional Review Board of the College of Food Science and Technology, Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety, Guangdong Ocean University, Zhanjiang, China, and the Laxmi Narayan College of Pharmacy, Department of Pharmacology, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, India. Informed consent was obtained from all participants involved in the study. All procedures performed were in line with the Declaration of Helsinki.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request, especially for those in need of the data for further research.

Consent to participate

Informed consent was obtained from all individual participants included in the study. Informed consent was obtained from all participants prior to their inclusion in the study. Participants were fully informed about the nature, purpose, and potential risks of the research, and their participation was voluntary.

Consent to publish

All authors have given their consent for the publication of this manuscript. Additionally, informed consent was obtained from participants for the publication of any identifying information and data.

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In Vitro and In-Vivo Analysis of the Combined Extract of Tabernaemontana divaricata and Mangifera indica Leaves for Enhanced Anti-Inflammatory and Antioxidant Properties

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Abstract

Ethnopharmacological Relevance: Tabernaemontana divaricata

Aim of the Study:

Materials and Methods

Results

Conclusion

Figures

1. Introduction

2. Material and methods

2.1 Collection and identification of plant material

2.2 Chemicals and reagents

2.3 Extraction Process of leaves of Taebermontana divaricata and Mangifera indica

2.4 Qualitative Phytochemical Screening

2.4.1 Test for Alkaloids

2.4.2 Test for Glycosides

2.4.3 Test for Tannins and phenolic compounds

2.4.4 Test for flavonoids

2.4.5 Proteins and amino acids

2.4.5 Sterols and terpenoids

2.5 Chromatographic analysis of the extracts

2.6 FT-IR analysis of the extracts

2.7 Total Phenolic Content

2.8 Preparation the combined extracts for the antioxidant and anti-inflammatory effect

2.9 Evaluation of Anti-oxidant Activity

2.10 Animal

2.11 Experimental protocol

2.12 Evaluation of anti-inflammatory action

3. Results and Discussion

3.1 Extraction yield

3.2 Preliminary phytochemical Screening

3.3 Chromatographic and spectral analysis of extracts

3.4 FTIR results

3.5 Total Phenolic Content

3.6 Pharmacological evaluation

3.6.1 DPPH radical scavenging assay

3.6.2 Anti-inflammatory activity

4. Conculsion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1